Citation
Structural Genomics of Fragaria--Wild and Cultivated Strawberries

Material Information

Title:
Structural Genomics of Fragaria--Wild and Cultivated Strawberries
Creator:
Tombolato, Denise C
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (220 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Horticultural Sciences
Committee Chair:
Folta, Kevin M.
Committee Members:
Settles, Andrew M.
Chandler, Craig K.
Peres, Natalia
Graduation Date:
8/11/2007

Subjects

Subjects / Keywords:
Diploidy ( jstor )
DNA ( jstor )
Genomes ( jstor )
Genomics ( jstor )
Incubation ( jstor )
Nucleic acids ( jstor )
pH ( jstor )
Polymerase chain reaction ( jstor )
Species ( jstor )
Strawberries ( jstor )
Horticultural Science -- Dissertations, Academic -- UF
ananassa, annotation, colinearity, dna, extraction, fragaria, gene, genetic, genome, genomics, haplotype, iinumae, isolation, linkage, mandshurica, mapping, markers, molecular, nilgerrensis, nubicola, pair, prediction, ssr, strawberry, structural, vesca, viridis
Genre:
Electronic Thesis or Dissertation
born-digital ( sobekcm )
Horticultural Science thesis, Ph.D.

Notes

Abstract:
The extensive phenotypic variability and complex genetic makeup of the cultivated strawberry Fragaria X ananassa permits advances in plant improvement, a factor breeders have exploited to great benefit. However, the introgression of specific characters is complicated due to the cumbersome genetics and limited knowledge of genome structure and function of genes relevant to traits of interest. The present study represents the first genomics-level insight into strawberry genome structure and explores the hypothesis that a new type of molecular marker, the Gene-Pair Haplotype represents a transferable marker that may hasten linkage mapping in the diploid and octoploid strawberry. My research presents the findings of four related research activities. First, an efficient and unified method for genomic DNA isolation was derived from over 100 experimental tests and conditions. Next, 1% of the Fragaria genome was sequenced and functionally annotated, using a bioinformatics approach and computational tools. Over 120 kb of intergenic regions were sequenced using the Gene-Pair-Haplotype approach, allowing for some initial relationships to be formulated concerning the diploid subgenome contribution to octoploid strawberry. Finally, Gene-Pair Haplotypes were used to add a suite of alleles to the growing Fragaria linkage map. These findings provide a starting point for further analyses of the strawberry genome. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2007.
Local:
Adviser: Folta, Kevin M.
Statement of Responsibility:
by Denise C Tombolato.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Tombolato, Denise C. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2007 ( lcc )

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Full Text







Table 4-2.
Primer
name


continued
Template


17022 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'
27F10 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'
29G10 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'
32L07 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'
34D20 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'
40M11 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'


PCR
product
size (kb)


1.4
1.4
1.4 & 1.0
1.4

1.4
1.5 & 1.4

1.0
1.5
1.0
1.0
1.8
1.0
1.0




0.7
0.7
0.7


2.7
1.9


Clone # Vector


library
678
668
653

655


library
2039-1
2040-1
2041-1

2043-1
2046-1
2046-2
library


2049-1
2050-1
2051-1


library
640
1090-11


pJET1
pJET1
pJET1

pJET1



pJET1
pJET1
pJET1

pJET1
pJET1
pJET1



pJET1
pJET1
pJET1



pJET1
TOPO


E. coli Sequence
strain obtained
from
forward
end (bD)


XL1-Blue
XL1-Blue
XL1-Blue

XL1-Blue



XL1-Blue
XL1-Blue
XL1-Blue

XL1-Blue
XL1-Blue
XL1-Blue



XL1-Blue
XL1-Blue
XL1-Blue



XL1-Blue
TOP10


Sequence
obtained
from reverse
end (bp)


Full clone
Full clone
Full clone

Full clone


Full clone
Full clone

Full clone

Full clone



Full clone
Full clone
Full clone



Full clone


2.7 647 pJET1 XL1-Blue No seq
2.7 993 TOPO TOP10 No seq
2.7 1000 TOPO TOP10 No seq
I attempted to amplify fragment from the octoploids 'Carmine', 'Diamante', 'Rosa
Linda', and 'Sweet Charlie', but amplification was not observed for any of them
2.0 1826-3 pJET1 XL1-Blue library
2.0 1827-3 pJET1 XL1-Blue Full clone
2.0 1828-4 pJET1 XL1-Blue Full clone
2.0 1829-1 pJET1 XL1-Blue Full clone
2.0 1830-3 pJET1 XL1-Blue Full clone
2.0 1831-5 pJET1 XL1-Blue Full clone
2.0 1832-2 pJET1 XL1-Blue Full clone


library


1088-1


TOPO


TOP10









first step in this process, that is, to test if intergenic variability could be used to assign GPH loci

to the diploid linkage map.

In all cases the GPH loci were assigned to the linkage map using a CAPS marker approach.

Here amplicons were digested with a restriction enzyme that corresponded to sequence variation

in the parental lines. A mapping population was treated with identical conditions to reveal the

genotype of the specific F2 plant. Analysis of segregation with isozyme, morphological and

molecular markers allowed assignment of these GPH loci to the diploid linkage map.

The assignment of these loci to the current map is important for two reasons. First, it

demonstrates that the GPH is a viable marker- in this case based on a single restriction site.

Other variable characters certainly exist in these regions that will complement the detection

noted by this restriction site. In the future, these GPH loci will likely serve as anchors for the

octoploid linkage map, because their likely variability supercedes that which is possible from a

simple SSR or other marker used for diploid mapping.

This study places markers on linkage groups I, VI, and VII, with several independent

markers in the latter. The next step is to translate these markers to an octoploid mapping

population. This will immediately bring relevance to the endeavor because GPH loci stem from

or are located near genes of known function. In this study GPH 17022 is localized near F3H

whereas 73122 associates with chalcone synthase, two genes necessary for fruit color production

and protective leaf pigments. A breeder with an interest in improving fruit color or possibly

increasing plant survival in high light environments may find such loci useful in breeding

selections.

The localization of the CHS gene determined by the GPH approach was different from the

linkage group to which the gene was assigned when intron length was used to map it in a F.









were adopted to purify the DNA from the guanidine thiocyanate: DNA adsorption to a silica

column and dialysis of the DNA preparation. DNA purified by the first method rendered

tractable DNA, whereas DNA remained unsuited for enzymatic reaction after dialysis. When

isolated by the "strawberry protocol" proposed by Manning, DNA was also intractable even after

treatment with proteinase K and dialysis. Therefore, it is possible that the co-purified guanidine

thiocyanate or other inhibitors are retained in the dialysis tube. A modification of DNA during

the extraction procedure was considered as a possible explanation to enzyme activity inhibition,

but the fact that previously intractable DNA purified by a silica column permits amplification by

PCR refutes this idea.

The disappearance of an absorbance peak at 230nm when incubation was carried out at

higher temperatures (figure 2-6) may be explained by the solubilization of sugars. At lower

temperatures, the sugars are present and are not solubilized by the extraction buffer, therefore are

carried throughout the remaining steps of the DNA extraction protocol. Their solubilization in

the early phase favors production of a purer product.

When considered together it is clear that many variables have no effect on yield. Whereas

many protocols alter CTAB concentration, Na concentration, method of precipitation, additional

organic extraction and use of affinity matrices, it is clear that concurrent physical and chemical

disruption of cells is the most critical parameter in the generation of pure genomic DNA suitable

for downstream manipulations.












GAGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAGCATTACAA
TGGAAGGCCAAGTGCTCAAGTAGTTAAACAAGAAGAGCATGCTCGCAAGTTGATGGAAGCTCAAGAGAGTAGAGAGA
GAGCTAGGAGAATTGCTTCTTTTACAAGTCGGGTAGCTGATTTGCAGCGAG


>GPH10 ananassa clone20
GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT
CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT
AGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTGGAGAA
AGCTTTCGGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTGATTAAAAGACAGGATTAATGTCAGTGAGGTTT
GGTTG GTTAACTGATGGGTTAAGGTCATAGGTTCAAACCTCACGACATATGTAGGGTGTATGAATTAT
TAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACGGCCTGATCACTCGACATATGTTGATATACGCCCA
ACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTTTTTTAAAATAATTAAATAACTATTTACGTTGTTT
AACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAGCTGAGCAAGAGTACGAGAACAAAAGTATGAGCTA
CATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAA
AAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCATATCATCCGCTTGATCATATATGCGGGT
GTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAATAAAACCCACACG
ACGCCGTTTTGCTCCAATCCCCCTTTCTTCAACCCCATAGTCGCCTCAGCTCAGTTCCATTTGTCTCAGATGCG
ATGGCCTCCGGCGACCCAATCTCCGACTACACCCAAACACATCGCATTGTCCTTCTAATCGACCTCAACCCACTCCT
CCATCTCCAAGATCCAACCCAATTCCTCACCTCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTTCTCTCT
CTTCCTCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCTCCTCTCCTCTCCTCTCCGCCTCCAAGCTCCCGT
CTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACATATCGATCCCTATCTCAAACCCTGGCGTCTCTCTCG
TTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAACGCTTGTTGCGGCTGCGATGCGGCAGCTGGTACATGA
TTACGCTTGGGAGCAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGGTACGTTTTCGAATTGCTGTGGTTTGAGGT
CTAATTTGGCTGTTGTGTTTTTACCGGCGTGTCAATTTGTGAATGAGTTCTTGAATTGTGAGTTGAATTGTGAGGGT
TTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAGGCATATGTGTATAGAGA
TATTCAATTGAGTTGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTG
TTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGCTTGGTTCGGCTCTTGTT
CCATTTGGTTTGATTTATCCAGAGATTGGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAG
AGCGCATTTGAGTCTTGAGATATCAGATGTAAAGGGGATGCCTTTGGAGTGCAAGTTTTGTGATCTTGAGTTGGCTG
ATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATCGCTTGTTTTCGGTGGAAGGCATGAACTCGCAGACAAGAGGT
CATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAATGGAGTGTCGAAGATTCAGGTTAAGGCTTTGCAGAAGGA
TAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCTGATTCTGGTCTATGAAGTTTCAGGAAAAGATGGAAAAG
AAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAGTGCTATCAAGTGAAATTGGGTGAGTTTGTACCGAGGAA
ATTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTTTCTATTTCAAATG
ATAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATTTTTGTTATGGATGAAGGA
ATTCACCCTCATAAAAAAGGGCATGGCATTGGTGCAGAGAATAAGGGTCAGTCTCGTCCAAAGATGAAGAATGAGAT
GTGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTG
AGATTGATGGAAAGAAAAAAAAGTAGCGAAAGAAGTTCACATTCACTCAAAGATCTCACCCGGAGTTCTTTCTGTAAG
GCAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAA
ATTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAGTCTAAGGTGCACCAGGAAA
AACAAAAGGAGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCGTCATCTGGTTCAGCT
GGAGAAATTTCTTTCCCTGTCGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAGATTACAAACCTCAGAAGA
TTTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCACACATC
GGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCACAACAACCCCTTCAGAAGATCAAACTCCTGTA
AAATCTGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGATATGGTTGC
CAGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCCTGGATGTGAAGGCTTTACTTCAGAAATAATAGTTC
GAGAGTATCCTTTCATTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCTACTTTAATGCTA
TGTAAACTTTGCCCCTTGTTACTGTTACACTTTTCCTTCACTAGCACAAAGATATGAATTACAGATACTTTTCCGGA
TGGAGATTTTACAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTGAAACATATTTGCACG
CTTTTGGAGACCATTCGTGCTCGGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGG
AAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTATTTTCTAATGAAATTT
GTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAATATATGTTTATGGTTTGCCTGTT
AATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATTTGTATGCCCTCCCAA
AAGGCCTTTGGGGGTAGTATGAAGAAGGGAGACATTGACCGTCAAAACTATTATCTCCTTATTTTACGTACAAAATT
GATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTCCTTTGCATAATTTCA
TATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATGCATACAACTTTT











GCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACTGCCCAAATTTATGTGA
ACACACTGCATTTATGTTTGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTTTCAGGTACTGTCA
GACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATGATGAGGAAGAACTCCCTA
ATAATTTATCCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATGAGGTGGGTGAAAATAGT
AGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAACATTACAATGGAAGGCCAAGTGCTCA
AGTAGTTAAACAAGAAGAGCATGCTCGCAAGTTGATGAAAGCTCAAGAGAGTAGAGAGAGGGCTAGGAGAATTGCTT
CTTTCACAAGTCGGGTAGCTGATTTGCAGCGAG



Polymorphic segment of GPH10: fragment between primers 10PPR1 and 10AB22


>10PPR1AB22 vesca
AACGGAGAAGAAGACTGTCGACATTTTAAGAGAAGTTTAGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT
TATCTGAAAGACAAGTTTAATATCAGCCGTTGGATCATATTACGGCCCTGATCGCTCGACATAATTCGATATATATA
TATA TATATTTTTTTT ATCATATACATATATTTTTTTTGAATTAATTAAATGAGTATTT
AGATCGCTTAAAAAGATAAACAATCGAAATTGGTTTAAGAACACCATAGGAGCAAGAGTATGAGAACAAAAGTATGA
GCTACACTGTTTGCTCATCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATAAACAATTAAACATTACA
AAATCAAATTACCTAACAATGAACCATTTTCAGACATGTAAAATCATAAAATTAAAAGGTTCGAGTCGCATATGAGT
TTGTCGAGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGTAACGAGTCAAACGAGCTAAAAACGAGT
CGAATAAAAATCGGGCACCATCAATATCGAGACTATGTAAGAGCCGAGGAGTAAAATAATAACAAACTCGTTATCTA
AAAGACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCCT
CATTCCCAATTTTCATTCCCACCAAAAACAAAACC


>10PPR1AB22 nubicola
AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT
TATCTGAAAGACAGGTTTAATATCAGCCGTTGGATCATATTACGGCCCTGATCGCTCGACATAATTCGATATATATA
TATATTATTTTTTTCTAAAAAAAAAAATCGATATACAGTATATTTTTTTTGAATTAATTAAAGTATTGTAGATCG
CTTAAAAAGATAAACAATTGAAGTTGGTTTAGAAGCATCATAGGAGCAAGAGTACGAGAACAAAAGTATGAGCTACA
CTGTTTGCTCGTCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATAAACAATTAAACATTACAAAATCA
AATTACTTAACAATGAACCATCTTCAGACATGTAAAATCAGAAAGTTAAAAGGTTCGAGTCGCATATGAGTTTGTCG
AGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGTAACGAGTCAAACGAGCTAAAAACGAGTCGAATA
AAAATCGGGCACCATCTATATCGAGACTATGTAAGAGCCGAGGAGTAAAATAATAACAAACTCGTTATCTAAAAGAC
AGGTTTAATATCAGCCCTTGGACCATATGTACGGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCCTTCC
AATTTTCATTTCCCACCAAAAACAAAACC


>10PPR1AB22 mandshurica
AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAGTTTAGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT
TATCTGAAAGACAGGTTTAATATCAGCCGTTGGATCATATTACGGCCCTGATCGCTCGACATAATTCGATATATATA
TATTATTTTTTTCTAAAAAAAAAAAAATCGATATACAGTATATTTTTTTTTGAATTAATTAAATGAGTATTTAGATC
GCTTAAAAAAAATAAACAATCGAAGTTGAATTAGGAGCACCATAGGAGCAAGAGTATGAGAACAAAAGTATGAGCTA
CATTGTTTGCTCGTCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATAAACAATTAAACATTACAAAAT
CAAATTACTTAACAATAAACCATCTTCAGACATGTAAAATCAAAAAGTTAAAAGGTTCGAGTCGCATATGAGTTTGT
CGAGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGTAACGAGTCAAACGAGCTAAAAACGAGTCGAA
TAAAAATCGGGCACCATCAATATCGAGACTATGTAAAAGCCGAGGAGTAAAATAATAACAAACTCGTTATCTAAAAG
ACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCATT
CCCAATTTTCATTCCCACCAAAAACAAAACC


>10PPR1AB22_nilgerrensis
AACGGAGAAGAAGACTGTCGACATATCTAGAGAAGTTTAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG
TTATCTGAAAGACAGGTTTAATATCAGCCGTTGGATTATATTCCGGCCCTGATCTCTCGACATATGTTGATATACGC
CTGACTCAAATTCTATATACATTTTCGAAAGAATTTTTGTTGTTGAAGTAACTAAATGACTATACGATTGAAGATAG
ATTAAGAGAAACATAGCAACTGAGTAAAAAGTATGAGAACAAAAGTATGAGCTACATTGTTTGCTCCTCGGTCTGTT
TATATGGAGAAAATTTGTGATGTTTTAAATTATCTAATAACGAATC
ATCTTTAGACGTACGTACAAAATCAGAGAGTTAAGAGATTCGAGTTGCTCAAATACCATATTTTACTTGACTTAACA












TCAGATTCAGAGAGCAGATCAGAGATAGCGTACAAGTGACCTAAGAAACAAAACAAAATTCCAACAAGATCGCAAAC
ATTCGAGATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAAGAGAATAAAGGGATCTGGAG
AATTAGGGGTTAGAGGTGACCTTAAGAGTTTGGGTGAAACACAACTGGGGAAGACAGAGACAGAGGAGGAACTGCGA
AGATCTATCTGAAACCAAACAAAGTAAAAGGGTTTAGCTGTCAGTAACGAGCTCCTAACCGTCCATCTCCAATCTT
GTCAGGGGTGATCCTACGCGTCCACTTGTCCTTTCCCAGTTCTAACTATGTAGACGCTAGCTGCGGATTGTTATTAT
GTTTTGGATAGAATACCTTTGCAAAATAGGAAGCTCCTCCTTGTTTTTCTGCAAGAGAAAGGCCAAAATATCTGACC
ATTCCGACGCCGGAGCTTCCTCAGAAAGCCAGTACCATCCGCAACATCGATGCCAGGCCTTGCGAGGTTTGCCTCCG
CTTCTTTGGATTGTGTTTTTCGTGGTTTAGGAGATTGTTGAACAAAAAAGAAAAACATATATGATAAATGAATTATC
AAATTAATTAATCAAGGTGACGATACAAGATGAGAACACCAAAGGTTCAATAGTGTGTACTCTCAAGCCTAATACTA
ACACAACAAAGAAAGATTCTATCCTTCCATTCCCAGATCAAAAACCACTCTAATGTACCGCCGGCAAAGTGCTGCTA
ATAGAATTGACATTGTAAGTAGGGGATAGTGTCACGAGCAGCTCTAGGAGGTAGTGTCATCGTGACACTTTATTGGG
GTGGATGCTAAGGGGGTTCAGGTTATGAGCAAGCATGGGGGGTAAGGGGGATATCTACTGGCATTTTGAATGACAAT
GTTGTAAATGAAAACTTATATTTCAAGGTATTTTGATTTAATATTTAGAAAAACTGTAACATCAAAAGGGGTTCAAT
ACATTTGCCGACATATTTTTATGAGGTTTTTATGAATTAGTTATGAGAGATGGTTTTCCTTGGACTATTTAGATTTT
GATGTTTCCTTAACACACATTATATTTCTCCATTTTCTTGTAAGTAATTTTCTGTATCACTTAAAAACATTTCTTAC
TCTTCCCAGAAACATCTCCAAACATCCCTAAACCGATAGCTCTAACATGTCAATGTCAATAGATGAAAGATCAACCT
AAATGGTACCATATGTCCATACATAAAAAGACCCAAAAAGAAAATAAATAAGCACCTTCATTTTTAAGCGCCATAAA
AAGTAGAGAAGAATACAAGGTTTGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTCGGAAACAATGCTA
ACCACCACCACTGCCACTCTCAGCTGCTCCTCCTCCTCTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACC
AAACCCAATCTCCCTCAGATTCTCCTCCACATTACAGCTAACCAAAACCAGAACCAGACCAACCCTTAAAACTCTCA
CTCGCCAAAAATGCCAGCTCCCTGCTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCCTCC
ACGGTGCCGTCGGAGATGAAGGCGTGGGTGTA


>GPH23 iinumae clone2
CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACGACCTTTACAGTGAGAGTGTGACCAGAG
GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGATAAAA
GACCTGCCAGAATCCACGCCACCAAACTCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCAT
CAACATGCAATAATGTGGGTCCATAAGAACCATGAGTATGACATAGAGTCTTCAAGCTTCGATTTCCTTATTTGCTT
CGAAAGAAGCAAGTTCAGAGTCCACACAACCAGAATATAGATCTCAAATTTAATAAACATATTCCTAAGAACCTAA
AGCAATATAAAATCGTAACTGGACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCAAAAACAAACTTCAAGT
TCAGATTCACAGAGCAGATCAGAGATAGCATACAAGTGACCTAAGAAACAAAACAACATTCTAACAAGATCGCAAAC
ATTGGAGATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAAGAAACTAAAGGGATCTGGAG
AATTAGGGGTTAGAGGTCACCTTAAGAGTTTCGGTGAAACACAACACAACTGGGGAGACAGAGACAGAGGAGGAACT
GCGAATATCTATCTGAAACCAAACAAAGTAAAAAGGGTTTAGCTGTCAGTAACGAGCTCCTAACCGTCCATCTCCAA
TCTTGTCAGGGGTGATCCTACGCGTCCACTTGTCCTCTCCCAGTTCTAACTATGTACACGCTAGCTGCGGATTGTTA
TTATGTTTTGGATAGAATAGAATACCTTTGCAAAATAGGAAGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAA
TATCTGACCATTCCGACGCCGGAGCTTCCTCAGAAAGCCGGTTCCGTCCGCAACATCGATGCCAGGCCTTGCGAGGT
TTGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTTTAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGAT
GAATGAATTATCAAATTAATTAACCAAGGTGACAATACAAGATGAGAACACCAAGGGTTCAATAGTGTGTACTCTCA
AGCCTAATACTAACACAACAAAGAAAGATTTCTATCCTTCCATTCCCAAATCAAAAACCACTACAATGTACCGTCTA
ATTGAATTGACATTGTAAGTGAGAGATAGTGTCACGAGCTGCATTGGGAGATAGTGTCATCGTGACACTCTATGAAG
GGGATGCTTAAGAGGGTCGCATCAATGACAAACATGAGGGCAAATAGAAGGTCTACTGGCATGTCGAATGACAATGT
CGTAATTAGTTAAGTGAAACTTATATTTCAAGGTACTTTGACTTAGTATTTAGAAAAACTGTAACATCGAAAGGAGT
TCAATACATTTGACGACATATTTTTATGAGGTTTCTATAAATTAGTTATGAGAGATGGTTTTCCTTGGACTATTTTG
ATTTTGATGTTTCCTTAACACACATTATATTTAAAGTAATTTTCCGTATCACTTAAAAACATTTCTTACTCTTTCCA
GAAGCATCTCCAAACATCTCCCTAAATGTCAATGTCAATAGATGAAAGATCAACCTAAATGGTACCATATGTCCATA
CATAAAAAGACCCAAAAAGAAATAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAGTAGAGAAGAATATAAGGTT
TGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTCGGAAACAATGCTAACCACCACCACTGCCAGTCTCA
GCTGCTCCTCCTCCTCTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACCAAACCCAATCTCCCTCAGATTC
TCCTCCACATTACAGCTAACCAAAACCAGAACCAGAACCAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCA
GCTCCCTGCTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCCTCCACGGTGCCGTTGGAGA
TGAAGGCGTGGGTGTA


>GPH23 iinumae clone5
CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACGACCTTTACAGTGAGAGTGTGACCAGAG
GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGATAAAA









carbohydrates (Westphal et al., 1952; Westphal and Jann, 1965) and nucleic acids (Kirby, 1956).

Chaotropic agents denature proteins by increasing the solubility of nonpolar substances in water

(Voet et al., 1998). Hofmeister (Hofmeister, 1888) defined the series of anions and cations with

increasing protein destabilizing properties when he measured the concentration of various salts

needed to precipitate proteins from whole egg white (translated by (Kunz et al., 2004)).

According to the Hofmeister series, urea, guanidinium, thiocyanate (Sawyer and Puckridge,

1973) and perchlorate (Wilcockson, 1973) are extremely chaotropic agents. Thus, high

concentrations of urea (Settles et al., 2004), guanidine hydrochloride (Logemann et al., 1987),

and guanidine thiocyanate have been used in isolation of RNA (Cox, 1968; Chomczynski and

Sacchi, 1987) and DNA (Chomczynski et al., 1997).

Chemical or physical means such as precipitation by isopropanol, ethanol, butoxyethanol

(Manning, 1991), acetone (Vogelstein and Gillespie, 1979), adsorption to silica (Vogelstein and

Gillespie, 1979), paramagnetic particles (Anonymous, 1980, 2001; Koller and al., 2001), and ion

exchange resin (QIAGEN Anion-Exchange Resin manual) can be utilized to retrieve DNA from

solution. The resin is coated with diethylethanolamine (DEAE), and DNA recovery is due to

interaction between negatively charged phosphates of the DNA backbone and positively charged

DEAE groups. In the case of silica columns, DNA is recovered from solutions because it tends to

adsorb to silica in the presence of chaotropic salts, such as sodium iodide (Nal) (Vogelstein and

Gillespie, 1979), guanidine thiocyanate, and guanidine hydrochloride. The binding capacity

depends on the solution's ionic strength and pH, being higher in concentrated solutions and at

pH<7.5 (GeneClean Manual). Silica columns have been used to eliminate polysaccharide

contaminants, and the ratio A260/230 increases as polysaccharides are removed (Abdulova et al.,

2002).









Table 2-3. DNA yields (pg DNA) from ten strawberry genotypes. Plant tissue incubation with
the extraction buffer was carried out at 40C for 5min. Averages of 2 replicates, 200mg
tissue each, extracted by 5ml buffer.
Genotype _g DNA/200mg tissue
F. vesca cv Yellow Wonder 127
F. vesca cv Alexandria 59
F. virginiana 54
F. chiloensis 0.85
F. x ananssa cv Diamante 0.65
F. x ananssa cv Strawberry Festival 50
F. x ananssa Laboratory Festival #9 52
F. x ananassa cv Camarosa 100
F. x ananassa cv Sweet Charlie 64
F. x annanssa cv Quinault 55


Table 2-4. Impact of interactions between maceration methods and incubation temperatures on
DNA yield and purity. The ratio between absorbance at 260nm and 230nm (A260/230)
estimate contamination by polysaccharides, whereas the ratio A260/280 estimate
contamination by proteins. Pure samples have both ratios equal to 1.80.
Yield ig DNA/50mg tissue A260/230 A260/280
4C 600C 40C 600C 40C 60C
slurry 31 38 1.02 1.78 1.71 1.91
no slurry 3.8 8.8 0.61 1.46 1.67 1.95









predicted restriction pattern for F. vesca 'Pawtuckaway". An unexpected fragment of 1249 bp

was observed for F. vesca, raising a concern that the putative single locus was in fact two loci.

The other possibility was that the higher molecular weight band was a different F. vesca allele

from the same locus. Had that been the case, a heterozygote should have been observed

containing the female allele (1249, 300, 251bp) and the male allele (702, 335, 308, 251 bp). Such

an individual was not observed, as 1249bp band cosegregated with the 758 and 429 bp bands.

The presence of the 1.25 kb band was attributed to partial restriction digestion and the scoring

was therefore carried out based solely on the expected 758 and 429bp bands versus the 702 and

335bp bands. Figure 5-2 shows banding pattern for digested amplicons of 34D20 and 72E18.

GPH40M11 is a dominant marker and amplifies a band only for the pistillate parent, F.

vesca. Since the PCR amplification was precluded for half of the F2 population for some reason,

this raised a concern about wrongly scoring individuals as homozygous F. nubicola. Thus,

amplification patterns for all other 7 loci were compared, using a primer pair as positive control.

Individuals for which amplification was observed in all those primer pairs but not observed for

40M11 were scored as homozygous for the F. nubicola allele.

The majority of the GPHs investigated were assigned to linkage group VII, as shown in

figure 5-3.

Discussion

Gene pair haplotypes are intergenic, multiple character signatures that define suites of

variability between two genomes. The purpose for these markers is to provide a complex field of

discrete variation that can be related to a specific subgenome donor with the goal of eventually

mapping genes to specific subgenomes of the octoploid strawberry. This chapter outlines the









information for the above-mentioned species than for Fragaria. The availability of strawberry

nucleotide sequences was so scarce in 2004 that, if one searched for "Fragaria" in public

databases, only 58 gene sequences were retrieved (Folta and Davis, 2006). In 2007, this number

jumped to over 20,000 sequences, of which 50% are Expressed Sequence Tag (EST) sequences.

Collaborative work between the laboratories of Drs. Thomas M. Davis (University of New

Hampshire), Kevin M. Folta (University of Florida), Jeffrey L. Bennetzen (University of

Georgia), and Phillip SanMiguel (Purdue University) have added an additional 50 genomic DNA

sequences, constituting slightly less than 2 megabases of genomic information. The sequences

are derived from a Fragaria vesca 'Pawtuckaway' genomic library and represent 1% ofF.

vesca's 200Mbp haploid genome (Folta and Davis, 2006). Due to its minute genome size and to

the facts that F. vesca is the most geographically predominant diploid Fragaria species (Folta

and Davis, 2006) and it is a plausible ancestor of the cultivated, octoploid strawberry (Ichijima,

1926; Davis and DiMeglio, 2004), this diploid serves as a valuable model for development of

molecular markers and comparisons amongst several Fragaria species, as well as other genera of

the Rosaceae family.

This study aimed to annotate the newly sequenced parcels of the F. vesca genome. This

represents the first opportunity to explore the gene distribution and the composition of the

Fragaria genome, which, at 200 Mbp, is comparable to the 157 Mbp (Bennett et al., 2003)

genome size of the model plant A. thaliana.

Materials and Methods

Dr. Thomas M. Davis at the University of New Hampshire used fosmids (CopyControlTM

pCC1FOSTM from Epicentre) as vectors to produce a F. vesca genomic library with 8x coverage.

The theory is that if the genome was digested into 35kb fragments, approximately 45,000

colonies would be necessary to represent the 200Mbp haploid genome 8 times. Fosmid vectors













>11D02_ananassa 7 unspecific
NNGGGACGTTTTGCAGATACTGCTGTGTGACCAAACCCTAAATAAACCCCACCATCCAGCCTTGCCACCTCTATTGC
ATGCCTTTTGATCGTTTCCTCCTCACACTTGTTTTCTTTCTCTTCCTCCCCCAAAACAAGAACCCAAAGTCCCCCAA
AACGCATCATATATAGATACAGACGCAGCGAGTGTTATTATTAACCCGAAGAAAAACCCAAAGAGCTAATCGACAGA
GAAGAAGAAGAAAGAGGCTTTATATAAAGAGAGAAAACGCTTGCTGTTGATTCAGAGCAACCAGCCCTTCTCTTTTT
CCCTTCTTCTCTCTGTGTGTAATATTTCAATGGCCGTTGAAGCTCGGCACCTCAATCTATTCCCCTCTCAACAACTC
TTCTGCAACAACAGGTACTGATCCTCACTTCATCTGGAGTTGAAC


>11D02_ananassa 9 unspecific
GAGCTGCTGTGTGAACCAAAAAAGAGAAAGACAGAAAAAAGAGCAGGAGGATGGAGTTGCCAAAAAGGCTGATCTGG
TGCGGCAAGGTTGATTTGTGTTTTGGTCCTTACTGATTTGTGTCCATTGGATTGCTTATAAATGACGTGTCAGCTTG
TTAGTGATGTCCAAGCAGACAAGCCAAATCCTATGAAAAGAGAGTCAATTACATATAAGTCTATTTTGAGACATTTT
CTCCCATATAGTTTCATCCAAACATTTTTACCCATATAAGTCCACCTAAAGCTAAATAGAGTAGTATATTAGATATT
AAAGTTTAACATAATTACAGGTTGTCATCTAGCAAAAAAAAAAAAATATTTCTCTTATTTACGAATATACCACTAGA
GTAAAAGGTCAACAACCACCCTCCTCTCAAAAAAAGTCTACGGTCGACTTCCAGCGATGTTTCCAATTAACTTTCGG
TGAGGATTTTGGTTCACACAGCAGCTC



>17022 vesca
AAAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATTTGACAACAGACA
TAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCG
TAGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTT
CCCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATATTACGATCCCCTTATC
ACTGTAGGGCTTCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATAGTAGCCTAGTTTAGTTTCTTATGCT
GAAGCAAAATATGTAATCACCTACGCTACAGAATAGTGTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATGA
AGAACGGTACCAGTTACCCAACATAATCATAGTTATTTTGGCCTATTGATATTTTGATTAACGTGTAATTGATCGCT
ACTTGAATGATGTATATTATGAATGGCACTATTTAATATTTTGGGCTGCTACCTACTCTTCAACAAACTCTAATTAA
TTAACCAAACATCAGTGTCACAAGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAACCTAC
CTCCCAAAAATAGTTACTTTAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGAT
TTGTTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTCTGGTCATCAGTGTCAAACTAG
AAGAACCGTGAATTGCGACCCCTCAGAATGTCAAAATGAGATCACTGTGATTCCTTTTAAAATTTTAACAGCGATTC
TTCTACAAAAGATGGACTAAATTCCACCTTGTACTGTACAAAAAACGAGTTTGAGTAGTGGGAATCGTTCCAATATA
TTTCTGCTCTGTTTACCAATTGCCAGGATGATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAGAATG
AGAAGAAAGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGC
AGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTTGG
AACAATATCATTGATTTCAGATTTTGTCTCAGATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATG
GATTCAGAAAATTTGCTACAAAAGCCCATAACTTGTAAACATCATCGAAGTTTGTGAGGAAACCC


>17022 viridis
TAAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATCTGACAACGGACA
TAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCG
TAGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTT
CCCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAAGGCAAATATGACTCTATATATC CGATCCCCTTATC
ACTGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTCTAGTTTCTTATGCT
GAAGCAAAATATGTAATCACCTAGGCTACAGAATAGTGTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATAA
AGAACGGTACCAGTTACCCAACATAATCATACTTGTTTTGGCCTATTGATATTTTGATTAATATGTAATTGATCGCT
ACTGGAATGATGTATATTATATTATGAATGGCACTATTTAATATTTTGGGCTGCTACCTACTCTTCAACAAACTCTA
ATTAATTAACCAAACATCAGTGTCACAGGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAA
CCTACCTCCCAAAAATAGTTACTTTAAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATT
CTGGATTTGTTACTCGCTAGTATGTGTTGAACTTTGTTCTCTTTACAAAGACAAAAGGACTTTGGTCATCAGTGTCA
AACTAGAAGAACTGTGAATTGCGACTACACCAGGATGCCTTTGGTCACTTACCAACCTCAAGAAAAGGACCCCCTCA
GAATGTCAAAATGAGATCACTGTGATTCCTTTTAAAATTTTAACAGTGATTCTTCTACAAAAGACTAAATTCCACTT
TGTACTGTACAAAAAACGAGTTTGAGTAGTGGGAATCGTTCCAATATATTTCTGCTCTGTTTACCAATTGCCAGGAT
GATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAATGAGAAGAAAGAACTCTACAAGAATCCAAAG










Table 5-1. continued
Putative Gene Function or
Primer
EST gb number
34D20Fb RNA recognition motif
34D20Rc cysteine-type peptidase
38H02F serine/threonine kinase
38H02R exportin
40M11F
40M FF-box protein
40M11Fc
40M11R transposase (E > 1013)
40M11Rc expressed protein (E > 10-9)


40M11Fb
40M11Fd
40M11Rd
40M11Rb
63F17F
63F17R
72E18Fb
72E18Rb
73I22F
73I22R
GPH1Oa
GPHlOb
GPH1Oc


secretary protein SEC14

ATPase
phospholipase D
unknown protein
actin
elongase
chalcone synthase A
chalcone synthase B
unknown protein
unknown protein
unknown protein


Sequence 5' to 3'
GCAGAAAGAAACTGATGTGCTT
CGCAGTCGTAAAAATTCGTCT
CCAGGCCTAAGCTTGTCATC
AAGGCATTGAAATCATTCTACCA
ACACAGGTCATTGGGTCCAT
TTGACCCGGATAACATGGAT
GTGTTGCACAAGTCCATTCG
CTGACAGCGAATCAATCTGC
GGCCTTCTTGACATTCCAGT
CAACATTTTGGTGGCCTTCT
CGGCCTATGAAACCACAGTT
TGGGGTTGTTGGAAAGAGAG
CGCTCTATGGAAGGGACAAG
TTAAGGGGTCTGTTGATGTGC
GCTAGGGAAAACAGCTCGTG
TGGGTTTGGTTTTGGGATAA
CAAGCCTGAGAAGTTAGAAGC
GAAAGTAGTAGTCGGGGTATGT
GGCTTCTTCTTGTCCGGCAGC
GAACTCCAGGTCAGATCTTCG
CTCGCTGCAAATCAGCTACC


Extension
Tamealmng (C) Time
Time60 3'30"
60 3'30"


53, 54, 60


2'30"


2'30"
4'


Table 5-2. Fragment sizes of parental amplicons digested with restriction enzymes
Locus Restriction Enzyme Amplicon estimate fragment sizes (bp)


Non-digested Digested


17022FRb

34D20FbRc



40M11FdRd
63F17
72E18FbRb
73122


Rsal

Alul



Dominant marker
HaeIII
HhaI
Pvull


1,374

2,050



3,100
1,266
2,620
3,000


F. vesca
486,413,292,83,
67,28,5
1249,758,429,
300,251,107,69,
48,26
present
992,234,40
1,400, 800, 300
2,200, 1,000, 600


F. nubicola
511,414,293,
83,67,28,5
702,335,308,
251,107,69,
48,45,41,26
absent
840,234,40
2,300, 300
2,200, 1,500









is not coding in Fragaria; ii, the gene prediction is correct, but the putative amino acid sequence

is not represented in the protein database because the transcript is not translated (RNA genes in

fosmid 15B13, for example), or because the protein has not yet been described; iii, the amino

acid sequence is indeed represented in the database, but it is not conserved with Fragaria, so the

E value threshold chosen as a threshold is too stringent. If a less stringent threshold is used (E

value 10-10, rather than 10-15), the number of BLASTX hits increases from 129 to 166 and,

therefore, software specificity rises from 55 to 70%.

Half of the ESTs that were identified in genomic regions for which no gene was predicted

(figure 3-3) were detected in fosmids that either contained sequence similar to chloroplast DNA

(11D02 and 32L07) or to ribosomal RNA (26S in fosmid 15B13). One of the ESTs displayed

identity starting in the first nucleotide of the fosmid insert. Perhaps the gene predictor failed to

perceive this ORF because the query sequence did not contain transcription initiation signals.

The other half of the ESTs that were identified but not predicted was similar to genomic

sequences from other species, and the reason why the gene prediction software failed to predict

them is not clear. This may suggest some facet ofFragaria gene structure that is not recognized

by other conditioned algorithms. The detection of putative genes through homology-based

similarity search reveals the need to utilize various homology search methods in combination to

ab initio gene prediction for the optimum genome annotation. This finding is exceedingly

important as the genomes of peach and apple will soon be sequenced. Accurate genome

annotation will depend on the capacity to adapt current gene prediction methods to these

genomes.









































































-GCTCCTCCT
-GCTCCTCCT
QGCTCCTCCT
-GCTCCTCCT
\GCTCCTCCT


CCTC
CCTC
CCTC
CCTC
CCTC









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

STRUCTURAL GENOMICS OF Fragaria-WILD AND CULTIVATED STRAWBERRIES

By

Denise Cristina Manfrim Tombolato

August 2007

Chair: Kevin M. Folta
Major: Horticultural Science

The extensive phenotypic variability and complex genetic makeup of the cultivated

strawberry Fragaria xaananssa permits advances in plant improvement, a factor breeders have

exploited to great benefit. However, the introgression of specific characters is complicated due to

the cumbersome genetics and limited knowledge of genome structure and function of genes

relevant to traits of interest. The present study represents the first genomics-level insight into

strawberry genome structure and explores the hypothesis that a new type of molecular marker,

the Gene-Pair Haplotype represents a transferable marker that may hasten linkage mapping in the

diploid and octoploid strawberry.

My research presents the findings of four related research activities. First, an efficient and

unified method for genomic DNA isolation was derived from over 100 experimental tests and

conditions. Next, 1% of the Fragaria genome was sequenced and functionally annotated, using a

bioinformatics approach and computational tools. Over 120 kb of intergenic regions were

sequenced using the Gene-Pair-Haplotype approach, allowing for some initial relationships to be

formulated concerning the diploid subgenome contribution to octoploid strawberry. Finally,

Gene-Pair Haplotypes were used to add a suite of alleles to the growing Fragaria linkage map.

These findings provide a starting point for further analyses of the strawberry genome.









small RNAs (snoRNAs, microRNAs, siRNAs, piRNAs), and long RNAs (Xist, Evf, Air, CTN,

PINK).

The second challenge for annotation is to ascertain or predict gene function, how gene

products might interact, and how they are regulated (Salamov and Solovyev, 2000). Gene finding

can be accomplished by similarity-based or ab initio gene prediction software. Similarity is

defined by the NCBI glossary as "the extent to which nucleotide or protein sequences are

related."

Similarity-based algorithms provide information on alternative transcription (Li et al.,

2006), translation start sites, and slicing and are more specific than ab initio. However, the latter

is more sensitive than the former because it does not bias findings based on prior descriptions

(Birney et al., 2004).

Similarity-based algorithms like GeneWise (Birney et al., 2004) predict genes by testing

putative translation products for similarity to known proteins. A nucleotide comparison against

cDNA, to an expressed sequence tag (EST), or a protein database using the Basic Local

Alignment Search Tool (BLAST) are also similarity-based gene predictions Non-coding rRNAs

are also identified using this approach (Stein, 2001). In contrast, the ab initio approach attempts

to predict genes from sequence data without prior information on gene characterization. Most

gene predictors attempt to define a gene using neural networks (modeled according to the

learning process in cognitive systems), rule-based systems (algorithms that use an explicit set of

rules to make decisions), or hidden Markov models (HMMs). HMMs are statistical algorithms

typically utilized in natural language processing. In gene prediction, they are trained with known

gene structures (Stein, 2001; Yandell and Majoros, 2002). A Markov model is a statistical model

in which the system being modeled is assumed to be a Markov process, i.e., a stochastic












CTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCACGTGGTATTTGATAGCCTCCACTTCCATCAG
GACTTCGCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA


>72E 18_nilgerrensis
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNAAAAAAGAGAGAGAGATTACAGATCTANGCGACGAACAATGAGAAGGAATGAGAGGCAGAGAGAAGAGATGAGGA
AGTTGACCTTTGTGAATGAGAGTGAGTGAGAGAGAGAGATCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGT
TTGTGAGTTGAGGCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATTTATGG
ACCGCGTCCATTGCGCCCTTGTGGGGCCATTACAGCTCCTTCCGCTGTTCCAGTCATTTTTTTCTCCACCTTCTGCC
CCATTCCCTTCCCTTTTCTCCCAATTCTTTCTCAACTCTTCTTAAACCTAATTGCATTTTCATTTTANTGCTTANAT
CAATATGATTAAGAAGCTCCATTTTGTCAACACAAGGCAACAAGGACNTANGGGAGCATGTCGATCATCGTTCCGGT
CACTTTCGTATATAATTTGGACTTAAAATGGTTGATCGATCGTAAAATTTGAAATGACGTTTTGGTATGATATTTGT
AAAGAGGACATAATTTACTATGGAACGAAGAGTATAGGTGAAAGTGTCATCCCACACATTTTAAAAGAGCTTTAATG
TAGGGGTAATGAGCACAACTACAAGCTGCATCCTATAAGGGATCAATCAGAACATTAAACAACGTAAAGAGGAAGGT
ATTTGCTTTACACAACCTTATAAAATGATGAGGATCTACTCAAAATCCAGACTACCTGGTTGGCAAAATTAGATTCC
TGCCTGTAACCAGCTAGGCATTGGCAATGCATAATAGCTAGAGCTAACCATAGGTGGGAGACTCATCATTGAGATCA
TAGGAAAAAAAATGATGAAAACAAGCAACAGTTATTCGAAAGCAAGTACAGAAGGGATTGTTCATTAAGTGTTCACC
AAGTCACAGCTTAGGGCATTCTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATAT
TTAGTCATGTTGTTTAAAGCCACAACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCA
GATGACAAAACAGTTCAAATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTT
ATTACAAAGTCTAAGCAGAACACTAACATAAATATTGAAATTGGATAAATATGCGATCTGAACTTCTTCACGTTGAT
GACCTATCATAGGAAATGGAATTGAACACTTGACACCAAAGAGAACAACGAAGGTAGCCTCGCCAATCACTTCTACA
AGAATGGGGGTAGAATCACCCATCTACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAG
GGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACTCATATGGATATGTTCTGGCGATTGCAG
AATATAATTATGTATACAAATATGCATGTACAGAGCTTCTACATACAAACTCATACGAATACTTGTAAATTTAGGCA
ATTTAATTCCAATAAAGGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAAGACAAAACATTTAAGCAAAAGAA
GAGCAGTAGAAGTTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAATGTAGCATAGAGGAGTGTACCTTCT
TTAAACGGCGGTGCTTTCCTAGGGCCCAGTTGGTCATTATAGAAGCAGCAACTGCAGCAAAAAGATAACCAGCAACC
GTCTGTGTTGCAATATTAAAACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAG
AAAACCACGAGGTATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAATATGGCAATACAAGT
TCGCGATTTGATTTATTATCCCAAAACCAAACCCA


>72E18 mandshurica
GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAGACCCGGCCTAAACATTAACATCAAAATCAGTCCTTGAGA
TTCAACATGCATAACAAAGACAATAAAGGGTACAAAAACAACCACTCAAACAATCACAACATAATATCATTCAATAC
CTTGACCATTCCGGTTCCATTATCACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAAC
ATACAACCACAATCAAATGCTACATTCACACAACAAAGAAATAGACATTCAAAGACAAAACACAAACACACTACTAAC
GTGGCACGGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACATAGTTCTCTCACAATAGTAAAGAAACGA
TCGTTGACAATCAAAAGGCATCGAAAGCTAGTAAAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTA
TATACATAAAACCCTCAAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAAAAATTCAAG
AAAAAAAGAGAGAGAAAATTACAGATCTAAAGCGACGAACAGTGAGAAGGAATGAGAGGCAGAGAGAAGAGATGAGG
AAGTTGACCTTTGTGAATGAGAGTGAGTGAGGGAGAGAGAGAGAGAGATCGACGACGAAGCAGAGCGAAAGAGACGA
GTGTGGTGTTTGTGAGTTGAGGCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTT
GATTTATGGACCGCGTCTATTGCGCCCTTGTGGGGCCATTACAGCTCCTTCCGCTGTTCCAGTCATTTTTTTCTCCA
CCTTCTTCACCTTTTTGCCCCTCAGTCCCTTCCCTTTTCTCCCAATTCTTTCTCAACTCTTCTTAAACCTAATTGCA
TTTCCCTAATTGCATTTTCATTTTAGTGCTGAGATCAATATGATTAAGAAGCTTCATTTTGTCAACACAAGGCAACA
AGGACACAAGGGAGCATGTCGATCATCGTTCCAGTCATTTTCGTATATAATTTGGGCTTGAAATGGTTAATCAATCG
TAAAATTTAAAATGACGTTTTGATATGATATCTATAAGGAGGACATAATTTACTTTATATGTCAGGTTTTAATACGG
AAGGAAGAGTATGGGTGAAAGTGTCATCCCACACATTTTAAAAGAGCTGTAATGTAGGGTAATGAGCACAACTGCAA
GCTGCATCCTATAATGGATCAATCAGAACAATAAACAACGTAAAGAGGAAGGTATTTGCTTTACACAACCTTATAAA









BamHI C
BamHI C


EcoRI
EcoRI
InDel RFLP
between-genomes
polymorphisms


(GA)8
(GA)6


T (GA)10
A (GA)10
SNP SSR
within-genomes
polymorphisms


A-genome haplotypes


B-genome haplotypes


Figure 4-1. An idealized GPH locus. Arrows represent primers designed to amplify the
intergenic spaces of a GPH. The combination of polymorphisms within (SSR, SNP)
and between subgenomes (InDels, change in restriction sites) define each haplotype.


Figure 4-2. Fragaria species and their geographical locations









CHAPTER 5
GENE-PAIR HAPLOTYPES: FUNCTIONAL AND TRANSFERABLE MARKERS AS
NOVEL ADDITIONS TO THE DIPLOID Fragaria GENETIC LINKAGE REFERENCE MAP

Introduction

Strawberry (Fragaria x aananssa Duch.) is an economically valuable fruit crop, with

average consumption of over 7.3 pounds per capital in 2005 in the United States (FAO STAT).

The demand tends to increase due to public awareness of the potential health benefits of

strawberry: small fruits have been shown to have high content of antioxidants (Wang, 2006),

polyphenols and micronutrients that may play a role in human health.

Despite of the great importance of strawberry, knowledge of its genetic composition is

very modest. The cultivated strawberry is octoploid, complicating development of molecular

markers and construction of genetic linkage maps. Researchers have resorted to utilizing wild

diploid strawberries to generate the first linkage relationships, in the hope of extending the

findings to octoploid genomes. The first genetic linkages identified showed relationships

between fruit color (Williamson et al., 1995) and runnering (Yu and Davis, 1995) to the

shikimate dehydrogenase and phosphoglucoisomerase loci, respectively. These associations were

shown in Fragaria vesca, a diploid that has been proposed to be a possible "A type" genome

donor to the cultivated strawberry (Potter et al., 2000). The first indirect evidence ofF. vesca as

a genome contributor to the cultivated octoploid comes from cytological studies by Ichijima in

1926, where he showed the formation of 21 bivalents and 7 univalents during the pairing

between F. vesca (then called F. bracteata) and F. virginiana, the pistillate parent to F. x

ananassa.

The first genetic linkage map developed for strawberry was constructed using Randomly

Amplified Polymorphic DNA (RAPD) markers developed for an F2 population derived from a

cross between two subspecies of the diploid F. vesca: ssp. vesca 'Baron Solemacher' (red-


























andshl
iridi
c anal



esca
andshl
iridi;














GPH10_ananassa clone CAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGGTACGTTTTCGAATTG 1241
GPH10_ananassa clone20 CAGGTGATCTGCGACGCCGTGGCGGCGGAGACCGGTACGTTATCAATTG 1372
GPH10_ananassa clonel8 CCGGTGATCTGCGACGCCGCGGCGGCGGAGACCGGTACGTTATCGAATTG 1325
GPH10_ananassa clonel9 CCGGTGATCTGCGACGCCGCGGCGGCGGAGACCGGTTTATCGTTG 1324


GPH10ananassa clone CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAAT 1291
GPH10ananassa clone20 CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTTACCGGCGTGTCAAT 1422
GPH10ananassa clonel8 CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTCACCGGCGTGTCAAT 1375
GPH10_ananassa clonel9 CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAAT 1374


GPH10ananassa clone TTGT GAATGAGTTCTCTTGAATTTTGAG------------GGTTTGGAGGAT 1329
GPH10ananassa clone20 TTGTGAATGAGTTCTTGAATTGTGAGTTGAATTGTGAGGGTTTGGAGGAT 1472
GPH10ananassa clonel8 TTGTGAATGAGTTCTTGAATTGTGAG------------GGTTTGGAGGAT 1413
GPH10_ananassa clonel9 TTGTGAATGAGTTCTTAATTGTAG------------GGTTTGGAGGAT 1412


GPH10ananassa clone TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAATGTGGATGA 1379
GPH10ananassa clone20 TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGA 1522
GPH10ananassa clonel8 TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAATGTGGATGA 1463
GPH10_ananassa clonel9 TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGA 1462


GPH10ananassa clone GGCATATGTGTATAGAGATATTCAATTAGTCGGGTTGATGTGAGGTATG 1429
GPH10ananassa clone20 GGCATATGTGTATAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATG 1572
GPH10ananassa clonel8 GGCATTTGTGTGTAGAGATATTCAA AGTTGGGTTGATGTGAGGTATG 1513
GPH10_ananassa clonel9 GGCATTTGTGTGTAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATG 1512


GPH10ananassa clone GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1479
GPH10ananassa clone20 GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1622
GPH10ananassa clonel8 GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1563
GPH10_ananassa clonel9 GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1562


GPH10ananassa clone GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1529
GPH10ananassa clone20 GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1672
GPH10ananassa clonel8 GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1613
GPH10_ananassa clonel9 GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1612
**************************************************


GPH10ananassa clone TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1579
GPH10ananassa clone20 TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1722
GPH10ananassa clonel8 TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1663
GPH10_ananassa clonel9 TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1662
**************************************************


GPH10ananassa clone GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAGA 1629
GPH10ananassa clone20 GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAGA 1772
GPH10ananassa clonel8 GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGTTTAGA 1713
GPH10_ananassa clonel9 GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGTTTAGA 1712


GPH10ananassa clone GCGCATTTGAGTCTTGAGATCGGATGTAAAGGGGATGCCTTTGGAGTG 1679
GPH10ananassa clone20 GCGCATTTGAGTCTTGAGATATCAGATGTAAAGGGGATGCCTTTGGAGTG 1822
GPH10ananassa clonel8 GCGCATTTGAGTCTTGAGATTCGGATGGAAAGGGGATGCCTTTGGAGTG 1763
GPH10ananassa clonel9 GCGCATTTGAGTCTTGAGATCGGATGGAAAGGGGATGCCTTTGGAGTG 1762


GPH10ananassa clone CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1729
GPH10ananassa clone20 CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1872
GPH10ananassa clonel8 CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1813
GPH10_ananassa clonel9 CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1812






169









LIST OF REFERENCES


Abdulova G, Ananiev E, Grozdanov P (2002) Isolation and purification of nuclear DNA from
excised cotyledons of Cucurbitapepo L.(zucchini). Bulg. J. Plant Physiol. 28: 3-11

Ahmadi H, Bringhurst RS, Voth V (1990) Modes of inheritance of photoperiodism in
Fragaria. J. Amer. Soc. Hort. Sci. 115: 146-152

Akiyama Y, Yamamoto Y, Ohmido N, Oshima M, Fukui K (2001) Estimation of the nuclear
DNA content of strawberries (Fragaria spp.) compared with Arabidopsis thaliana by
using dual-stem flow cytometry. Cytologia 66: 431-436

Albani M, Battey NH, Wilkinson MJ (2004) The development of ISSR-derived SCAR markers
around the Seasonal Flowering Locus (SFL) in Fragaria vesca. Theoretical & Applied
Genetics 109: 571-579

Aljanabi SM, Forget L, Dookun A (1999) An improved rapid protocol for the isolation of
polysaccharide and polyphenol-free sugarcane DNA. Plant Mol. Biol. Rep. 17: 1-8

Anonymous (1980) IEEE Transactions on Magnetics 16: 387-490

Anonymous (1998) Montreal Protocol on Substances that Deplete the Ozone Layer. United
Nations Environmental Program (UNEP).

Anonymous (2001) Journal of Magnetism and Magnetic Materials 225: 1-314

Antonius K, Ahokas H (1996) Flow cytometric determination of polyploidy level in
spontaneous clones of strawberries. Hereditas 124: 285

Arnau G, Lallemand J, Bourgoin M (2003) Fast and reliable strawberry cultivar identification
using inter simple sequence repeat (ISSR) amplification. Euphytica 129: 69-79

Arulsekar S, Bringhurst RS, Voth V (1981) Inheritance of PGI phosphoglucoisomerase and
LAP leucine aminopeptidase isozymes in octoploid cultivated strawberry. J Amer Soc
Hort Sci 106: 679-683

Ashley MV, Wilk JA, Styan SMN, Craft KJ, Jones KL, Feldheim KA, Lewers KS, Ashman
TL (2003) High variability and disomic segregation of microsatellites in the octoploid
Fragaria virginiana Mill. (Rosaceae). Theor Appl Genet. 107

Barakat A, Matassi G, Bernardi G (1998) Distribution of genes in the genome of Arabidopsis
thaliana and its implications for the genome organization of plants. Proc Natl Acad Sci U
SA 95: 10044-10049

Bedbrook J, Gerlach W, Thompson R, Flavell RB (1980) Emergent Techniques. University of
Minnesota Press, Minneapolis









restriction digestion (figure 4-6) and sequence was obtained for the full clones (figure 4-7). The

primers flaking the most polymorphic region (10PPR1 and 10AB#22) were utilized to amplify

that region from all six diploids included in this study. A cladogram based on this polymorphic

region is shown in figure 4-9.

72E18

72E18 was the only GPH sequenced that presented polymorphism in the number of repeats

in SSRs.

Estimations of Relatedness from Sequence Variation

Cladograms are branching diagrams assumed to be an estimate of a phylogeny where the

branches are of equal length. Therefore, cladograms show common ancestry, but do not indicate

the amount of evolutionary "time" separating taxa (information from the http://www.ebi.ac.uk/

website). In this study the use of cladograms generated from multiple sequence alignments

provide an outstanding means to gauge the relatedness between strawberry genomes. When

compared against each other, the use of cladograms depicts the relative divergence between

similar sequences, and thus is a useful estimate of SNP frequency between the alleles in F. x

ananassa and the putative diploid subgenome donors. The following cladograms derive from

all GPH that contained at least one allele representing the octoploid strawberry compared to all

cases where products could be amplified from diploids. The results indicate that octoploid

alleles cluster together, as do diploid alleles. The most related diploid to octoploid alleles is

consistently F. iinumae, and surprisingly, alleles closely matching F. vesca were not detected for

any of these GPH loci.

Relatedness may also be assessed by studying the order of insertion-deletions and SSRs.

Presumably, a set of similar indels or SSRs may be conserved between the diploid subgenome

donors and the modern cultivated octoploid. The presence and order of these features provides









CHAPTER 2
DNA EXTRACTION FROM RECALCITRANT SPECIES

Introduction

Strawberry (Fragaria x aananssa) is an important crop worldwide, and it supports many

regional economies in the United States. However, relatively little is known about the genes that

govern agriculturally important traits or their expression. Contemporary genomics tools have the

potential to accelerate study of strawberry and bring additional resolution to strawberry gene

form and function. Strawberry belongs to the genus Fragaria, a genus that includes a number of

species of varying ploidy with a small haploid genome size. These facets make strawberry an

excellent candidate for genomic studies representing the Rosaceae family. Because it is easily

transformable, it is particularly well suited for translational-genomics studies.

Any genomics effort, whether translational, structural or functional, is generally dependent

on a reproducible and effective means to isolate quality genetic material. Although protocols

have been streamlined over the last several decades, it is challenging to isolate large amounts of

quality DNA from strawberry (Manning, 1991; Porebski et al., 1997). A similar problem has

been encountered in other species. Plants like cotton (Katterman and Shattuck, 1983; Dabo et al.,

1993; Chaudhry et al., 1999; Li et al., 2001), sugarcane (Aljanabi et al., 1999), conifers (Crowley

et al., 2003), tomato (Peterson et al., 1997), grape (Collins and Symons, 1992; Lodhi et al.,

1994), and the rosaceous chestnut rose (Xu et al., 2004) have been reported to be recalcitrant to

DNA extraction. The high content of polysaccharides and polyphenols either limit DNA

isolation or inhibit downstream enzymatic reactions.

The DNA Extraction Procedure

A typical DNA extraction is accomplished by three basic steps: lysis of the cell, removal of

proteins, and separation of nucleic acids from other cellular compounds. Cell lysis is easily









(Milbourne et al., 1998). The map published in 2004 had 78 markers and new microsatellite loci

were added later, totaling 182 markers (Sargent et al., 2006).

Strawberry belongs to the Rosaceae family, to which the horticulturally important peach,

cherry, apple, raspberry, and rose also belong. Although SSRs are markers transferable between

mapping progenies within and between species (Dirlewanger et al., 2002) (Hadonou et al., 2004),

they are generally not transferable between genera. The challenge in developing transferable

markers resides in the fact that markers are, by definition, placed on polymorphic regions of the

DNA and, to be transferable, such markers are must be located on conserved regions. A recent

study (Sargent et al., 2007) explored intron length polymorphisms, having PCR primers

anchored in flanking exons that were conserved across Prunus and Malus, and thus generated

highly transferable markers. In addition, because these markers were gene-linked, they also

provided functional information.

A new approach to development of transferable and functional markers was explored by

this research. The innovative mapping tool, named "Gene-Pair Haplotype" (GPH) consists of a

stretch of intergenic space and takes advantage of its rich polymorphism for the development of

markers. GPHs are PCR-amplifiable, with PCR primers anchored to exons of adjacent genes,

making these makers transferable between species where microcolinearity is maintained. A

significant degree of conservation between Fragaria, Medicago and Arabidopsis has been

demonstrated (T. M. Davis, personal communication) suggesting that these same intervals might

be easily transferable between rosaceous crops.

This investigation aimed to introduce the gene-pair haplotype concept as an innovative

mapping tool, thereby increasing the number of transferable and functional markers genetically

linked to the existing F. vesca x F. nubicola diploid reference map.












nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnTAAGAACTTGATGTTATTTGCTTGATGAGTTGAT
GGAGGATTACATATGAGGTTTGGTTATGTTTTTAGGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTG
CCTAAGGAGGCCGAGGAGTACCTTGTCTCGCATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAA
AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCATGTGTTCTAGTTTCTGTT
TGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCAT
TGTATAATTCGGTTTTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAAATCTTCAGTTGCACC
TGATCAGCTTACACGAAAGAGAATGCCGGATGTGATAAATTCAGGTGTCAATGACCCTCCACAAAAGAGATCATTGG
ATGTAAGTATCATATGCTACATGGAACTTTTGTAGTATGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTC
GGGTTTTTATTATAATTTTTCAGCAGCCAATGTCTTCTTTGGTTAATTCAGGCTTCAAAATAGCAGCCAATGTCTTCTTTGCACCTCATCAGCTT
ACGCAAATGAGATTGCCGGATGTGGTAAATTCAGGTGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTAT
CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAGTTTGGCTGGATTATGGA
GTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGATTTTTGGCTTCCAGTTTTTCTTCACATAAGCATTT
TAAAGCTGGTCATTGTAATTGAACTCGAATAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATC
ACAATTTCGTATTTTATGATCAACACCGAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAAGTGGATTTCTT
AATATATGCCTTGTCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGG
ATTATCTGGTGCAGGAGCTGTACTCCGTGCTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAGCATTGTGAATT
TTTTTTTATATATATTTTTGTTTTTGTAAAAATGGTTTTATAACATTGGGGTTACTATAGTTGCACCGGCTGCGGGA
AGGTGTGTGCAACGGCA


>GPH5_nilgerrensisclonel9
CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACTGGAAGATCAT
CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT
GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG
AAAAGAACTTGCTTGCGCTTTCTCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT
ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT
CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT
TGGTGTTCAAAGTCGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAACAGAAGGTAATATGCATG
ATATAAATACCAAGTTAATTGTACAATGATATTTGTAATCAGTGAAAATAATGAAAATCTTTATAACAAAATTTCAG
TTATCCTTCCATTGCTGTGTGAACTGTTACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAG
ACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAGAAGAC
AAAGAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCAT
GTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAACACATATTGGACCATTGGGCT
TAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTATAGAAACAGAGATTCTCTGTGATCTTCA
CCATTGAGGAGTCAAGTTACACAGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGG
TTCTTCAAAGTTTGAAACTTTAAGCTTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTG
GTTGGCATATATAAAAGCTTGAAGGATTGCCAAACCAAGCTGGTTCCTCGGTAAAGCTTTGATCTTTTAAGCCTTTT
ATAATTTGATTACCCTTATTGTTTTATCAAnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnGTTTTTCTAAGAACTTGATGTTATTTACTTGATGAGTTGATGGAGGATTAC
ATATGTGGTTTGGTTTTGTTTTTAGGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGG
CCGAGGAGTACCTTGTCTCACATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAAAGATGGTCTG
TTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATTTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTA
TTTTCATGGCATGTGGCGTGGAGCTAGTTGCATATGGTATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGT
TTTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTGATTCAGGGTTCAAAATTGCACCTAATCAGCTTACA
CGAAAGAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAGATCGCTGGATGTAAGTATCAT
ATGCTACATGGAACTTTTGTAGTTTGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCAGGTTTTTATTAT
AATCTCTCAGCAGCCAGCGTCTTCTATGGTTAATTCAGGCTTCAAAATTGCACCTTATCAGCTTACACGAATGAGAT
TGCCGGATGTGGTAAATTCAGNGTGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGCATCTTATGCTACAT
GGAAATTTTATTCTGGCCAGATTGGTATGAAAATCCAGATACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATC
ACTTGTTTATTGTATTTATTCCGCAAATGATGTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGA
TAATTGTAATCGAACTCGAGTAATT CTACTGGTGTAAGTTGCCTTGTGTC CCCACCACTAAGATCACAATTTCG
TATTTTATGATCAACACTGAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAAGTGGATTTCTTAATATATGA
CTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGG
TGCAGGAGCTGTACTCCGTGCTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGGAT









selected from each plate with transformants containing PCR products from diploids. Because

several different alleles were sought for the octoploid, 30 colonies were selected from the plates

that had transformants with inserts amplified from 'Strawberry Festival'. The tested colonies

were streaked on a separate LB/ampicillin plate during the set up of the colony PCR reactions.

The PCR products were resolved in 0.8% agarose gel with lx TAE buffer. PCR-confirmed

transformants were grown in 3ml LB broth containing 50[g amplicillin/ml for approximately 4

hours at 37C, with agitation at 220 rpm. Plasmids were extracted from 1.5ml culture by the

alkaline lysis method, followed by 24:1 chloroform extraction. Isolated plasmids were

resuspended in 50[l of deionized water and 5 [il were digested with 1 unit of restriction

enzymes: EcoRI or XbaI/XhoI for amplicons ligated to TOPO or pJET1, respectively.

Transformants carrying distinct alleles were detected by digestion with EcoRI. The digested

bands were resolved in 2% Metaphor/1xTBE or 2% agarose/lxTAE. Clones with similar

restriction patterns were grouped into different classes and a representative clone of each class

was sent to DNA sequencing facilities. A list of primers generated for sequencing reactions can

be found in Appendix C, whereas the sequences generated are included in Appendix D.

Sequences obtained were analyzed for conservation between diploid and octoploid alleles.

Alignments were performed using the global alignment tool ClustalW available at the European

Bioinformatics Institute's website at http://www.ebi.ac.uk/clustalw/. Except for the penalty for

gap extension, which was set at 0.05 instead of the default 6.66, all other penalty settings were

the default ones: gap open: 15; end gap: -1; gap distance: 4.

Results

Considering the number ofFragaria ESTs available at the time this study was initiated

(approximately 1,500 ESTs), and the estimated 26,000 genes in the Arabidopsis genome (Sterck

et al., 2007), if micro-colinearity indeed existed, the chance that two adjacent genes would be










Predicted
Number of Genes
Fosmid
ab Similari
initio ty


Protein Hit


EST
Hits
(gb no.)
CX6616
57.1
C08169
31.1


Fosmid
Insert Size
(bp)


Putative Gene Distr (kb
between genes)
ab initio Similarity-
Sit based
based


peroxidase
unknown


9 3


ATP synthase,
mitochondrial
X

X

X
Release Factor 2,
chloroplast
X


37,961 4.2


DY668
653.1

DW342
667.1
DW344
738.1
+ DW346
600.1


BQ1055
41.1


heat shock binding

hydrolase
leucyl-tRNA
synthetase
leucyl-tRNA
synthetase
leucyl-tRNA
synthetase
leucyl-tRNA
synthetase
zinc finger family
20G-Fe(II)
oxygenase
integrase


senescence-
associated
26S ribosomal RNA
(not a protein)
X
senescence-
associated
X

X


37,707 4.7


- DY670
360.1
+ DY671
649.1

- CX6613
47.1
CA8540
88.1

- CX6613
47.1

CX6614
21.1


23,212 3.3 11.6


7
8
11D02
1

2
Not
predicted
3


12.7


8 8


7 2


7

8

9
13103
1
2

3
4AA



5

6

7

8

15B13
1
Not
predicted
2

3

4

5









de la Cruz M, Ramirez F, Hernandez H (1997) DNA isolation and amplification from cacti.
Plant Molecular Biology Reporter 15: 319-325

Degani C, Rowland LJ, Levi A, A. HJ, Galletta GJ (1998) DNA fingerprinting of strawberry
(Fragaria x annanssa) cultivars using randomly amplified polymorphic DNA (RAPD)
markers. Euphytica 102: 247-253

Degani C, Rowland LJ, Saunders JA, Hokanson SC, Ogden EL, Golan-Goldhirsh A,
Galletta GJ (2001) A comparison of genetic relationship measures in strawberry
(Fragaria x annanssa Duch.) based on AFLPs, RAPDs, and pedigree data. Euphytica
117: 1-12

Deng C, Davis TM (2001) Molecular identification of the yellow fruit color (c) locus in diploid
strawberry: a candidate gene approach. Theor Appl Genet. 103: 316-322

Dirlewanger E, Cosson P, Tavaud M, Aranzana M, Poizat C, Zanetto A, Arfis P, Laigret F
(2002) Development of microsatellite markers in peach [Prunuspersica (L.) Batsch] and
their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theor
Appl Genet. 105: 127-138

Diwan N, Bouton JH, Kochert G, Cregan PB (2000) Mapping of simple sequence repeat
(SSR) DNA markers in diploid and tetraploid alfalfa. Theor Appl Genet. 101: 165-172

Doleiel J, Bartos J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size
of trout and human. Cytometry 51A: 127-128

Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf
tissue. Phytochemical Bulletin 19: 11-15

Durbin M, Learn Jr G, Huttley G, Clegg M (1995) Evolution of the Chalcone Synthase Gene
Family in the Genus Ipomoea. Proc Natl Acad Sci U S A 92: 3338-3342

Fay EW (1903) Latin etymologies. The American Journal of Philology 24: 67

Fedoroff N, Wessler S, Shure M (1983) Isolation of the transposable maize controlling
elements Ac and Ds. Cell 35: 235-242

Fedorova NJ (1946) Crossability and phylogenetic relationships in the main European species
of Fragaria. Doklady Akademii Nauk SSSR 52: 545-547

Feldmann KA (1991) T-DNA insertion mutagenesis in Arabidopsis. Plant J. 1: 71-82

Feldmann KA, Marks MD (1987) Agrobacterium-mediated transformation of germinating
seeds of Arabidopsis thaliana: A non-tissue culture approach. Mol Gen Genet 208: 1-9

Flavell RB, Bennett MD, Smith JB, Smith DB (1974) Genome size and the proportion of
repeated nucleotide sequence DNA in plants. Biochem Genet 12: 257-269











TTTCGATAGGATTGCAGTAATTTTTTTTGGACAGTATTACGGGACACTGTGACAGCTTTAGAGTTTGAATCTTAGGT
TGGATGATTTAAGTATCTTAGTTGAATGGATGTTATGACATATTGGTCATTAGTATTAGAGTTATGAGAAAGAGAAA
ATAAAATGAAAATACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAATGCAATAGATTGACAAAGAAAT
GAAGACCTAAAAAAACCATTGCATTAATGCAATAGTGTTGATATTCCAATCTCTCCTGAATAGTATTACAACTCTCC
TGGACAAGTCGTAACTGTGGGGGGTAATGGTGTAAACAAACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTG
CTGGGCAGACATAGCACCCCATAAATCATATCAGGTGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAA
ACCTAATTTTGTCTAAAGAATGCTAAAATCGAACACTCCCAAGCAACCGAATCTTCTGTTCCCCTGCTTTAGTATGT
TGTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGGAGTTACAGCATGAAGCAGCTCGTCTCTTGT
TGCAGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATGAAGCNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN


Clones from unspecific amplification
The size fragments amplified with primers for the vector were not the expected, i.e., they had
lower molecular weight than the that had been amplicon cloned.


>1 1D02nilgerrensis unspecific
ATAAAACCTTAAAACCAGTTTTGGAATATCTAATAACAATCACAATAAACTTCTGATAATGAGATATTAATCCTCAC
ATATCTTATTCAACAAGNCCTTAAACAACATTTCAAACCTCACTTAAAAATATTCCTCCACTTTTCATAGGTGTGAC
AATAGGTGCAGTTTTTCCCGTTTGCGCAAAAACTTTTCCCCTCATTGAAATATTTACATTTTTTCTTCTTATAGAAA
GAGTCACGAGGCACCGGAGCTTCCACCGCTGGCTCAGGATGCTGCTGTGCGAACGCCGCCTAATGATGGGGAGGACA
AACCGCCTGGAGATGCTGAGGATAACTCTGATAACCCGCTTGAGGATAGAGAGAATAACTCACCTGAGGATGGGGAT
GATAACTCTGATAACCCGCCTGGGGATGGAGAGGATAATCAATATCCATAAATCAACACAAACCATAAATTGTTCAA
ATAAGTTATTGGTGTGAACCAAATCATAAAACCTTAAACCAGTCTAGAAATGTCTAATAACAAGCACAATAAACTTC
TGGTAATGAGGCATTAATCCTCACATCTTATTCAACAGGCCTTAAACAACATCTCAAACCTCACTTCATCTGGAGTT
GAAC


>11D02 F ananassa 3 unspecific
AAAGACGTTTTGCAGATGTTCACTCCAGATGAAGTGAGGGAGTGTGATGCCATTAAATGGCAGCAACTATAAGACTT
GGAGAACTGAGTTAGACCTGAACCTAGCTCAGCAGAATGCAGATTGGTGTCTAAGTGTTCCAATGCCTACTGAACTT
GGCGCAGCTAGAGATAATTGGCTGAGAGCTAATAAGATCTGTAAGCTTACCATTATACGGACTATGACAGATGTTGT
GAAAGGTGGTATCCCTGAGAAAGAGGTAGCTAGTGAGTTTTTGGAGGCCATAGCTGAGAGGTTTGCTGTGAGTGACA
AGGCTGAAACCAGCATGTTGCTGGATCAGCTGCATAGCATGAAGTATGACATGAAGCTGAACATTAGGGAGTATATC
CTGAAGATGATAGACATAGCCTCTAAGCTTACTGCCCTTAAGATGACCATAGAGGAGGACTTGGTGGTTATGCTGGT
TCTGAAATCCCTGCCTGTAGAATTTGATCAGCTGAAAACTGCCTATAACACCCAGAAGGATAAGTGGTCGTTGAATG
AGTTAATTGCTGTGTGTGTCCAGGAGCATGAGAGGATTAAGAGGGTAACCATTCACCTAGTTACCACCAAACCTCAG
TGGAACAAAGCTGAGAAGAAAACTGGTCCTTCTAAGAACTTGGGAGTTGGCAAGAAGGCCATGAAGGTCAGTGGTAA
CAAGGGAGGCATTAAGTGTTTTTTCTGCAAGGTTAAGGGTCACATGAAACCTGACTGTGGCAAGTATAAGACCTGGA
AAACTATGATAGGAAATGAACCAGTCAAAAACTTTTATGTTTAGGTTAATTTTAGCTAATGTTCCAACTGAAACTTG
TAGTTTT


>11D02 R ananassa 3 unspecific
ATCTAAAGGCAGCGTGTGACCAATCCAAAGGGTTGTACTTCTACTTGCTTTCTTTTTAGATTGTTTAGCAAATTTAC
CCTTAACACACTCGATGCAATCCACCAAATCAGAAAAGTCTAAATCCGGCAAAATTCCGCTGTTTTACCAACAATTT
TAATCTCTCCTTTGAGACATGTCCAAGTCTCCTGTGCCAAAGAAAAGCAGACTTCTCATTCATTTTTCTTTTATTTC
CAGTAATAGAATCATCAACACCATTCTCAATCAACAAAACTTCAGTGTGGCAAGTATTAGGTAAAGACCAGTAATCA
TCAATAAACTGAGCAGTAGCAAGATAATGAGAAGCACGACTTATTTTCATTCCATAAGAATCAATCAAAAACTGACA
ATTGTCTTTAACCAAAACAGCAACTGAAATTAAGTTCCTAGACATGGAAGGAACATAATACACTTGCTCTAAAACTA
GAAAAACTTCTGGCCTCAAAACTAACTTAACAAAACCAACTGCTTCTATTTCTACTCTCGTGCCTTCTCCAACATGA
ATCCTGACTTCATTTTCCCTTGAACTCAACAGGCTTTGAACCCTCTTGTAAAGAATAGATAATGTGCCCCTGTGAAC
CTGTATCACACTCCCAGTAACTGTATAACATTTCTTTAATTCACCACCAACTTAGAAATTCCCTTTTATACCATTAC
CTTCCAATCCTTCAATCTAATATATTTATAACTAACCCAAGATTGTTCCCATAACCTCCCTAAAAAAAATATAATA
TGAACTCTCTTTTTATCTCATCATTAGCAATGACTATCTCC
















0.7k 1.5K1
1.2kl
0.5k 1.o K
0.7kl
0.5kl
0.3k 0.3k
0.3kI


Figure 5-2. Amplicon restriction patterns for GPHs 34D20 and 72E18. M: molecular weight
marker; U: uncut amplicon; PI: female parent, F. vesca, P2: male parent, F. nubicola;
H: heterozygote.















2710 vesca TTCATCACAACAGCTAAGCAGCTCATG-ATTCCTTTAAACACAACAAAAAAA---CCC 115
27F10 mandshurica TTCATCACAACAGCTG GCAG CTCATG-ATTCCTTT CACACACA AA--CCC 115
F10 nubicola TTCATCACAACAGCTGAAGCAGCTCATG-ATTCCTTTAAACACACA AA --CCC 114
F10 iinumae TTCATCACAACAGCTGAAGCAGCTCATG-ATTCCTTTAAACACAA-AAA --CCC 113
2710 ananassa TTCATCACAACAGCGAAGCAGCTCATGGACTCCTTAACAACA-AAAAA--CCC 113
2710 viridis TTCATCACAACAGCTAAGCAGCTCATG-ATTCCTTTAAACACAAAAAAAAAAACACCC 116


27F10vesca ACAGTCAAAATGAGGAAATGAACAATACCCAAGTCATGAACACACAAAATTCAGTAAAAA 175
27F10 mandshurica ACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGAACACACAAAATTCAGTAAAAA 175
27F10 nubi cola ACAATCAAAATAGAAAATGAACAATACCCAAGTCATGAACACACAAAATTCAGTAAAAA 174
27F10 iinumae ACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGAACGCACAAAATTCAGTAAAA 173
27F10 ananassa ACGATCAAAATGAGGAAATGAACAATACCTAAGTCATGAACACAAAAATTCAGTAAAAA 173
27F10 viridis ATAATCAAAATGAGGAAATGAACAATACCCTAGTCATGAACACACAAAATTCAGTAAAA 176
********** ************** ********** ******************


2710 vesca AGTAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 235
210 mandshurica AGTAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 235
F10 nubicola AG-AAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 233
F10 iinumae AGAAAAAAGGGATCCGCTTCAATACAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 233
2710 ananassa AGAAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCACCCCTTTGGAGACAAAT 233
2710 viridis AGAAAAAAGGGATCCGCTTCAAGCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 236


27F10vesca TTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATG 295
27F10 mandshurica TTCGTTGCTTAATGTAATAAGCAACAAAAAAATCAGCTCGGCTGGATCAAAGCCCAGATG 295
27F10 nubicola TTCGTTGCTTAATGTAA AAAAGCCAAATTCAGCTCAGCTGGATCAAAGCCCAGATG 293
27F10 iinumae TTCGTTGCTTAATGTAATAAGCAACAAAAA-TCCAGCTCAGCTGGATCAAAGCCCAGATG 292
27F10 ananassa TTCGTTGCTTTGAATAAGCAACAAAAA-TTCAGCTCAGCTGGATCAAAGCCCAGATG 292
27F10 viridis TTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATG 296
****************************** ****** ********************

2710 vesca AAAAAGATTAAAACTTTAAACAAGAAAATAAAGATCAGAGAAAGAAAATATGATGGGTAG 355
F10 mandshurica AAAAAGATTAAAACTTTAAACAAGAAAATAAAGATCAGAGGAAGAAAATGATGGGTAG 355
F10 nubicola AAAAAGATTAAAACTTCAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGTAG 353
F10 iinumae AAAAAGATTAAAACTTTACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGATGGGTAG 352
2710 ananassa AAAAAGATTAAAACTTTACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGGTGGGTAG 352
2710 viridis AAAAAGATTAAAACTTTAAACAAGAAATAAAGATCAGAGGAAGAAAAATGATGGGNAG 356


27F10 vesca ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCAGTGAAGGACTGA 415
27F10 mandshurica ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCAGTGAAGGACTGA 415
27F10 nubicola ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCAGTGAAGGACTGA 413
271F10 inumae ATCGGGAGAGATAAAATTACCAGAATCTGAAGTGGGGGAAGTGAATCAGTGAAGGACTGA 412
27F10 ananassa ATCGGGAGAGATAAAATTACCAGAATCTGAAGTGGGGGAAGTGAATCAGTGAAGGACTGA 412
27F10 viridis ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCCGTGAAGAAGTGA 416


27F10 vesca GTTGGTGGAGTCTTGGGAGATCTGAGATATAGCTCAAAGCCGGCG-AAGGATGCGCGG 474
27F10 mandshurica GTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG-AAGGATGCGCGG 474
F10 nubicola GTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG-AAGGATGCGCGG 472
F10 iinumae GTTGCTGGAGTCTTGGGAGATCTGAG------CTCTAAAGCCGGCG-AAGGATGCGCGG 464
2710 ananassa GTTGCTGGAGTCGTGGAAGATCTGAG------- C ------CCGGCG-AAGGATGCGCGG 457
2710 viridis GTTGGTGGANTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCGCAAGGATGCCCGG 476
ease ease we *** ********* e ****** ******** ***


27F10 vesca CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAGATGAGAATA 534
27F10 mandshurica CGCAGGATAGGAGGGAACAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATA 534
27F10 nubicola CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATA 532
271F10 inumae CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATA 524
27F10 ananassa CGCAGGATCGGAGGGAAAAGGGTGCGTAGGATAACCCAACCAATGAACCAAATGAGAACA 517
27F10 viridis CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCACTCCANGAACCANATGACAATG 536







184












GGTTTTAANTAGGGAAATGGAGGAGTGNGGGTGAAAAGTGTCCATCCCACACGTTTTAAANGAGCNTTAANGNAGGG
NAANGNGCAGNAACNGCAAGCTGCATNCCTNTAATNGGATCAATCAGAACATTAAGNCAACGTAAAGAGGAAGGTAT
TTGCTTTACACAACCTTATAAAATGATGNGGATNTACTCCAAAGTGAGGACTACCATGGTCGGCAAAATTAGTTCCT
GACTGTAACCAGCTAGGCATNGGCAATGCATAATAGCTATAGCTGACTATAGGTGGGAGACTCATCATTGAGATCAG
AGAAAAACNAAGATGAAAGAAAGAAATGATGAAACAAGAATAGTTATTCGAAAGCAAGTACAGAAGGGATTGTTCAT
GAAGTGTTCACCAAGTCACAGCTTAGGGCATTCTTAGAAGCAACAAGCTTGCCAACTTCCATTTACTTGTTTCAAGT
TCATGNTGATATTANCCATCCAACGAAAACATCCAAAGGTNCTGTGACTGAAAGCTCAAGGGGATATCNGTGTTTAA
AGCCACTACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCAAATGACAAAACAGTTCT
AATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCA
GAAAACTAACATCAATATTGAAATTGGATAAATATGCGATCTGAACTTCTTCACGTTGATGAGCTATCGTAGGAAAT
GGAATTGAACACTTGACACCAAAGAGAACAACGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGGAGGTAGAATC
ACCCATCGACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACAAAAGATCA
ATATTGCATGCAAAGAGCTTCTACATACAAACTCAAATGGAGATGTTCTGGCGCTTGTAGAATATAATTATGTATAC
AAATATGCATGTACAGAGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAAG
GTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAAAAAAAGACAAAACATTTAAGCAAAAGAAGAGCAGTAGAAG
TTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAGTCGTATAGAGGAGTGTACCTTCTTTAAACGGCGGT
GTTTTCCTAGGGCCCAATTGGTCATGATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACTGTCTGTGTTGCA
ATATTAAAACCCAACCACTGATAAATCTCAGTTGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCACGTGG
TATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTTGAT
TTATTATCCCAAAACCAAACCCA


>72E18 iinumae
GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAACCCGGCCTAAACATTAACATCAAAATCAGTCCTTGAGA
TTCGACATGCATAAAAAAGACAATAAAGGGTACAAAAACAACCACTCAAACAATCACAACATAATAGCATCCAATAC
CTTGACCATTCCGGTTCCATTATCACACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCTAC
ATACACCACAATCAATGACACCAATGCCTCATCGACACAACAAACAAATAGACATTCAAAAACAAAACACAATACAC
GATGCTAACATTTCCCTAATCTCTCCTCCATCAACTAAAATCTCCATTCCAAATCACACACTACTACACAGAAACCA
AAGCATGATTCAAAACAAACCAAGAACATCTACATAGTCCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAA
AGGCATCGAAAGCTAGTAAAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTATATACATAAAACCCT
CAAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATTAAAAAAAAATCAAGAAAAAAAGAAGAGAG
AAAATTACAGATCTAAAGCGACGAACAAATGAGAAGGAATGAGAGACAGAGAGAAGAGATGAGGAAGTTGACCTTTG
TGAATGAGAGTGAGAGAGAGAGAGAGATCGAAGACGAGGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG
GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATTTATGGACCGCGTCCATT
GCGCCCTTGTGGGGGCCATTACAGCTCCTTCCGCTGTTCCAGTCATTTTTCTCCACCTTCTTCACCTTTCTGCCCCT
CGTTCCCTTTCCCTCTTCTCCCAACTCTTCTTAGCCTAATTGCATTTTCATTTTAGTGCTTAGATCAATATGATTNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNCCGAAAGCTCAATGGGATATCTGTGTTTAAAGCCACAACATGACTAAATATA
ATTGCTCCCAATTTCTAAAGTTACATTCGTTTTGTACAAATGACAAAACAGTTCAAATTGACTGCATAAATAGATTA
CTCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAA
CTGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTGAACACTTGACACCAAA
GAGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGGGGGTAGAATCACCCATCGACGTGGATACTTGGG
TCTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTA
CATACAAACTCAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAGAGCTTCC
ACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAAGGTGAGTTTAAATAGACCAAGATG
TTAGCTAAAAAAAACAGACAAAACATTTAAGCAAAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAACATATTT
GGGTTGGAGGACAAAGTAGTATAGAGGAGTGTACCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCATG
ATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAAACCCAACCACTGATAAAT











ATACGAGTCAAACGAGCTAAAAACGAGTCGATTAAATCTAAGAGCCGAGGAGTAAAGTAATAACAAACTCGTTATCT
AAAATACAGGTTTAATATCAGCCGTTGGATCATATATACAGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCC
TCATTCCCAATTTTCATTCCCACCAAAAACAAAACC


>1OPPRIAB22 viridis
AACGGAGAAGAAGACTGTCGACATTTCTAGAGAAAGC AGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT
TATCTGAAAGACAAGTTTAATACCAGCCGTTGGATCATATTACTGCCCTGATCGCTCGACATAATTCGATATATATA
TATATATTATTTTTTTCTAAAAAAAATAAATCGATATACAGTATATTTTTTTTTGAAGTAATTAAATGATTATTTAA
ATCGCTTAAAAAGATAAACAAGAAGTTGGTTTAGGAG AGGAGCAAGAGTATGAGAACAAAAGTATGAGCCA
CACTGTTTGCTCTTCGGTTTGTTTATACAGAGAAAATATAAAAGTGATGTTGTAGAAACAATTGAACACTAAAAAAT
CAAATTACCTAACAACGAACCATCTTCAGACATACAAGATCAGAAAGTTAAGAGGTTCGAGTCGCACATGAGTTTCT
TGAGGCGATCAAATACCACAGTTTACTTGACTCAACAACTTTACGCATACGAGTCAAACGAGCTAAAAACGAGTCGA
ATAAAAATCGGGCACCATCAATATCGAGACTATGTAAGAGCCGAGGAGTAAAAAATAATAACAAACTCGTTATCTAA
AAGACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTGTGATTCGAAAACCGAGTTAACCCGCCAAACCCTCC
TTCCCAATTTTATTTCCCACCAAAAACAAAACC


>10PPR1AB22 iinumae
AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAG GCTTTGAACTTTGAAGTAGTGTAGGATAATAACAA
ACTCGTTATCTAAAAGACAGGTTTAATATCAGCCGTTAGATCCTATTAAGAGCCGAGGAGTAAAATAATAACAAAGT
CGTAACCTAAAAGCGGCTTCATATCATCTACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCC
AAACCCAATTTTCATTTCCCACCAAAAACAAAACC


>10PPR1AB22 ananassa clone2
AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCATCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG
TTATCTAAAAGGCAGGTTTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGC
CCAACTCAAATTCGATATATATTTTCGATATACATATATTTTATTTTTTTAAAGTAACTAAATGACTATGTACATCG
TTTAACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCATAACAGCTGAGAAAGAGTACGAGAACAAAAGTATGAG
CTAAAACAAATAGAGAAAATATAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCC
GATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCATATCATCCGCTTGATCATATATGCGGGTGTGAT
TCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACC


>10PPR1AB22_ananassa clone7_(samerestrictionpattern as clone20)
AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAAGTGGAGTGTAGGGTAATAACAAACTCG
TGATTAAAAGACAGGATTAATGTCAGTGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTTC
AAACCTCACGACATATGTAGGGTGTATGAATTATTAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACG
GCCTGATCACTCGACATATGTTGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTT
TTTTAAAATAATTAAATAACTATTTACGTTGTTTAACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAG
CTGAGCAAGAGTACGAGAACAAAAGTATGAGCTACATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAG
AAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCG
GCTTCATATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCA
ATTTTCATTTCCCACCAAAAATAAAACC


>10PPR1AB22 ananassa clonel8
AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCT GCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG
TTATCTAAAAGACAGGTTTAATATCGGCCGTTAGATCACATTACGGCCCTGATCACTCGACATATGTTGATATACGC
CTAACTCAAATTCGATATATATTTTCGATATACATTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTC
GATATACATTTTTTTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAA
GAGCACCATGACAGAGTACGAGAACAAAAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATG
TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACAAA
CTCGTAACCTAAAAGCGGCTTCATATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCG
CCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACC


>10PPR1AB22 ananassa clonel9
AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCT GCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG
TTATCTAAAAGACAGGTTTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGC









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40M11


vesca CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGA 60
mandshurica CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGA 60
************************************************************


vesca TGCATCAGAACAGTATGTGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTC 12(
mandshurica TGCATCAGAACAGTATGTGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTC 12(
************************************************************


vesca ATTAATCTGCAAACAATAAGATTTTTTAGGCAAAGCAGAACTATGAGTTCCCCAAACTAA 18(
mandshurica ATTAATCTGCAAACAATAAGATTTTTTAGGCAAAGCGGAACTATGAGTTCCCCAAACTAA 18(



vesca TAGCTTTCAAACAAGTAGAGGAGCACATTTACTAAAGATACCTTTGCCTGCTGCTCTTCA 24(
mandshurica TAGCTTTCAAACAAGLAGAGGAGCACATTTACTAAAGATACCTTTGCCTGCTGCTCTTCA 24(



vesca CTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTT 30(
mandshurica CTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTT 30(
************************************************************


vesca TTGAGGGGAAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACA 36(
mandshurica TTGAGGGAAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACA 36(
************************************************************


vesca GTTCCCTTCGGTCTCCCATCACCATCAAATACAATAAATTTCAATATATTTAACAAAAAA 42(
mandshurica GTTCCCTTCGGTCTCCCATCACCATCAAATACAATAAATTTCAATATATTTAACAAAAAA 42(
************************************************************


vesca ATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATTGTTCAATATATCATTTGA 48(
mandshurica ATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATTGTTCAATATATCATTTGA 48(



vesca AATTAACAACTTTTATTCTTCTAGTCAACCACATTTCGCAGCTACTTGTTAACTCATAA 54(
mandshurica AATTAACAACTTTTATTCTTCTAGTCAACCACATTTTGCAGCTACTTGTTTAACTCATAA 54(
************************************ ***********************


vesca ACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTT 60(
mandshurica ACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTT 60(
************************************************************


vesca CACAACAAT CCAACAAAAAGAT CAAAAAAACCATCCAAAACTCATGCACAACATAAT 66(
mandshurica CACAACGAATCCAACACAAAAGGATCAAAAAAACCATCCAAAACTCATGCACAACATAAT 66(



vesca CAACCAAATATTTTAACCACAAAAACAAGCACAATTCTCCAAAGTACA AAATGGG 72(
mandshurica CAACCAATATTTTAACCACAAAAACAAGCACAATTCTCCAAAGTAC AAATGGG 72(



vesca CTTTAGACACCAGGAAGGCATATCAAACCGGCCCACACACGTTAAAGGGATACAAAGATC 78(
mandshurica CTTTAGACCAGAAGGCATATCAAACCGGCCCACACCGTTAAAGGGATACAAAGATC 78(



vesca TCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAG 84(
mandshurica TCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAG 84(
************************************************************


vesca CTCCTCAGATGTCGGAGAGACCCATCTGAACCCAAGTCAACTGCACTGTTACAGCAACTA 90(
mandshurica CTCCTCAGATGTCG AGACCCATCTAACCCAAGTCAACTGCNNNNNNNNNNNNNNNN 80(



vesca CAAAACGCAAAGATAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAGATGAG 96(
mandshurica NNNNNNNNNNNNNNNNNNNNNNNN--------------------- 92-







196












From fosmid 10B08


>FvescaParent 10B08Fb
GTATGTCCTATTGATTAGATCGTAAATGATTAATTAGATAGGTAATTACTTTCTTGAAAGCGGGCAAGCCGTGATAG
TCTTGAAAGAGAGCGAGCTTTCTGAAGATGGATTTCCCGTTTGTTTTGAGCCCCGCCGCGTCTGGGTTGCTTGCAAC
CCACGACTCAAGTAGGTCAAGCGAAAGCTGCAAACAAGTGTGTTCATGACCATCAGTACATAGTCTGGAAGTTCAAT
GCTTACCTAATCAACACTACTCAAGTCAAGTGTCCAGTACTGATCGACCTACTTAGACCACGTACGTCATATATTTT
TCTTCTATATATTTTCAAGGCTAGAATGAGAACTAAGAACCTAGCTAGCTAGCTAACCTGATTCTCTGCAAGTCCCA
TCTGGATGATTCCGGTGGGATTTTTAACTTGATCATACGGGTTCTTCTCGTATTCTTCCCACCCTAGGAAGTATGAC
GAGTTCTGGCCATGAGAGTCGCAGCTAGCTTTCTTCGACAACATTTTCGAGCAGTTAAACAGATCAAGAGCTTTTCA
ATTCTGAGAGAGAGAGGGACAGAGGAGCAAAGAGAGCTAGACGTAGATAGAGAAGGTTTTGTAAGCAGCTCAGTTTG
GTTTGTGGAAAATATTTAGGTAAGGCTGCATGGATATAAATAGGTGCTGTTGATTCGTTTTACTTATTAGCTTAAAC
AAACACATATGAGTTGGACCTCATCCGAATCTTTTATCTTAATTCTACTCGTACTTTTTTTTTTTTTT


>FnubicolaParent 10B08Fb
AGTTAGTTAATAATATGTCCTATTGATTAGATCGTAAATGATTAATTAGATAGGTAATTACTTTCTTGAAAGCGGGC
AAGCCGTGATAGTCTTGAAAGAGAGCGAGCTTTCTGAAGATGGATTTCCCGTTTGTTTTGAGCCCCGCCGCGTCTGG
GTTGCTTGCAACCCACGACTCAAGTAGGTCAAGCGAAAGCTGCAAACAAGTGTGTTCATGACCATCAGTACATAGTC
TGGAAGTTCAATGCTNACCTAATCAACACTACTCAAGTCAAGTGTCCAGTACTGATCGACCTACTTAGACCACGTAC
GTCATATATTTTTCTTCTATATATTTTCAAGGCTAGAATGAGAACTAAGAACCTAGCTAGCTAGCTAACCTGATTCT
CTGCAAGTCCCATCTGGATGATTCCGGTGGGATTTTTAACTTGATCATACGGGTTCTTCTCGTATTCTTCCCACCCT
AGGAAGTATGACGAGTTCTGGCCATGAGAGTCGCAGCTAGCTTTCTTCGACAACATTTTCGAGCAGTTAAACAGATC
AAGAGCTTTTCGATTCTGAGAGAGAGGGACGGGG AGAGAGCTAGACGTAGATAGAGAAGGTTTTGTAAGCA
GCTCAGTTTGGTTTGTGGAAAATATTTAGGTAAGGCTGCATGGATATAAATAGGTGCTGTTGATTCGTTTTACTTAT
TAGCTTAAACAAACACATATGAGTTGGACCTCATCCGAATCTTTTATCTTAATTCTACTCGTACTTTTTTTCTTTTT
TCTTTTTCATGTGCATAAGCAAATGCATTTCCGATTAAACATAAAAATGTACTGTCGAAACATCATTTCCAGCCAAA
TCCAAACATTCGCTCTAAAAAAGCTACATTTGCTATTAGATTCAATAACACAAAACCAAGCAAACATTAATCTTCAT
AAATACCAAAATTGGCCTCAATACCCAACTTAAAAACGACCTCAGTCCAGAGGAACCTCAACGTCTCCGTCGGTGAT
CTCCTGCTGCAAGTCCTTGGGGTCCTTCCCATCCAC


>11D02 vesca
GAGCTGCTGTGTGAACCAAATGGTACAGAGAAGCCGTTTGCCAAACCTACCCATGATCCAATCAAATGCATAAACTT
TAAGAACTCAAATACCAAACGATCAAACATAATGACTGAAATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAA
AACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCAGCCATT
CCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAATTTCATACAGA
AGTCTTCCCACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATAGTATAAGAAGGA
GATGAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTG
GTTTCCTTTCAGTCTCTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCAAT
TTTCTTTTCTAGGTGATTTTTTTTCTTTTATAATTAATTTGGTTTTATTTTTCCAAATAATACCTGAAAGACTTTTT
TTTTCGATAGGATTGCAGTAATTTTTTTTGGACAGTATTACGGGACACTGTGACAGCTTTAGAGTTTGAATCTTAGG
TTGGATGATTTAAGTATCTTAGTTGAATGGATGTTATGACATATTGGTGATTAGTATTAGAGTAATGAGAAAGAGAA
AATAAAAT GAAAATACAGTACT GGCAATAAACACAATACGGT GGAGCAAT CAACAAT GCAATAGATT GACAAAGAAA
TGAAGACCTAAAAAAACCATTGCATTAATGCAATAGTGTTGATATT CCAATCTCTCCTGAATAGTATTACAACTCTC
CTGGACAAGTCATAACTGTGGGGGGTAATGGTGTAAACAAACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTT
GCTGGGCAGACATAGCACCCCATATATCATATCAGATGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTA
AACCTAATTTTGTCTAAAGAATGCTAAAATCGAACTCCCAAGCAACCGAATCTTCTGTTCCCCTGCTTTAGTATGTT
GTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGGAGTTACAGCATGAAGCAGCTCATCTCTTGTT
GCAGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATGAAGCTGCTCGTCTGG
AGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAGGTCCCAACACTTACCAGGTAGGTTAGTCTCTT
CTGCGTCGAGTAACCATGCGGGCACCTGGTGAGAAAAGCGTAACATCTCTCTTCTCGGAATCCATAGAATGGCGCTT
CTGTCCGTATCAGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGGTGAGACACAAACACCAATCTCTTC
AGAGTAATCATCAAGAACCTCCACCATTTGATATTGGTGCCTCACTTCATCTGGAGTTGAAC


>11D02 viridis









were developed by Kim et al. (Kim et al., 1992) to address undesirable recombination during

cloning in multicopy cosmid vectors. Due to the single-copy F-factor replicon, DNA inserted

into fosmid vectors underwent a lower rate of rearrangements and deletions than did fragments

inserted into cosmids.

In order to annotate the newly available F. vesca sequence, a complement of ab initio and

similarity-based approaches was utilized. Preliminary screening for putative genes was executed

by using the gene prediction software FGENESH (accessible at http://www.softberry.com) for

each of 26 fosmid insert sequences, using Medicago as the gene model. Subsequently, a series of

different types of sequence similarity searches were performed using BLAST algorithms

(http://www.ncbi.nlm.nih.gov/BLAST/), as illustrated in figure 3-1.

The amino acid sequences from each gene predicted by FGENESH were used as query

sequences against the non-redundant protein sequences database for "all organisms" using the

BLASTP algorithm. Significant similarities between a query sequence and a sequence in the

database, termed "hits", were indicated by an expectation value (E value) lower than 10-15. (The

lower the E value, the more significant is the score because the E value ultimately represents

how likely two sequences are of being similar by chance alone.) The threshold of 10-15 was

defined based on thresholds used in the Arabidopsis genome annotation (The Arabidopsis

Genome Initiative, 2000), where BLASTP E values < 10-20 and 10-10 were adopted to identify

protein families and functional roles between different organisms, respectively.

The BLASTP results that produced significant hits were used to guide the subsequent

BLAST interrogations because they determined which nucleotide fragments should be further

analyzed. Though the entire 30-45kb sequence could conceivably be analyzed at once, it is more

convenient to do the analysis in sequence parcels. The response to a BLAST submission of









32. CTAB buffer concentrations of 2% (T91), 6% (T92), and 20% (T93) were tested. The
slurry was formed by breaking down 400mg of tissue in liquid nitrogen first, then adding 2 ml of
buffer for further grinding. Once homogenization was achieved, another 8ml of buffer were
added and the mixture was incubated at 650C for 30min. The 10ml of buffer were split into 2
tubes (treatment replications) and 5 ml of chloroform:octanol were added to each tube. Nucleic
acids from centrifugation upper phase were precipitated by isopropanol and the dry pellet
resuspended in lml TE pH 8.0. Samples were quantified by NanoDrop.
33. Because homogenizing tissue in buffer seemed to have a positive effect on DNA
recovery, an experimented was set up to test Polytron homogenizer speeds (half maximum
speed-T95-T98; full speed-T99-T103) and duration of homogenizing treatment (no
polytron-T94; 5 seconds-T95, T99; 15 seconds-T96, T100; 30 seconds-T97, T101; 60
seconds-T98, T102; 120 seconds-T103). Enough Laboratory Festival #9 tissue for all
treatments (2g) was ground in liquid nitrogen and, by adding an aliquot of the buffer, ground to a
paste consistency. The paste was divided into 10 tubes and enough buffer to reach 5ml was
added to each tube just prior to treatment with Polytron. Samples were incubated at 650C for
30min. Downstream steps from incubation were as described above.

The final strawberry DNA extraction protocol is listed bellow.

Strawberry DNA Extraction Protocol

CTAB extraction buffer 100ml
2% CTAB 2g
1.4M NaCl 28ml of 5M NaCl
100mM Tris-HC1, pH 8 10ml of 1M Tris
20mM EDTA pH8 4ml of 0.5M EDTA
1% BME Iml
diWater to 100ml

Tissue-to-buffer ratio = 40 mg/ml. For 12-ml tubes, maximum tissue processed is 200 mg.

* Grind 200 mg of liquid-nitrogen frozen leaves (young or unexpanded) in mortar-and-pestle
* Add 2 ml extraction buffer to ground sample, macerate in mortar until consistency of paste
is achieved. Transfer the paste to a 12-ml tube, and add 3 ml buffer
* Homogenize utilizing a Polytron at full speed for 2 min
* Incubate for Ih at 650C, with intermittent agitation
* Add equal volume (5ml) of 24:1 chloroform:octanol
* Mix by shaking vigorously
* Centrifuge at 4,000 rpm, 5 min
* Transfer the upper, aqueous phase to a new 12-ml tube
* Precipitate DNA with equal volume of 70% isopropanol
* Mix by inverting the tube several times
* Centrifuge at 4,000 rpm, 5 min
* Discard the supernatant
* Air-dry nucleic acids pellet









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40: 1127

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Li L, Li W, Guangqiang Hu XH, Wenjie Li, Jian Li, Zhanwei Liu, Long Li,
Jianping Liu, Qiuhui Qi, Jinsong Liu, Li Li, Tao Li, Xuegang Wang, Hong Lu,
Tingting Wu, Miao Zhu, Peixiang Ni, Hua Han, Wei Dong, Xiaoyu Ren, Xiaoli
Feng, Peng Cui, Xianran Li, Hao Wang, Xin Xu, Wenxue Zhai, Zhao Xu, Jinsong
Zhang, Sijie He, Jianguo Zhang, Jichen Xu, Kunlin Zhang, Xianwu Zheng, Jianhai
Dong, Wanyong Zeng, Lin Tao, Jia Ye, Jun Tan, Xide Ren, Xuewei Chen, Jun He,
Daofeng Liu, Wei Tian, Chaoguang Tian, Hongai Xia, Qiyu Bao, Gang Li, Hui Gao,
Ting Cao, Juan Wang, Wenming Zhao, Ping Li, Wei Chen, Xudong Wang, Yong
Zhang, Jianfei Hu, Jing Wang, Song Liu, Jian Yang, Guangyu Zhang, Yuqing
Xiong, Zhijie Li, Long Mao, Chengshu Zhou, Zhen Zhu, Runsheng Chen, Bailin
Hao, Weimou Zheng, Shouyi Chen, Wei Guo, Guojie Li, Siqi Liu, Ming Tao, Jian
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the Rice Genome (Oryza sativa L. ssp. indica). Science 296: 79-92












nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnACTGGTGTAAGTTGCCTTGTGTCACCTCCACTAAGATCACAATTTCGTATTTTATGATCAACACT
GAATACCTATGTCTAGTGTCATGATTATAGTCATGTGAAGTGGATTTCTTAATATATGCCTCATCTATGTCTTCATT
CAGCAATCCTGCATACTTGAGTTTGATGGTGCTTCAAAAGGAAATCCTGGACCATCTGGTGCAGGAGCTGTACTCCG
TGCTGAAGATGGGAGTGTTGTATGTGGATTTCATGAAAACATTGTGAATCTTTTAGGATATATATTTTTGTTTTTGT
AAAAATGGATCTCTTTATAACGTTGGGGTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAACGGCA


>GPH5 ananassa clone7
CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA CCGGACCGGAAGATCAT
CGGAATCAGATTTGTTCGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGAGAAGATCCTAAGGAGGAACTCCGAGTCT
GGTTTAGAATCCTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG
AAAAGAGCTTGCTCTTGCTTTCCACCTACTCACAGCACGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT
ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT
CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT
TGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTTCCATTTGAAACAGAAGGTAATATGCATG
ATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAATAAGTGAAAATAATGACAATCTTTATAACAAAATTT
CAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCACACACAAGAGCAACAACACCAAACAAACAGAAC
CAGACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGA
CAAAGAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCA
TGTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGC
TTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTGTAGAAAATACTGTGATCTTCACCATTG
AGGAGTCAAGTTACTCAGCCATGAAGTCAAGGTCAAGCCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTCTTC
AAAGTTCGAAGCTTTAAGCTTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGC
ATATATAAAAGCTTGAAGGATTGCCAAAACCAAGCTGGTTCATCGGTAAAGTTTTGATCTTTTAAGCCTTTTGTAAT
TTGATCACTCCCATTGTTTTATCAATTTTTGATTTCCCATTTGATTACATTACTGGGTCTTGTTTATTTTGTTGAAA
TAACTATGCCCTTTCGTTCTAGCATGCAACTGAAATTTACTGCTAGATTGTATTGTTGTGCCGTTATGGTGTTCATT
ATGTAAAAGAGAATGAATTCTGGTGGTGGGTATAGAGTACCTCCCTGATTTTTTATGAGATACTATGCTTCTGGAAA
ATGTTATAAGATGAAAACTAGTTTTTCTAAGAACTTGATGTTATTTACTTGATGAGTTGATGGAGGATTACGTATGT
GGTTTGGTTTTGTTTTTAGGTATTCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGG
AGTACCTTGTCTCACATGGGCTTAAGAATGCTTCATGTACTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTTGGA
AGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATTTCATGTGTTCTAGTTTCTGTTTGGGCATCTGTTATTTTCA
TGGCATGTGGCGTGAAGCTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCAGTTTTTTA
TTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCGCCTAATCAGCTTACACCAAAG
AGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAGATCGCTGGATGTAAGTATCATATGCTA
CATGGAACTTTTGTAGTTTGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTT
TCAGCAGCCAGCGTCTTCTATGGTTAATTCAGGCTTCAAAATTGCACCTGATCAGTTTACACGAATGCGATTGCCGG
ATGTGGTAAATTCAGGTGTCAATTACCCTCCACAGAGGACATTGCCGGATGTAAGTATCTTATGCTACATGGAAATT
TTGTTCTGGCCAGATTGGCATGAAAATCCAGACACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTT
TATTGTATTTATTCTGCAAATGATGTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGT
AATCGAACTCAAGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACACTAAGATCACAATTTCGTATTTTAT
GATCAACACTGAATACCTATGTCTAGTGTCATGATTATAGTCATGTGAAGTGGATTTCTTAATATATGCCTCATCTA
TGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGTGCTTCAAAAGGAAATCCTGGACCATCTGGTGCAGGAG
CTGTACTCCGTGCTGAAGATGGAAGTGTTGTATGCGGAGTTCATGAAAACATTGTGAATCTTTTAGGATATATATTT
TTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAAC
GGCA





>GPH10 ananassa clone2
GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT
CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT
AGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTGGAGAA
AGCTTTCATCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTTATCTAAAAGGCAGGTTTAATATCAGCCGTTAG
ATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACA
TATATTTTATTTTTTTAAAGTAACTAAATGACTATGTACATCGTTTAACAAAAGAAACAATTGAAGTTAAATTAAGA
GCACCATAACAGCTGAGAAAGAGTACGAGAACAAAAGTATGAGCTAAAACAAATAGAGAAAATATAGAGGCGATGTT










APPENDIX B
In silico ANNOTATION AND DISTRIBUTION OF Fragaria vesca GENES

Under each fosmid name is a list of numbered potential genes predicted by FGENESH.
The nucleotide intervals that had protein hits by BLASTP were used for a similarity search
against the non-redundant Viridiplantae, protein database using BLASTX. The best matches
identified by the algorithm are listed under "Protein Hit". Threshold value was 10-15. Letter "X"
under "Protein Hit" denotes no similarity was detected in the protein database. Under
"Orientation", "+" signs signify that the query sequence is translated in the same direction it was
input, where negative orientation signifies that the complement strand is translated. "EST Hits"
are sequences of DNA for which an EST was detected within Rosaceae, with a minimum length
of 100 nucleotides and 95% identity.
Gene distributions were calculated by dividing each fosmid insert size by the number of
genes either predicted by FGENESH or identified by similarity to the non-redundant
Viridiplantae protein database.
Simple Sequence Repeats (SSRs) with at least 5 repeats of a motif are represented by
color-coded triangles:
A in FGENESH-defined genic sequence; kin FGENESH-defined intergenic sequence


Predicted Putative Gene Distr (kb
Number of Genes EST Fosmid between genes)
Fosmid Insert Size
ab Similari Hits n i Similarity-
Protein Hit C (bp) ab initio based
initio ty O (gb no.) based
01L02 13 7 40,302 3.1 5.8
1 unknown
2 X
3 A pectin lyase +
4 unknown
5 beta-glucan binding
6 enolase
7 x
x
919 unknown 436.1
10 X
11 X
12 L X
DY670
13 unknown
952.1
05N03 8 5 34,611 4.3 6.9
ATP +
1 A binding/adenylate
cyclase
2 X
SSenescence- + CX6614
associated 21.1
DW248
4 hypothetical 990.1
990.1









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Tuskan GA, Difazio S JS, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S,
Rombauts S, Salamov A, Schein J, Sterck L, Aerts A, Bhalerao RR, Bhalerao RP,
Blaudez D, Boerjan W, Brun A, Brunner A, Busov V, Campbell M, Carlson J,
Chalot M, Chapman J, Chen GL, Cooper D, Coutinho PM, Couturier J, Covert S,
Cronk Q, Cunningham R, Davis J, Degroeve S, Dejardin A, Depamphilis C, Detter
J, Dirks B, Dubchak I, Duplessis S, Ehlting J, Ellis B, Gendler K, Goodstein D,
Gribskov M, Grimwood J, Groover A, Gunter L, Hamberger B, Heinze B,
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Rpf2 gene. Theoretical and Applied Genetics 94: 1092 1096












GAAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTAGTAATCATGGAGATGATGAAGAGCGTAATGATGATGATG
TTAATGAACCTTGGTATAGAGAAGAGACCGTTATGCCTCAGCAGACGAATTTACGACTNNN


>34D20 ananassa
NNNNNNNNNAACTGATGCGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTCATGGGATAAAGGAAGTA
TTAATGTTAGGCATCCTAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC
GAATTTGATTTCTACAAGGTGATCAACATGCCTGCACACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT
TATGTGAAT CTGCATATTATCTATATCTCATTGGGATATTATCTATG TATAA TCAGAACTACCAGGAAAGATTATCGGC
AAGAAGGTGTTT TGCATGCATGCAGCACAAAATGCTATGGGCCAATTTCCCTTGCAAAATGCTATGGTGTAATG
ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA
ATGGATTTAGTACTTCCATTAACTAGTAACTGAGGAATACAATTGCACTCTGTGTTTTCCATGCACAGGAGAACTGC
AGGTGAACTATATGTCTATATAGATATGTCGTATGTTAGATAGGATATGTATGTGGGTGTGTATGAACTATAAG
TAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGCATGAGTCTCCCTCCCGGCCACACTAGAC
CACACTTTTGAACTGGCGGATTCCATCCGGCCTAGATCTTGTGCCGACTATCACAATAGTGTAAGTTGGTCCTCCTA
GCTATAGTTTCTAGTACTATTCTACTGATATCATGTTTCGTCTCAGCTTTTGACAATGGAATATGATGGATATGGAA
TGAACAAAACCTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTTTCTTT
ATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGCATTCTGCTGATTTAGGATTTAGGATTGCACTACTTGT
ATAGTTGTATTGATCTAAATTTTTCCTAGTTAACAGAGGATAGGACTCCGGCTGACCTTATCCTACAAGGAAACAGA
AACGTACAATTAACGGATTCACAAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATT
GTCCAGTACTGGAAGAGCTATATTTATCTGATAACAGAAAGTGCGTACTTGCTGGTTCATACTCAATATGGATCCGA
AGGTCCTTAGTTCCCAAGGTGTTGACCTGAGAATGAGCGACTTGGATCTTCTTAGAGGCCCTTATTACTTAACCGAT
AGCATCATTCAATTCTATTTCACTTATCTTACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTC
TGATTTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGA
AAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG
TATTTCAGAAAATCAAACGCATTGGTACATTACGACAGCTTGGGGGGTAACAATAGTTTGGATGCTAGGAAAATGTA
TACAGCATTCAAGAAACTTGTGGCAGTTCCAGCAACACAAGCACCAACTCCAGCTGGGAGTAGTAGTTTGGTTACCA
ACAACAGTTCTACAATGGGACACGAGTGCTACTCTACGCAGTCGCGGCGGTTTATAGACCATACCAAGATAATGCTT
CGGGTTGGGGGTTTTGTTGTCAAATACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATCATCATCCTTC
GGAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTAATCATGGAGATGATGAAGAGCTTAATGATGATGATGTTA
ATGAACCTTGGTATAGAGAAGAGACTCTTATGCCTCGGCAGACGAATTTTACGACTGCG



>40M1ll vesca
CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGATGCATCAGAACAGTATG
TGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTCATTAATCTGCAAACAATAAGATTTTTTAGGCAAA
GCAGAACTATGAGTTCCCCAAACTAATAGCTTTCAAACAAGTAGAGGAGCACATTTACTAAAGATACCTTTGCCTGC
TGCTCTTCACTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTTTTGAGGGG
AAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACAGTTCCCTTCGGTCTCCCATCACCAT
CAAATACAATAAATTTCAATATATTTAACAAAAAAATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATT
GTTCAATATATCATTTGAAATTAACAACTTTTATTCTTCTAGTCAACCACATTTCGCAGCTACTTGTTTAACTCATA
AACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTTCACAACGAATCCAACA
CAAAAGGATCAAAAAAACCATCCAAAACTCATGCACAACATAATCAACCAAATATTTTAACCACAAAAACAAGCACA
ATTCTCCAAAGTACAAAAAGAAATGGGCTTTAGACACCAGGAAGGCATATCAAACCGGCCCACACACGTTAAAGGGA
TACAAAGATCTCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAGCTCCTCA
GATGTCGGAGAGACCCATCTGAACCCAAGTCAACTGCACTGTTACAGCAACTACAAAACGCAAAGATAGAGAGAGAG
AGAGAGAGAGAGAGAGAGAGAGAGAGAATATTACACAGGAAAAAA ATCTGGGATCAGAGTGATGATGCTGTG
TTTGTGTTTGAGTTTGAAACTGAAGCCCAACCACGACCAACACGTGCTATTTGTAAGCCGAACCCATCTGCCTTCCT
TTCCTTCCATGTCTGTTCTGTGGTGTAGACTTTTCGGACAAAGCTTCCGTGGTGTCGTTTTTTTCCCAGGGTGGAAA
GGTCTGAACTGCCCCGGGGGACAACGGCGTGGTGTGGTGTGGCCGCTGCCCTTTTGAGGAAATTCACGTGGATTATG
GTGTGTCCTGCTTGTACTGTTGTCGGAGTTTACTAGGAACAATGAAATCATATCTATTTCTCATAAAAGGAAGCGTA
TTCTTTTTTATTAACCTTTTATTAATTACCTAGATTAGCTATTTCAAGTCAAAATTCATATATCGAAATATATGCTT
CTTGTCGCTAGACGCTATATTAGGTAACGAACATTTTAGGTAGTCGAACTGGTAGGCAGCGGCGGAGCTAGGATTTG
TTATTAGAGAGGTTAAGATGTAAAAGGTAAGTTTATGTATATGACACTTATTTATATATTTTTCCATTTTTAATGTA
AAATTCTATTATGATTTGGAAATCTGAGAAATATCTTATTTTCATTTGAGAAATGTCTTATTTTCATATGAGAAATG
GTGTGTGTGGGAGCTTTTGGGGGGGGGGGGGGAGCTAAAGGCATTTTTCTATTTATATTAGTTGTTTAGAAGAAAA
AGAATGAACTATTTGGACATGACCCCTCGACCCAGCATGACCTGATTTTGAGATTAGAAGGGCCAAGTAACCAAAA










Predicted o Putative Gene Distr (kb
Number of Genes EST Fosmid between genes)
Fosmid a Insert Size
ab Similari Protein Hit Hits n Similarity-
SProtein Hit (bp) ab into
initio ty O (gb no.) based


x
x
x
heat shock


36,230 4.0


6
7
8
9
52E09
1
2
3 A
4
5
6 A


28,318 4.7


+ DY672
511.1


unknown
cyclin-like F-box
X
cyclin-like F-box
cyclin-like F-box
cyclin-like F-box

Arf GTPase
activating
heavy metal
transport/detoxificati
transposase


phospholipase D
unknown
binding
X
X
X

hydrolase
hydrolase
reverse transcriptase
hydrolase
X
hydrolase

unknown
transferase
spliceosome-
associated
unknown
actin 7, actin 11

glycoprotein-like


ribosomal L24/L26
X
X


DV438
212.1


+ DY669
358.1
+ DY675
437.1
+
+

+

DY670
963.1


40,183 5.0


9 8


36,293 3.0


8 a
9
63F17

IAA
2
3

5A
6
72E18
1
2A
3
4

A
6


7
8
9
10
11
12

84N10
3IA

3


6 3


12 11


8 2


20.1














4.1-6.2 1


E
C
r 2.3-4.0



c 2.0-2.3
o
1)
-o
1.7-1.No amplification
E EAmplification
C
0

4 1.5-1.6
0)
u
C

-o
L 1.1-1.4
-o


0.5-0.6


0 10 20 30 40 50
Number of samples observed

Figure 2-5. DNA contamination by carbohydrate (estimated by the ratio between absorbance at
260nm and 230nm) and its influence on PCR outcome. Absorbance at 230nm and
260nm wavelengths were observed for 94 samples from a genetic linkage mapping
population. The A260/230 ratio was calculated for each sample and the ratio data
were grouped into 7 categories, varying from 0.5 to 6.2. Most samples presented ratio
in the 1.7-1.9 range (1.8 is the optimum for DNA purity from carbohydrates).
However, even within the purest DNA category, amplification by PCR was not
observed for 1/3 of the samples. Therefore, contamination by carbohydrates may not
be considered the sole responsible for the polymerase inhibition.


























































































gerrensis HV\AG/\ GGG/\GT AIO GG TA i LOAilenae G
idshurica AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAA(
)icola AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAG(


CATGATTATAC
CATGATTATAC


;CTG 26'
;CTG 26'
;TTG 26:


GATATATAT
GATATATAT
GATATATAT
GATATATAT
GATATATAT
-ATATATAT
FATATATAT
-ATATATAT









because sequences utilized for similarity search were from F. x ananssa, this method is better

than the annotation ofF. vesca genome method to address questions of diploid subgenome

contributions to the octoploid. Primer pairs designed for gene pairs detected through this method

amplified the octoploid, whereas most (8 out of 11) of the primer pairs generated through F.

vesca genomic sequence did not amplify alleles from the cultivated strawberry. This study

further supports the likelihood ofF. iinumae as the B genome donor to the octoploid.

The approach based on gene prediction to identify gene pairs, had a higher amplification

success rate and it is useful to characterize intergenic regions, serving as a tool to detect

polymorphisms between diploids. Chapter 5 showed how this approach was successfully

employed to create molecular markers in the Fragaria diploid reference map.

We have described the development and mapping of 8 markers, linked to at least one gene of

known function. Therefore, this investigation proved the concept that putative intergenic regions

may be used as functional markers. In addition, because the markers are designed for conserved

sequences across different taxa in Viridiplantae, there is great potential for transferability and use

on comparative mapping to appreciate Rosaceae structural genomics.










Predicted o Putative Gene Distr (kb
Number of Genes EST Fosmid between genes)
Fosmid a Insert Size
ab Similari Protein Hit Hits n Similarity-
Protein Hit (bp) ab initio
initio ty 0 (gb no.) based
4 ATP binding
5 x
6A x
7 X
8 kX


Totals
Means
Sample
Standard
Deviatio
n


235
9


129


905,491
34,827


2.1 2.4


4,426












CTAACTCAAATTCGATATATATTTTCGATATACATTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTC
GATATACATTTTTTTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAA
GAGCACCATGACAGAGTACGAGAACAAAAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATA
TAGAGGCGATGTTGTAGAAATAATAGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACAAA
CTCGTAACCTAAAAGCGGCTTCATATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCG
CCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACC


>10PPR1AB22 ananassa clone20
AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG
TGATTAAAAGACAGGATTAATGTCAGTGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTTC
AAACCTCACGACATATGTAGGGTGTATGAATTATTAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACG
GCCTGATCACTCGACATATGTTGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTT
TTTTAAAATAATTAAATAACTATTTACGTTGTTTAACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAG
CTGAGCAAGAGTACGAGAACAAAAGTATGAGCTACATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAG
AAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCG
GCTTCATATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCA
ATTTTCATTTCCCACCAAAAATAAAAC



>GPH23 ananassa clone3
CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACGACCTTTACAGTGAGAGTGTGACCAGAG
GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGATAAAA
GACCTGCCAGAATCCACGCCACCAAACATCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCA
TCAACATGCAATAATGTGGGTCCATAAGAACCATGTGCATGACATAAGATTCCTCAAGCTTCGATTTCCTAATTTGC
TTCAAAAGAAGTAAGTTCAGAGTCACTCAAACCCTAATATAGATCTCAAATTTAATGAAACATATTCCTAAGAGCCT
ACACAAATATAAAATCGTAACTGAACTTAATCTGAAACTGTCGTATAAATTGTAAATCGATCAAAACCAAACTTCAT
GTTCAGATTCACAGACCGTATCAGAGATAGCATACAAGTGACCTCTGAAACAAAACATAATTCCAACAAGATCGCAA
ACATTCGAGATTAAATACGATGAGCTATGAGACAACTATTCCATGCAAATCTAACAAAAAAGAATAAAGGGATCTGG
AGAATTATGGGTTAGAGGTGACCTTCAGAGTTTGGGTGAAACACAACTGGGGACGACCTACACCGAGGAGGAACTGC
CAAAATCTATCTGAAACCTAACAAATAAAAAGGGTCTATCTGTCAATAAACGAGCTCCCTATCGTCCATCTCCAATC
TTGTAAGGGGTGATCCTTACGCTTCCCTTTGTCCTCTCCCCCATCTACTAAGTAGACGCTAGCTGCGGATTGTTATT
ATGTTTTGGATAGAATACCTTTGCAAAATTGGAAGCTCCAGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAAT
ATCAGACCGTTCCGACGCCGGAGCTTCCTCAGAAAGCCTACCATCCGCAACATCGTTGCCAGGCCTTGCGAGGTTTG
CCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTATAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGATGAA
TGAATCATCAAATTAATTAATCAAGGTGACAAAGAAAGATTATATCCTTCCATTCCTAAGTCAAAAACCATTACAAT
GTACCGCCGGCAAAATGCTGCTAATAGAATTGACATTGTAAGTGGGGGATAGTGTCACGAGCTGCAATAGGAGGTAG
TGTCATCGTGACACTCTAGAGGTGATGCTTAAGAGGGTCACAAGGTCAATGGCAAAGCATGAGGGTTAAAGAGGATG
TTTACTGACATGTTGAAAGACAATGTCGTAATTAGTTAAAGTGAAGTACTGTGAAGTTAGTATTTCGAAAAACTGTA
ACATCGGAAGGGGTTCAATACATTTGACGACATATTTTTATGAAGTTATTAGGAATTAGTTACGAGAGATGGTTTTT
TCTTAGAATATTTTGATTTTGATGTTTCCTTGACACACATTATATTTCTCCATGTTCTTCTATGTATAAGTAATTTT
CTGTATCACTTAGAAACATTTCTTACTCTTTCCAGAAGCATCTCCAAACATCCCCCTAAACCAATAGCCCTAACATG
TCAATGTCACATGTCAATAGATGAAAGATCAACCTAAATGGTACCATATGTCCATACATAAAAGGACCCAAAAAAAT
AAATAAAGAAATAAATATGCACCTTCATTTTTAAGCGCCAGAAAAAGTAGAGAAGAATATAAGGTTTGAAGTGATCA
AGGGGATAAGCAGTTTAAGGTCGACTTGTTTGGAAACAATGCTAACCACCACCACTGCCACTCACAGTCTCAGCTCC
TCCTCCTCTGCTTCCCAACTCCCATCGCTCTTCCACTCTCTATCACAAAACCCAATCTCCCTCAGATTCTCCTCCAC
ATTAAAGCTAACCAAAACCAGAACCAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTGCTCTGA
GAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCCTCCACAGTGCCGTCGGAGATGAAGGCGTGGGTG
TA


>GPH23 ananassa clone4
CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACGACCTTTACAGTGAGAGTGTGACCAGAG
GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGATAAAA
GACCTGCCAGAATCCACACCACCAAACTCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCAT
CAACATGCAATGATGTGGGTCCATAAGAACCATGAGTATGACATAGAGTCTTCAAGCTTCGATTTCCTTATTTGCTT
CGAAAGAAGCAAGTTCAGAGTCACAAACCAGAATATAGATCTCAAATTTAATGAACATATTCCTAAGAGCCTAA
AGAAATATAAAATCGTAACTGAACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCAAAAACAAACTTCAAGT

















mandshl
nilger
viridi
iinumas
ananas




vesca
mandshl
nilger
viridi
gL-1 1


mandshl
nilger
viridi;


mandshl
nilger
viridi
iinuma
ananas;



vesca
mandshl
nilger
viridi


nandsh
nilger
viridi













9AC 996
9AC 996
GGC 984
CGC 976


ii ii ii


CATGAAGTCAAGGTCAAGCC
CATGAAGTCAAGGTCAAGCC


CGAAGC


TTAAG 1096


AACTTTAAGCGa
\GCTTTAAGCGq


GCTCCAAC(


CCGAA


CCCACC









156
















SeqL-eri-_fl of SO foiids


pFGENES eBL.ESwTprlasBAT wt i- -q




dpc w re daeatt ri p







Figure 3-1. Flowchart of genomic DNA sequence annotation scheme. The software FGENESH
was used with Medicago as the gene model to predict possible gene positions in the
genomic sequence. BLASTP algorithm was utilized as preliminary validation
FGENESH prediction, whereas BLASTX was used to determine coding sequence
orientation and assign tentative gene function. Putative homologs within Rosaceae,
conservation amongst various taxonomical families, as well as sequence repeats and
duplications were detected by different homology searches utilizing BLASTN.
Finally, putative genes that had not been predicted by FGENESH were identified by
searching similarities between large fragments of genomic sequence (containing or
not FGENESH-predicted genes) and Rosaceae EST.







Omin 5min 30min 60min

S 5min incubation +25min incubation +30mir incubatro
8vol 6volI 4vol 2voi
aliquot 2vol



Svoil W W
Add vol chloroform





Figure 2-1. Design of incubation temperatures and durations experiment. The scheme illustrated
above was followed for each of the incubation temperatures of 4, 20, 42, and 65C.
Samples for a specific temperature were ground and homogenized together to
decrease random variation between time points.





































































TCCTTAAGC
TCCTTAAGC
TCCTTAAGC
TCCTTAAGC



vGGAATTCAC
vGGAATTCAC
vGGAATTCAC
\GGAATTCAC









to collect only the largest-fruited plants, Freezier imported only female plants. About 50 years

later, the product of the pollination ofF. chiloensis by F. virginiana was observed in Germany,

Switzerland, Holland, and the Trianon gardens in France (Darrow, 1966).

F. x aananssa's nuclear genomic content can be traced to fifty-three founding clones

(Sjulin and Dale, 1987), whereas as few as seventeen cytoplasm donors are represented in the

cultivated strawberry (Dale and Sjulin, 1990). Wild accessions from the octoploid parents have

been used relatively recently in strawberry breeding programs for introgression of various

characteristics (Hancock, 1999), including day neutrality into California cultivars (Ahmadi et al.,

1990).

Although the identities of the direct ancestor ofF. x aananssa are known, their genome

constitutions and evolution are not. The present research investigated polymorphisms in the

intergenic regions of diploid species, as well as the cultivated octoploid to attempt to trace

ancestry and make inferences about the octoploid genome mode of inheritance.

Materials and Methods

Before the commencement of this study in the year 2004, virtually no Fragaria genomic

sequence was available. Therefore, it was necessary to develop a means to capture useful

sequences for analysis. Two different approaches were adopted: i, inference of gene adjacency

by putative micro-colinearity between F. x aananssa and Arabidopsis thaliana; ii, construction

and annotation of a F. vesca genomic library (discussed in Chapter 3).

Potential micro-colinearity was detected using the approach described in figure 4-3. This

approach was possible because the genome of Arabidopsis has been completely sequenced and

the genes were numbered in such fashion that their locus tags indicate their position on the

chromosomes. The hypothesis was that if two genes were adjacent in Arabidopsis, they would

also be adjacent in Fragaria. Similarity between F. x aananssa ESTs and A. thaliana transcripts















iridi


29G10









columns have been used elsewhere to eliminate polysaccharide contaminants, which is verified
by increase of the ratio A260/230 (Abdulova et al., 2002). The protocol used here was based on
Rogstad's article (Rogstad, 2003), which uses a CTAB extraction buffer and describes the
preparation of the silica binder. CTAB extraction buffer: 2% CTAB, 1.4M NaC1, 100mM Tris-
HC1 pH 8.0, 20mM EDTA pH 8.0, 1% 2-mercaptoethanol. 'Strawberry Festival' leaves were
ground (10mg-T44 and 100mg-T45) and 5 ml of extraction buffer were added. Incubation
was carried out at room temperature for 30 minutes. Equal volume of chloroform:octanol was
added, samples were centrifuged, the upper phase was transferred to a new tube, and 2.5ml of
silica binder were added. The mixture was agitated thoroughly for 5 min, then centrifuged. The
supernatant was discarded, and 4ml of silica wash (25% isopropanol, 25% ethanol, 100mM
NaC1, 10mM Tris-HCl pH 7.4, 2mM EDTA pH 8.0) were added, vortexed to resuspend the
silica. Samples were centrifuged, supernatant discarded, and a second wash took place. The silica
pellet was dried for 2 hours at 37C, and the DNA was eluted by lml of ultra pure water,
vortexed, and incubated at 650C for 5 min. After centrifugation, the upper phase was transferred
to a new tube, RNAse-treated, then DNA was precipitated by isopropanol.
The following protocols (18-22) attempted to extract DNA from nuclei isolated from leaf
tissue. Protocols 23-33 consist of variations of the protocol by Murray and Thompson and
utilized leaves (rather than isolated nuclei) for DNA extraction.

DNA Extraction from Isolated Nuclei

Nuclei were purified according to the procedure described by Folta and Kaufman [Folta,
2000] and nuclei were recovered from the 35/80 interphase of percoll gradients. Nuclei were
incubated with each extraction buffer at 650C for at least 10 minutes. The following buffers were
mixed to 50-150tl of purified nuclei in storage buffer as an attempt to extract DNA:
18. Qiagen DNeasy Plant Mini kit. Different volumes (501l-T46 and 150i1l-T47) of
isolated nuclei were processed according to manufacturer's directions.
19. Fulton's nuclei lysis buffer [Fulton, 1995], supplemented with 0.5% sodium bisulfite:
200mM Tris pH 7.5, 50mM EDTA pH 8.0, 2M NaC1, 2% CTAB. Two tubes, one 50tl nuclei
(T48) and the other containing 75p1l nuclei (T49), were incubated with 200 and 75p1l of nuclei
lysis buffer at 650C for 45min. Phenol:chloroform followed by chloroform extractions took
place, the upper phase transferred to a new tube, and DNA precipitated by isopropanol.
20. Peterson's procedure [Peterson, 1997]: 20% SDS was added to a final concentration of
2% and mixed with 50tl nuclei (T50) or 150tl (T51) by gentle inversion to lyse the nuclei. The
mixture was incubated in water bath at 650C for 10 minutes, cooled to room temperature, then
5M sodium perchlorate was added to reach final concentration of 1M. Sodium perchlorate is
used to dissociate nucleic acid-protein complexes [Wilcockson, 1973]. Following centrifugation,
the upper phase was transferred to a new tube using a large-bore tip. After a phenol
deproteinization step, the aqueous phase was dialyzed twice, the first overnight and the second
for an entire day, both into TE pH 7.0 at 40C. Samples were consecutively treated with 50[tg/ml
RNAse for 1 hour and with 150[tg/ml proteinase K. After extractions with
phenol:chloroform/isoamyl alcohol and chloroform/isoamyl alcohol, DNA was precipitated and
resuspended.
21. Guanidine thiocyanate buffer (4M guanidine thiocyanate, 100mM Tris-HC1, 10mM
EDTA, 0.5M NaC1, 1% sarkosyl, 1% sodium bisulfite) was used (750pl1) to extract DNA from
50p l nuclei (T52). The buffer/nuclei were incubated at room temperature for 10min and were










Predicted o Putative Gene Distr (kb
Number of Genes EST Fosmid between genes)
Fosmid a Insert Size
ab Similari Protein Hit Hits n Similarity-
Protein Hit (bp) ab initio
initio ty O (gb no.) based
38H02 7 6 31,669 4.5 5.3


1 X
transposon protein +
3 cytochrome P450 +
4 cytochrome P450 +
5 integrase +
6 serine/threonine
kinase
7 A exportin
38H05 11 1 32,050 2.9 32.1
1 X
2 X
Not X dbjlAB2
predicted 08565.1
3 retrotransposon
polyprotein
Not XdbjlAB2
predicted 08565.1
4 X
5 X
6 X
7 X

9 X
10 X
11 X
40B22 9 8 36,230 4.0 4.5
1 unknown +
2 cyclin-like F-box
3A X
4 cyclin-like F-box +
5 cyclin-like F-box
6A cyclin-like F-box
7 Arf GTPase
activating
8 heavy metal +
transport/detoxificati
on
9 MuDR family
transposase
40M11 9 5 31,718 3.5 6.3
1 X
2 cyclin I-like F box +
3 X
4 X














,andshurica AGTTCTCTCACAATAGTAAAGAAACGATCGTTGACAATCAAAAGGCATCGAAAGCTAGTA 4:
ilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 4:
iridis AGTTCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTA 4
inumae AGTCCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTA 4
nanassa AGTTCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTA 4




esca AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTACATGCATAAAACCCTC 4
andshurica AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTATATACATAAAACCCTC 4"
ilgerrensis NNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 4
iridis AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTACATGCATAAAACCCTC 4U
inumae AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTATATACATAAAACCCTC 5,
nanassa AAGAAACGATCTTTCAGATGGGAAATGCCCAAATTTGATTACTATATACATAAAACTCCC 5:




esca AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAA-TTCAA 5,
1andshurica AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAA TTCAA 5:
ilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNN 5
iridis AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAAAAAATTCA 5
inumae AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATTAAAAAAAATCAA 6(
nanassa AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACAG-AAAAAATTCAA 5'




esca GAAAA-AAAGAGAGAGA--AAATTACAGATTTAAAGCGACGAACAA-TGAAAAGGAATGA 6(
1andshurica GAAAA-AAAGAGAGAGA--AAATTACAGATCTAAAGCGACGAACAG-TGAGAAGGAATGA 51
ilgerrensis NNAAA-AAAGAGAGAGAGA--- TTACAGATCTAN-GCGACGAACAA-TGAGAAGGAATGA 51
iridis AGAAATAAAGAGAGAGA--AAATTACAGATCTAAAGTGACGAACAA-TGAGAAGGAATGA 6(
inumae GAAAAAAAGAAGAGAGA--AAATTACAGATCTAAAGCGACGAACAAATGAGAAGGAATGA 6;
nanassa GAAAAAAAAAAGAGAGAGAAAATTACAGATCTAAAGCGACGAACAA-TGAGAAGGAATGA 6:


esca GAGGCAAAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGGGAGAGA 6
andshurica GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGGGAGAGA 6
ilgerrensis GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGT--------GA 6
iridis GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGG--GAGA 61
inumae GAGACAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGT--------GGAGAGA 7
nanassa GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGA----------GTGAGGGAGAGA 6/


esca GAGAGAGAGATCGACGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 7
andshurica GAGAGAGAGATCGACGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 7
ilgerrensis GAGAGAGAGATCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 7(
iridis GAGAGAGAGATCGAAGACGAAGCAGAGCGAAAGACGAGTGTGGTGTTTGTGAGTTGAG 7;
inumae GAGAGAGAGATCGAAGACGAGGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 7'
nanassa GAGAGAGAGATCGAAGACGAAGCTGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 7,


esca GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 7/
,andshurica GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 7'
ilgerrensis GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 71
iridis GCGAA-GAATTGNACCNNNATANAGGAGTGNGATTGACNAGTTATCTCNGCNGNTTGATT 7
inumae GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 8:
nanassa GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 8(


esca TATGGACCGCGTCTATTGAGCCCTTGTGGGG-CCATTACAGCTCCTTCCGCTGTTCCAGT 8Q
,andshurica TATGGACCGCGTCTATTGCGCCCTTGTGGGG-CCATTACAGCTCCTTCCGCTGTTCCAGT 8;
ilgerrensis TATGGACCGCGTCCATTGCGCCCTTGTGGGG-CCATTACAGCTCCTTCCGCTGTTCCAGT 8;
iridis TATGGACCGCGTCCATTGTGCCNTTGTGGGG-CCATNACNGCTCCTNCCNCTGTNCCNGC 8'
inumae TATGGACCGCGTCCATTGCGCCCTTGTGGGGGCCATTACAGCTCCTTCCGCTGTTCCAGT 8!
nanassa TATGGACCGCGTCCGTTGCGCCCTTGTGGGG-CCATTGCAGCTCCTTCCGCTGTTCCAGT 81






200














GPH10_ananassa clone GGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGAGATTGAT 2328
GPH10_ananassa clone20 GGTCGCAAACTGGGCCTTCACCATCTATGCATTCTGCTGAGATTGAT 2472
GPH10_ananassa clonel8 GGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGAGATTGAT 2413
GPH10_ananassa clonel9 GGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGAGATTGAT 2411


GPH10ananassa clone GGAAAAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCAC 2378
GPH10ananassa clone20 GGAAAGAAAAAAGTAGCGAAAGAAGTTCACATTCACTCAAAATCTCAC 2522
GPH10ananassa clonel8 GGAAAAAAAAAAGTAGCAAAAAAGTTCACATTCACTCAAAGATCTCAC 24 63
GPH10ananassaclonel9 GGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCAC 24 61


GPH10ananassa clone CTGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGG 2428
GPH10ananassa clone20 CCGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGG 2572
GPH10ananassa clonel8 CTGGAGTTCTTTCTGTAGGCAGCATTCGATTTTCAGACTTACATTTGG 2513
GPH10_ananassa clonel9 CTGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGG 2511


GPH10ananassa clone AAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAGTTGAAATTT 2478
GPH10ananassa clone20 AAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAGTTGAAATTT 2622
GPH10ananassa clonel8 AAGAGGCTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAAATTT 2563
GPH10_ananassa clonel9 AAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAAATTT 2561


GPH10ananassa clone CTAAAATGCTGGATGAAACAGATTAAAAAACTGAATTATCCATACGGA 2528
GPH10 ananassa clone20 CTAAAATGCTGGATGAAACAGATTAAAAACTGAAGTATCCAATAACGGA 2672
GPH10ananassa clonel8 CTAAAATGCTGGATGAAACAGATTAAAAAACTGAATTATCCATACGGA 2613
GPH10ananassa colonel 9 CTAAAATGCTGGATGAAACAATTAAAAACTGAAGTATCCAATAACGGA 2611
**************************************************


GPH10ananassa clone GGAATCTAAGGTGCACCAGGAAAAACAAGGAGATGAGCAATAGGTTGG 2578
GPH10ananassa clone20 GGAGTCTAAGGTGCACCAGGAAAAACAAAAGGAGATGAGCAATAGGTTGG 2722
GPH10ananassa clonel8 GGAGTCTAAGGTGCACCAGGAAAAACAAAA GATGAGCTAGGTTGG 2663
GPH10ananassa clon9 GGAGTCTAAGGTGCACCAGGAAACAAAAGGATACATAGGTT 2661


GPH10ananassa clone ATTTGTTGCACCAAGAGAGCGAA CAGCCTGTCG TCTCATCTGGTTCAGCT 2628
GPH10 ananassa clone20 ATTTGTTGCACCAAGAGAGCGAACAGCCATGTCG TCTCATCTGGTTCAGCT 2772
GPH10ananassa clonel8 ATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCATCATCTGGTTCAGCT 2713
GPH10 ananassa clone9 ATTTGTTGCACCAAGAGAGCGAACCCAATGTCATCATCTGGTTCACT 2711


GPH10ananassa clone GGAGAAATTTCTTTCCCTGTGGCCTTTGGAGTACAGGATGAAGCTGCTCA 2678
GPH10ananassa clone20 GGAGAAATTTCTTTCCCTGTCGCCTTTGGAGTACAGGATGAAGCTGCTCA 2822
GPH10ananassa clonel8 GGAGAAATTTCTTTCTCTGCGGCCTTTGGAGTACAGGATGAAGCTGCTCA 2763
GPH10ananassaclonel9 GGAGAAATTTCTTTCTCTGCGGCCTTTGGAGTACAGGATAAGCTGCTCA 2761


GPH10ananassa clone GGAACATAGATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATA 2728
GPH10ananassa clone20 GGAACATAGATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATA 2872
GPH10ananassa clonel8 GGAACATAGATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATA 2813
GPH10ananassaclonel9 GGAACATAGATTACAAACCTCAAAATTTTTTCTTAATTTCTCTATA 2811


GPH10ananassa clone AGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCGCA 2778
GPH10ananassa clone20 AGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCACA 2922
GPH10ananassa clonel8 AGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCGCA 2863
PH10ananassa clon9 AGATCCAACAAGGGCTAGTCTAAGTAGTAGACTTGGGGGCATTCGCA 2861


PH10ananassa clone CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAACATAGCA 2828
PH10ananassa clone20 CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCAC 2972
PH10ananassa clonel8 CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAGCATAGCTC 2913
GPH10ananassa clonel9 CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAGCATAGCTC 2911






171














11D02 viridis TGATGCCATAGCATAATTCATTGCTGTATAAGGAGTCTTCCCCTAACTTGGTTTCCTTTCAGTGT 414
11D02_nubicola TGATAACTTTCAA TAGTATAAGAAGGAGATGAGGACAGTCTT 417
11D02_vesca TGATAACTTTTCAA TAGTATAAGAAGGAGATGAGGACAGTCTT 417
11D02_iinumae TGATAACTTTTCAATACTATAAGAAGGAGATGAGGACAGTTT 417


11D02_viridis TGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCCTAACTTGGTTTCCTTT- GTGT 474
11D02 nubicola TGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTGGTTTCCTTTCAGCCT 477
11D02 vesca TGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTGGTTTCCTTTCAGTCT 477
11D02 iinumae TGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCTTAACTTGGTTTCC--------- 468


11D02 viridis CTTCGACCTTCTTCTAAAACGACGGAGTCGGTGAAACTGTGCAAGTCTTCTTGTGA---- 530
11D02 nubicola CTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCA 537
11D02 vesca CTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCA 537
11D02 iinumae --------------------GACGGAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCA 508


11D02 viridis ATTTTCTTTTCTAGGTGATTTTTTTTTTCTTTTAATTAATTTGGTTTTATTTTTCCAA 590
11D02 nubicola ATTTTCTTTTCTAGGTGATTTTTTTT--CTTTTATAATTAATTTGGTTTTATTTTTCCAA 595
11D02 vesca ATTTTCTTTTCTAGGTGATTTTTTTT--CTTTTAATTAATTTGGTTTTATTTTTCCAA 595
11D02 iinumae ATTTTCTTTTCTAGGTGTTTTTTTTT--CTTTTATAATTAATTTGGTTTTATTTTTCCAA 566


11D02 viridis ATAATACCTGAAAGACTTTTTTTTTTTTTTTTGATAGAAATACCTAAAAGACTTCATAA 650
1D02 nubicola ATAATACCTGAAAGACTTTTTTTTC-------GATAG ---------- -- 625
11D02 vesca ATAATACCTGAAAGACTTTTTTTTT-------CGATAGGA-------------------- 628
11D02 iinumae ATAATACCTGAAAAGCTTTTTTTTTT----- CGATAGAAATACCTGTTAAGACT----- 616


11D02 viridis AAGCTGTTAAGGCTTCATTTAGGATTGCAGTAATTTTTTTTGGACAGTATTACGGGACAC 710
1D02 nubicola ----------------------GATTGCAGTAATTTTTTTTGGACAGTATTACGGGACAC 663
11D02 vesca ------------------------TTGCAGTAATTTTTTTTGACAGTATTACGGGACAC 664
11D02 iinumae -------TAAGACTTCATTTAGTATTGCAGTAATTTTTTT-GGACAGTATTACGGGACAC 668


11D02 viridis TGTGACAGCTT--GAGTTTGAATCTTAGGTGGGATGATTTAAGTATCTTAGTTGAATGGA 768
11D02 nubicola TGTGACAGCTTTAGAGTTTGAATCTTAGGTTGGATGATTTAAGTATCTTAGTTGAATGGA 723
11D02 vesca TGTGACAGCTTTAGAGTTTGAATCTTAGGTTGGATGATTTAAGTATCTTAGTTGAATGGA 724
11D02 iinumae TG--ACAGCTTTAGAGTTTGAATCTTAGGTTGGATGATTTAAGTATCTTAGTTGAACGGA 726


11D02 viridis TGTTATGACATATTGGTGATTAGTATTAGAGTTATGAGA------ AAATAAAATGAAAAT 822
11D02 nubicola TGTTATGACATATTGGTCATTAGTATTAGAGTTATGAGAAAGAGAAAATAAAATGAAAAT 783
11D02 vesca TGTTATGACATATTGGTGATTAGTATTAGAGTAATAGAAAGAGAAAATAAAATGAAAT 784
11D02 iinumae TGTTATGACATATTGGTACTTAGTATTAGAGTTATGAGAA --- -AAGAAAAAATGAAAAT 782


11D02 viridis ACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAAAGCAATAGATTGACAA-G 881
11D02 nubicola ACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAATGCAATAGATTGACAAAG 843
11D02 vesca ACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAATGCAATAGATTGACAAAG 844
11D02 iinumae ACAGTACTGGCAATAAACACAATTCGGAGGAGCAATCAACAATGCAATAGATTGGCAA-G 841


11D02 viridis AAATGAAGACCTAAAAAAAACCATTGCATTAATGCAATAGTGTTGATTTTCCAATCTCTC 941
1D02nubicola AAATGAAGACCTAAAAAAA-CCATTGCATTAATGCAATAGTGTTGATATTCCAATCTCTC 902
D02vesca AAAT GACCAAAAAAA-CCATTGCATAATGCAATAGTGTTATATTCCTCTCTC 903
11D02 iinumae AAATGAAGACCTAAAAAAA-CCATTGCATTAATGCAATAGTGTCGATTTTCCAATCTCTC 900


11D02 viridis CTGAATAGTATTACAACTCTCCTGGACAAGTCATAACTGTGGGGGGTAATGGTGTAAGCA 1001
11D02 nubicola CTGAATAGTATTACAACTCTCCTGGACAAGTCGTAACTGTGGGGGGTAATGGTGTAAACA 962
D02 vesca CTGAATAGTATTACAACTCTCCTGACAAGTCATAACTGTGGGGGGTAATGGTGTAAACA 963
11D02 iinumae CTGAATAGTATTACAACTCTCCTGGACAAGTCATACCTGTGGGGGGTAATGGTGTAAACA 960






179









ACKNOWLEDGMENTS

I thank my parents Vadir and Marlene, and my brothers Eduardo and Ricardo, for their

teachings, advice, support, and, above all, for their unconditional love. Though not content with

my departure from Brazil, my family always supported my decisions. I appreciate their

confidence in my choices and me, for it reaffirmed my personal mission in moments of doubt.

I am grateful to my professor Dr. Kevin M. Folta, who accepted me as his student in an

altruistic gesture, and who has been a lato sensu adviser since. I thank the members of my

committee for the enjoyable discussions about my project and about science in general: drs. A.

Mark Settles, Natalia A. R. Peres, and Craig K. Chandler. I also wish to thank my laboratory

colleagues and friends drs. Philip J. Stewart and Amit Dhingra, Thelma F. Madzima, Stefanie A.

Maruhnich, Jeremy Ramdial, Dawn Bies, and Maureen Clancy, as well as project collaborators

drs. Thomas M. Davis and Daniel J. Sargent, for DNA sequences and plant material from the

genetic linkage mapping population.

Many people made special the almost-9 years I spent in Gainesville, while I pursued part

of my undergraduate training and two advanced degrees. I convey my gratitude to all those who

facilitated not only my adaptation to a new country and language, but also the discovery of who I

am and of matters I learned to be truly meaningful. I recognize Welch McNair Bostick III

("McNair"), whose short life was vastly fruitful. McNair caused positive impact into the lives of

whomever surrounded him: his wife and my friend Carmen Valero, his neighbors (including

myself), and his colleagues. I thank him for having shown to me the importance of treasuring the

time shared with loved ones, expressing honest opinions and making a difference in society.

I express my appreciation for the time and assistance granted to me by professors and

technicians with whom I worked since my arrival to the University of Florida: Richard D.















GPH5 viridis ATTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTGTAGTTGCACCGGCT 2744
GPH5 iinumae TTTTGTTTTTGTAAAAGTGGATCTCTTTATAACATTGGGTTTACTATAGTTGCACCGGCT 2734
GPH5 nilgerrensis TTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTATAGTTGCACCGGCT 2761
GPH5 mandshurica TTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTGCTATAGTTGCACCGGCT 2759
GPH5 nubicola TTTTGTTTTTGTAAAAATGG ----TTTTATAACATTGGGGTTACTATAGTTGCACCGGCT 2766
GPH5 vesca TTTTGTTTTTGTAAAAATGGATCTCTTCATAACATTGGGGTTACTATAGTTGCACCGGCT 2794




GPH5 ananassa clone2 GCGGGAAGGTGTGTGCAACGGCA 2778
GPH5 ananassa clone7 GCGGGAAGGTGTGTGCAACGGCA 2776
GPH5 viridis GCGGAAGGTGTGTGCAACGGCA 2767
GPH5 iinumae GCGGGAAGGTGTGTGCAACGGCA 2757
GPH5 nilgerrensis GAGGGAAGGTGTGTGCAACGGCA 2784
GPH5 mandshurica GCGGGAAGGTGTGTGCAACGGCA 2782
GPH5 nubicola GCGGGAAGGTGTGTGCAACGGCA 2789
GPH5 vesca GCGGGAAGGTGTGTGCAACGGCA 2817






GPH23: SNPs other than introduced by DNA polymerase are true. After preliminary


sequence alignment, the putative SNPs were verified by observation of unambiguous peaks in


the chromatograms.


GPH23 iinumae clone2 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACG 50
GPH23 iinumae clone CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACG 50
GPH23mandshurica clone3 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACG 50
GPH23 ananassa clone CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACG 50
GPH23 ananassa clone CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACG 50


GPH23 iinumae clone2 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100
GPH23 iinumae clone5 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100
GPH23mandshurica clone3 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100
GPH23 ananassa clone4 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100
GPH23 ananassa clone3 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100
**************************************************

GPH23 iinumae clone CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGAT 150
GPH23 iinumae clone CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGAT 150
GPH23_mandshurica clone3 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGTGTCTGCCATTTGAT 150
GPH23 ananassa clone4 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGAT 150
GPH23 ananassa clone3 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGAT 150


GPH23 iinumaeclone2 AAAAGACCTGCCAGAATCCACGCCACCAAAC-TCTTTAGCACAATCCAA 199
GPH23 iinumae clone5 AAAAGACCTGCCAGAATCCACGCCACCAAAC-TCTTTAGCACTAATCCAA 199
GPH23mandshurica clone3 AAAAGACCTGCCAGAATCCACACCACCAAAC-TCTTTAGCACCAATCCAA 199
GPH23 ananassa clone4 AAAAGACCTGCCAGAATCCACACCACCAAAC-TCTTTAGCACTAATCCAA 199
GPH23 ananassa clone3 AAAAGACCTGCCAGAATCCACGCCACCAAACATCTTTAGCACTAATCCAA 200


GPH23 iinumae clone TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 249
GPH23 iinumae clone TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 249
GPH23mandshurica clone3 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 249
GPH23 ananassa clone4 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATGATGTGG 249
GPH23 ananassa clone3 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 250





161

















SIPula i1 e gene
= Putative intergenic region


1kb


Figure 5-1. Fosmid 40M11 with primers designed on exons of FGENESH-predicted genic
regions.



Table 5-1. PCR primer pairs and amplification conditions used in this study
Putative Gene Function or Extension
PrimerEST gb number Sequence 5 to 3 Ta.eam.g (C) Time
Control F
CoFvFntrolnF Leafy CACTGCCAAGGAGCGTGGTG
FvLFYintron2F
control R variable variable
CoLnrol 3 Leafy TCAGTAGGGCAGCTGATG
FvLeafy3'
01LO2Fb EST AY573376 GAACCGTTCAAGTTCATAATTGG 54-65 1'30"-
01LO2Rb unknown protein AAGGGAGGACGTTCAATGT G 2'30"
01LO2Rc unknown protein ACGGAGATCGGGGACTTGT 54-58 2'30"
10B08F Leafy protein GGGCCAACTACATCAACAAGC
58-63 3'-4'
10B08R ACC synthase TGTTCTGTTGGGTGGACATGA
10BO8Fb ACC synthase TGCCATCGTTTCCATCAGTA
52 1'
10BO8Rb ribosomal protein CGCGAAGATCAT GAAGAACA
11D02F EST BQ105541 GAGCTGCTGTGTGAACCAAA
56-60 2'30"
11D02R heat shock binding protein GTTCAACTCCAGATGAAGTGAGG
17022F Oligopeptidase AAAATGGGTTGCACGAGTTC
17022Rb Putative protein GGGTTTCCTCACAAACTTCG
17022Fb Oligopeptidase GGTACCTCCAATGCAAGGAA
17022R Putative protein TTCATCAGAGAAGGCGGACT
22H18F EST DY646954 ACCAATGCTTGGACACACAC
52-65 2'30"
22H18R unknown protein GATGAAATTCCATGCTTGTGAC
22H18Rb unknown protein GGACTCCATGTAACACGGCTA 56-65 2'30"
27F10F kinase CCTGCAGGGTTTTTCATCAT
27F10R hypothetical protein T GGAAATGTATTCTGGTTCTCC 59
29G10F phenylacetaldehyde TGGCCTTGTTTCCTAAACTCTT
synthase 59 1
29G10R unknown protein AGAAGAAGGCAGCACCCAAT
30124F transferase TTGAGAGAGGTCTCCAAGCTC
30124R chromating remodeling CGGAAGATGGCAAGCTATTG 54, 59 4'
factor
32A10F i -12 CGGAGAGAACGATGGAGTTG 1
32A10Fb C CAAATGAATCAAGCTCAAGTG 52-62
32A10R pathogenesis-related protein ATTGTCGACCAGTGCAGCAA


SMC2 (Structural
maintenance of
chromosomes)


Exostosin


GAGTTGAAAAACGGGTCGAA
CCTTCCAAGGTCACCTCCTT
TTAGCCCGGTTATGGAGTTG
GAAGGTTCAAGGAGCATGGA
AGGAAAATGCGGGAGAAAGT
GAACGATTTCCGAGGTGTGT


RbRd


32L02F
32L03Fb
32L02Fc
32L02R
32L02Rb
32L02Rc


53-61


I LI I I


P4C 1















ubicola GT-----G-----TAGGATAATAACAAAGAAACTCGTTATCTGAAAGACA 90
landshurica GT-----G-----TAGGATAATAACAAAGAAACTCGTTATCTGAAAGACA 90
esca GT-----G-----TAGGATAATAACAAAGAAACTCGTTATCTGAGACA 90
1iridis GT ----- G -----TAGGATAATAACAAAGAAACTCGTTATCTGAAAGACA 90
ilgerrensis GT-----GGAGTGTAGGATAATAAC --- --AAACTCGTTATCTGAAAGACA 91
nanassa clonel8 GT-----GGAGTGTAGGATAATAAC---AAACTCGTTATCTAAAAGACA 91
nanassa clone20 GT-----GGAGTGTAGGATAATAAC---AAACTCGTAT-TAAAAGACA 90
nanassa clonel9 GT-----GGAGTGTAGGATAATAAC-----AAACTCGTTATCTAAAAGACA 91
nanassa clone GT-----GGAGTGTAGGATAATAAC----AAACTCGTTATCTAAAAGGCA 91
inumae CTTTGAAGTAGTGTAGGATAATAAC--------AAACTCGTTATCTAAAAGACA 96




ubicola GGTTTAATATCAGC---------------------CGTTGGATCATA---TT 118
landshurica GGTTTAATATCAGC---------------------CGTTGGATCATA---TT 118
esca AGTTTAATATCAGC---------------------CGTTGGATCATA---TT 118
iridis AGTTTAATACCAGC-------------------CGTTGGATCATA---TT 118
1ilgerrensis GGTTTAATATCAGC-------------------CGTTGGATTATA---TT 119
lnanassa clonel8 GGTTTAATATCGGC-------------------CGTTAGATCACA---TT 119
nanassa clone20 GGATTAATGTCAGTGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTT 140
lnanassa clonel9 GGTTTAATATCAGC-------------------CGTTAGATCATA---TT 119
lnanassa clone2 GGTTTAATATCAGC-------------------CGTTAGATCATA---TT 119
inumae GGTTTAATATCAGC-------------------CGTTAGATCCTA---TT 124




ubicola ACGGCCCTG ------ATCGCTCGACATA------------------- 140
landshurica ACGGCCCTG ------ATCGCTCGACATA------------------- 140
esca ACGGCCCTG ------ATCGCTCGACATA------------------- 140
iridis ACTGCCCTG ------ATCGCTCGACATA--------------------- 140
ilgerrensis CCGGCCCTG ------- ATCTCTCGACATA--------------------- 141
nanassa clonel8 ACGGCCCTG ------ATCACTCGACATATGTTGA-TATACGCCTAACT- 160
nanassa clone20 AAGGTCATAGGTTCAAACCTCACGACATATGTAGGGTGTATGAATTATTA 190
nanassa clone ACGGCCCTG------ATCACTCGACATATGTTGA-TATACGCCTAACT- 160
nanassa clone2 ACGGCCCTG-------ATCACT--------- ---------- 13z
inumae ---------------------------------------------------




ubicola -------------------------------------------------A 141
tandshurica -------------------------------------------------A 141
esca ------------------------------------------------A 141
iridis -------------------------------------------------A 141
ilgerrensis -------------------------------------------------T 142
nanassa clonel8 --------CAAATTCGATAT ------------ATATT------------ 177
nanassa clone20 ATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACGGCCTGATCA 240
nanassa cl 1onel9 --------CAAATTCGATAT ------------ATATT------------ 177
nanassa clone2- - - - - -
inumae --------------------------------------------------




ubicola TTCGATATATATATATA------TTATTTTTTTCTAAA----------AA 175
tandshurica TTCGATATATATATA--------TTATTTTTTTCTAAA--- --AA 173
esca TTCGATATATATATATATATATTTTTTTTTTTTCTAAA--------AA 181
iridis TTCGATATATATATATATA ---TTATTTTTTTCTAAA----------AA 177
ilgerrensis GTTGATATACGC---------------------CTGAC----------TC 161
lnanassa clonel8 TTCGATATACA------ TTTTTTTTTTAAGTAACTAAATGACTATTCGAT 221
lnanassa clone20 CTCGACATATGTTGATATAC-------GCCCAACTCAA -----ATTCGAT 278
nanassa clonel9 TTCGATATACA------TTTTTTTTTTAAGTAACTAAATGACTATTCGA 221
nanassa clone --CGACATATGTTGATATAC-------GCCCAACTCAA-----ATTCGAT 170
inumae -







175









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to be 157 Mb and thus -25% larger than the Arabidopsis Genome Initiative estimate of
-125 Mb. Annals of Botany 91: 547-557

Bennetzen JL, Kellogg EA (1997) Do plants have a one-way ticket to genomic obesity? Plant
Cell 9: 1509-1514

Bennetzen JL, Ma J, Devos KM (2005) Mechanisms of Recent Genome Size Variation in
Flowering Plants. Annals of Botany 95: 127-132

Bennetzen JL, SanMiguel P, Chen M, Tikhonov A, Francki M, Avramova Z (1998) Grass
genomes. Proc Natl Acad Sci U S A 95: 1975-1978

Besemer J, Borodovsky M (1999) Heuristic approach to deriving models for gene finding.
Nucleic Acids Res. 27: 3911-3920

Bies DH, Folta KM (2004) An effective substitute for triisopropylnaphthalenesulfonic acid in
the preparation of plant RNA. Anal Biochem. 333: 201-203

Birney E, Clamp M, Durbin R (2004) GeneWise and Genomewise. Genome Research 14: 988-
995

Bringhurst RS (1990) Cytogenetics and Evolution in American Fragaria. Hortscience 25: 879-
881

Bringhurst RS, Arulsekar S, Hancock JF, Voth V (1981) Electrophoretic characterization of
strawberry cultivars. Journal of the American Society for Horticultural Science 106: 684-
687

Bringhurst RS, Gill T (1970) Origin of Fragaria polyploids. II. Unreduced and doubled-
unreduced gametes. American Journal of Botany 57: 969-976

Bullock WO, Fernandez JM, Short JM (1987) XL1-Blue: A high efficiency plasmid
transforming recA Escherichia coli strain with beta-galactosidase selection.
Biotechniques 5: 376-379









M. Davis, and encompassed a 770-nucleotide region. Both PCR reactions were carried out for 35

cycles, with 550C as annealing temperature, and Imin as extension at 720C.

The original CTAB protocol designed by Murray and Thompson (Murray and Thompson,

1980) is extremely laborious, requiring a long centrifugation period in a cesium chloride (CsC1)

gradient. Since the aim of this project was to develop a rapid, practical method to extract DNA,

the CsCl step was omitted from all DNA extraction attempts. Further modifications of the

protocol were tested systematically to pyramid the beneficial aspects of each preparation into a

unified and effective means to generate high-quality DNA for downstream analysis as described

bellow:

* CTAB was tested at 1, 2, 6, and 20%

* Inclusion of one or combinations of the following reagents to prevent DNA oxidation:
0.01% -1% sodium (bi)sulfite, 5mM ascorbic acid, 1-4% PVP

* EDTA concentration from 10mM (as proposed by Murray and Thompson) to 200mM

* Tris concentration ranged from 50mM (as in original protocol) to 200mM. The pH was
adjusted to 8.0 by addition of HC1. In cases where boric acid was used to adjust the pH, the
Tris-borate solution was brought to pH 7.6 because at that pH, boric acid forms complexes
with polyphenols

* The original protocol removes proteins by treating the solution with 24:1
chloroform:octanol. Alternative deproteination methods tested were: 25:24:1
phenol:chloroform:isoamyl alcohol, 1M sodium perchlorate, and 150tg/ml proteinase K

* DNA was recovered by either adsorption to silica, or precipitation by ethanol, isopropanol,
2-butoxyethanol, or 5M potassium acetate. In Murray and Thompson's original protocol,
DNA is precipitated by decreasing salt concentration

* Attempts to remove water-soluble contaminants by adsorption to silica column (QIAGEN
DNeasy kit) and by dialyses of DNA solution into TE pH 7.0 at 40C

* Instead of adding buffer subsequent to grinding the plant tissue, an additional tissue/buffer
homogenization step was performed. An aliquot of the final buffer was used to either
produce a tissue/buffer paste in the mortar and pestle or Polytron homogenizer

* In place of the standard incubation in buffer at 50-600C for 20-30 minutes, incubation was
carried out at 4, 20, 42, and 65C for 0, 5, 30, and 60 minutes. In order to eliminate









LIST OF FIGURES


Figure page

2-1 Design of incubation temperatures and durations experiment ........................................33

2-2 Effect of incubation temperature and time on DNA yields.. ..........................................37

2-3 Effect of tissue-to-buffer ratios on DNA yields .......................................................... 37

2-4 Relationships between DNA yield, tissue-to-buffer ratios, and sample amenability to
am plification by PCR .................... .................... .... .. ........ .. ........ .... 39

2-5 DNA contamination by carbohydrate (estimated by the ratio between absorbance at
260nm and 230nm) and its influence on PCR outcome.. ...............................................40

2-6 Effect of interactions between maceration method and incubation temperature in the
absorbance at 220-340nm ............................................ .......................................... 4 1

2-7 The effect of Polytron homogenization on nucleic acid recovery...................................41

3-1 Flowchart of genomic DNA sequence annotation scheme.........................................52

3-2 Diagram of two fosmid inserts of variable length, with their putative proteins and
Sim ple Sequence R epeats (SSR s).............................................. ............................ 53

3-3 EST classes identified by homology searches between large genomic F. vesca
sequence and R osaceae E STs.. .............................. ... .......................................54

4-1 A n idealized G PH locu s......... ...... ........... ................. ............................ ..................... 70

4-2 Fragaria species and their geographical locations.................................... .................70

4-3 GPH design upon comparison between strawberry ESTs and Arabidopsis database. ......71

4-4 Subset of the alignment of GPH5 octoploid and diploid clones .............. ...............76

4-5 Diagrammatic representation of alignment of full GPH23 clones..............................76

4-6 EcoRI Restriction patterns observed for GPH10 clones from the octoploid
'Strawberry Festival', indicating four different allele classes .......................................77

4-7 GPH10 clones, 4 alleles from the octoploid Fragaria x aananssa ................................77

4-8 Subset of GPH72E18 alignment displaying SSR polymorphisms. ................................78

4-9 Cladograms ofF. x aananassa and diploid alleles for six independent GPH loci .............79








































Idshurica TTAACCGATAGCATCATTCGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGAT 143
Lgerrensis TTAACCGATAGCATCATT-----------CACTTATCTTACTTCCCATTACGATGATGAT 136,
lumae TTAACCGATAGCATCATTCGATTCTATTTCACTTATCTTACTTCCCATTATGATGATGAT 133
inassa TTAACCGATAGCATCATTCAATTCTATTTCACTTATCTTACTTCCCATTATGATGATGAT 136
ridis TTAACCGATAGCATCATTCGATTCTATTTCACTAATCTTAGTTCCCATTATGATGATGAT 139.
Dicola TTAACCGATAGCATCATTCGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGAT 137




sca ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGAT 149"
Idshurica ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGAT 149"
Lgerrensis ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCTGGATCCTGAGGAT 142.









(random) process in which the conditional probability distribution of future states of the process

depends on previous states. While in the Markov model one or more states can be directly

observed, in the hidden Markov model, they cannot. HMMs are popular because they are

relatively simple, and efficient methods that exist for training and testing HMMs, these being the

Baum-Welch and the Viterbi algorithms, respectively (Mark D. Skowronski, personal

communication). For a review on HMMs, refer to Rabiner, 1989 (Rabiner, 1989). Examples of

ab initio HMM gene prediction software are GenScan (Burge and Karlin, 1997), GeneMark

(Besemer and Borodovsky, 1999), and FGENESH (Salamov and Solovyev, 2000). When used to

annotate the rice genome, FGENESH was more sensitive and more specific than GeneMark and

GenScan (Yu et al., 2002).

Plant genomic annotation mechanisms gained favor in the year 2000, shortly after the

completion of sequencing ofArabidopsis thaliana, a widely used genetic, developmental and

physiological model for plants (The Arabidopsis Genome Initiative, 2000), followed by rice in

2002 (Yu et al., 2002). The initial annotation of the Arabidopsis genome was submitted by

numerous centers, each of them utilizing their own annotation method and terminology. The

genome has been re-annotated and classified using Gene Ontology terms as a solution to the

cumbersome handling of the information that had resulted from non-centralized annotation (Haas

et al., 2005).

Since the completion of the first draft of the rice genome, sequencing of many plants has

progressed: high-quality finishing of rice and deep draft coverage of maize, alfalfa (Medicago

truncatula, the model legume), tomato (Lycopersicon esculentum) (National Plant Genomics

Initiative, 2002), and black cottonwood (Populus trichocarpa) (Tuskan and Difazio S, 2006).

Despite the high commercial value of strawberries, there is extensive more nucleotide sequence












APPENDIX D
SEQUENCES GENERATED DURING CHARACTERIZATION OF "GENENPAIR
HAPLOTYPES"


>GPH4 ananassa 13
ACGAGGGCTTGGAAGAAAGGAGGTCAATTTGGTTAAGGTGTGTTGGAGTCGCCAAGTTGAGGGTGATGCATTCTTGG
GAGTTAGAGTCGGATATGAGGGCTAAGTACCCCAAGTTGTTTCTTTTTGAGTTAGTATCTTAAAATTTCGGGGACGA
AATTTCTTTAAAGAGGGTAGAGTGTAATACCCCAGAAATTTGATATTAGTTTCTAATTTTATTTAGGAATTTTTGAG
TTAGAAGTTAGCGTGTTTTGAAGTTTGAAGGAAGAACGGAAGGGTTCGGATGCATAAATTGCTGAACCGGTTTTATG
GTTCTGAAAGGTCAAGAGTTGACTTTCTAATCCGTTGGGTTTCTCGAGAAACTTCCTTCACGGAAGTTGTAGAGCAC
GACGATACGAGTTCGTAGACACGTGGCACGCGTAAAACGGACTTCGTATGAGAAAGTTATGGTCAGCAGAAGTTGTG
GCTTTTCGGGAATATTTAGGTTAAATAGGAAATTTTCGTTTTGGGTTCTATTATTTTTCAGAAATTCCTTTCTTCCC
CTTCTTCTCTCTCCCCGACCCCGAGAGAACCCAAGCTTCCCAGCCGACCCGACCCGGACCCGGTTGACCCCACCCGG
ATTTTCCGGCCATCTCCGGCCGACCCAGGCAACGGCACTGGTCGGGTTCTCTTCCTCTCCTCCGTCAGAGCTGACCT
GTGGCGGTGATGTGCGCCGTTTCGGTCCCGAGGTGGTGACCCGAAGCTCGGAAGTTCGGGTTGGGGT CGGA
TTCCTTCATCCGGCGGCGGCGACAAGGTAGGAGACCGATCGGGATAGAAACCCCTTGGCGTCTTGGTCCGATTGCTG
GTGGCCTTGAAGTGCGACACACGGCGGAGAGTGGCAGTGGTGGATCCTAATCTTTCCGGCGAGTTTCCGGCAAATTC
CCGGCCGATTTGGTTTCGACCTCAGGTATGGAAGTTGCTCTCCTTGCTCTGAGCTATATTTTTGGTGTTGGAAGTTT
GTCCGTTTTCGAAGGTTAGTGGGGTGGCGCGTGGGACCCACGTGCAGTCGCTAAGGGCAGCGCGTAGCGGCGCGTCC
GTAGGTGGTTGTGGTCTTTCTGTTGTTGGGC


> GPH4 ananassa 15
ACGAGGGCTTGGAAGAAAGGAGCCTTTGTCTGTGAATATGTTGGGGAGATAATGACCTACAAGTACTTGTATAATCG
GGGAAGACACACATACTCAATCACTGGATGCCGGTTGGGGCATTGCTTAGCCTTGTGTGTTTTTGGTTAAGCAGATC
ATATTTCCCAGAATTAGATCTGCATAATAATCCATTTTCACGCCAGTCATTTGGTGCCTGTGGTTTAGAACTTAGAT
TTCAGAAGTTCTATCAAGTTTGTCACTTCCTCACCTCTTGTGATGAGAGAAATTTTCAATTCGTTGATGTTGACAAA
GATCATCTGACTATAAATTTGCCCATGTAATCGTATTCTGTTTCTCCTAAAAATATTGTTCTTGTAAATTTGGGGAA
ATCCGGAAAAGGCTATACTGTCATTTGCTTCCTAACTTGTCTTGAGCAATGACCTAATGATTTTCCTATAGCTTTTG
TTGGTTTTTTTCTCGTTCTTTCTTTCTCTGACGTTATGTTTAATTCCCTCAACAACTCCAGATGCTATGATGAAAAC
TTGGTTGATATCCCAGTTCAAGTGGATACTCTTGCTCGCTATTATTACCATGTATGAATTTGGCTGCTTCCTTTCTA
AATGGTCTTTCTGTTGTTGGGC



>GPH5 vesca clone21
CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGGCCGGACCGGAAGATCAT
CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT
GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG
AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT
ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAGAAGCTT
CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT
TGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATATGAAATAGAAGGTAATATGCATG
ATATAAATATCTAGTTAATTGTACAATGATATTTGTAACCAGTGAAAATAATGACAATCTTTATAACAAAATTTCAG
TTATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAG
ACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGACAA
AGAATCCTTTTGTCATATGGATTGAATCTGAATTAGTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCATGT
TACATGTCTCATGATGTCTTCATTTGGTGACAAAAGCTAAATCTTAACCTGACCTAAGTATCAAGACATATTGGACA
ATTGGGCTTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTATAGAAAACACTCTGTGATCT
TCACCATTGAGGAGTCAAGTTACTCAGCCCTGAAGTAAAAGTCCAGTCAGTAGTGCAGTTGAGTTCAACTTGTTCTG
GGTTCTTCAAAGTTTGAAACTTTAAGCTTCGATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATG
TGGTTGGCATATATAAAAGCCTGAAAAGATTGCCCAAAACCCAAGCTGGGTTCATCCGAAAAAGTTTTTGAATCTTT
TTAAAGCCCTTTTTTAATAATTTGGAATNCCACCTTCCnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnGTATTAGATGAAAACTAGTTTT
TCTAAGAACTTGATGAGTTGATGGAGGATTACATATGAGGTTTGGTTATGTTTTTAGGTATGCAATCCTTCTGTAAG
















































886




937
936
929
928
936


987
986
979
978
984


1037
1036
1029










Predicted o Putative Gene Distr (kb
Number of Genes EST Fosmid between genes)
Fosmid a Insert Size
ab Similari Protein Hit Hits n Similarity-
SProtein Hit (bp) ab initio
initio ty O (gb no.) based


x
Secretory Protein
SEC14
X
ATPase
unknown
glycosyl hydrolase
X


- DY675
900.1
DY672
841.1


CX6621
88.1


5

6
Not
predicted
7
8
9
Not
predicted
43P07
1


retrotransposon
polyprotein
X
X
DNA cytosine-5-
methyltransferase
unknown
methyltransferase
small domain
X
X
X


10.9


- DY668
476.1
+ DY668
476.1
+


44J07 11
1
2AA
3


29,636 2.7


DY672
792 1


disease resistance
unknown


9 4


x
polyprotein
integrase
retrotransposon
protein


-DY671
343.1


DY650
877.1


DY669
025.1


34,817 3.9


43,641 4.4


I.


10 4


14.8


7

A
9
10


47H15
'A
2

4
5









Sargent DJ, Clarke J, Simpson DW, Tobutt KR, Arus P, Monfort A, Vilanova S, Denoyes-
Rothan B, Rousseau M, Folta KM, Bassil NV, Battey NH (2006) An enhanced
microssatellite map of diploid Fragaria. Theor Appl Genet. 112: 1349-1359

Sargent DJ, Davis TM, Tobutt KR, Wilkinson MJ, Battey NH, Simpson DW (2004) A
genetic linkage map of microsatellite, gene-specific and morphological markers in
diploid Fragaria. Theoretical and Applied Genetics 109: 1385 1391

Sargent DJ, Hadonou AM, Simpson DW (2003) Development and characterization of
polymorphic microsatellite markers from Fragaria viridis, a wild diploid strawberry.
Molecular Ecology Notes 3: 550

Sargent DJ, Rys A, Nier S, Simpson DW, Tobutt KR (2007) The development and mapping
of functional markers in Fragaria and their transferability and potential for mapping in
other genera Theor Appl Genet. 114: 373-384

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of Biological Chemistry 248: 8429-8433

Settles AM, Latshaw S, McCarty DR (2004) Molecular analysis of high-copy insertion sites in
maize. Nucleic Acids Research 32: e54

Sevag MG, Lackman DB, Smolens J (1938) The isolation of the components of streptococcal
nucleoproteins in serologically active form. J. Biol. Chem. 124: 425-436

Shapiro HS, Chargaff E (1960) Studies on the nucleotide arrangement in deoxyribonucleic
acids. IV. Patterns of nucleotide sequence in the deoxyribonucleic acid of rye germ and
its fractions. Biochim Biophys Acta. 39: 68-82

Sjulin TM, Dale A (1987) Genetic diversity of North American strawberry cultivars. J. Am.
Soc. Hort. Sci. 112: 375-385

Soltis PS, Soltis DE (2000) The role of genetic and genomic attributes in the success of
polyploids. Proc. Nat. Acad. Sci. 97: 7051-7057

Staudt G (1973) Fragaria iturupensis, eine neue Erdbeerart aus Ostasien. Willenowia 7: 101-
104

Staudt G (2003) Notes on Asiatic species: III. Fragaria orientalis Losinsk. and Fragaria
mandshurica spec. nov. Bot. Jahrb. Syst. 124: 397-419

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Syst. 126: 163-175

Stein L (2001) Genome annotation: from sequence to biology. Nature Reviews Genetics 2: 493-
503












17022 esca AATTTTAACAGCGATTCTTCTACAAAAATG-GACTAAATTCCACCTTGTACTGTACAAA 966
17022 mandshurica AATTTTAACATGATTCTTCTACAAAATAAACTATTCCACCTTGTACTTACAA 1009
17022 viridis AATTTTAACAGTGATTCTTCTACAAAAGA---CTAAATTCCACTTTGTACTGTACAAA 1014
17022 nubicola AATTTTAACAGTGATTCTTCTACAAATG-GACTATTCCACCTTGTACTTACAA 991
17022 iinumae AATTTTGACAGTGATTCTTCTACAAAAGATG-GACCAATTCCACCTTGTACTGTACAA 1009
****** **** ***************** ********* **************

17022 vesca AAACGAGTTTGAGTAGTGGGTCTTCCAATAT-ATTTCTGCTCTGTTTACCTTGCC 1025
17022 mandshurica AAACGAGTTTGAGTAGTGGGAATCGTTCCAATAT-ATTTCTGCTCTGTTTACCAAGTGCC 1068
17022 viridis AAACGAGTTTGAGTAGTGGGTCTTCCAATAT-ATTTCTGCTCTGTTTACCTTGCC 1073
17022 nubicola AAACGAGTTTGAGTAGTGGGAATCGTTCCAATATTATTTCTGCTCTGTTTACCATTGCC 1051
17022 iinumae AAACGAGTTTGAGCAGTGGGAATCGTTCCAATAT-ATTTCTGCTCTGTTTACCATTGCC 1068


17022 vesca AGGATGATACAAACATCTA-ACTCTACAGGACCCTTTTCTAGCAAAAGAA-TGAGSAGA 1084
17022 mandshurica AGGATGATACAAACATCTAAACTCTACAGGAACCATCTTCTAGCAAAAAAA-TGAGAAGA 1127
17022 viridis AGGATGATACAAACATCTAACTCTACAGGAACCCTTTTCTAGCAAAAAAA-TIGAGAGA 1132
17022 nubicola AGGATGATTCAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAA-TGAGAAGA 1110
17022 iinumae AGGATGATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAATGAGAAGA 1128


17022 vesca AAGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1144
17022 mandshurica AAGAACTCTACAAGAATC-AAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1186
17022 viridis AAGAACTCTACAAGAATCCAAAGTGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1192
17022 nubicola AGGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1170
17022 iinumae AAGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1188
**************** **** ************************************

17022 vesca AAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGA 1204
17022 mandshurica AAATTTGCTGCAGCCTCCACTGAGGAGCATCTCCAGCAAGAACAAAAGAATCAAACCAGA 1246
17022 viridis AAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGA 1252
17022 nubicola AAATTTGCTGCACCTCCACTGATGAGCTTCTCCAGCAAGTACAAAAl3ATCAAACCAGA 1230
17022 iinumae AAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGA 1248


17022 vesca TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1264
17022 mandshurica TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1306
17022 viridis TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1312
17022 nubicola TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1290
17022 iinumae TAAAATGGAAAATCTCCTCTCACGTCGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1308


17022 vesca ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAGGATGATG TTCAGAAAATTT 1324
17022 mandshurica ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTG 1366
17022 viridis ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAGGATGATG TTCAGAAAATTT 1372
17022 nubicola ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGAAGGATTCAGAAAATTTG 1350
17022i inumae ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTG 1368


17022 vesca CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1374
17022 mandshurica CTACAAAAGCCCATAACTTGTAAGGCATCATCGAAGTATTGTGAGGAAACCC 1418
17022 viridis CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1422
17022 nubicola CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1400
17022 iinumae CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1418





27F10

27F10 vesca CCTGCAGGGTTTTTCATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 59
27F10 mandshurica CCTGCAGGGCTTTTTATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 59
27F10 nubicola CCTGCGGG--TTTTTATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 57
27F10 iinumae CCTGCAGG-TTTTTCATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATCATATCATC 58
27F10 ananassa CCTGCAGG-TTTTT-ATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 57
27F10 viridis --TGCGGG-TTTTTCATCATGTAAGGACCTCCATTGTTCGGTAGCTTTATGCATATCATC 57





183














32L07 vesca TATATAGATGCATATTCACTATCAAGCTACCCAAGTATGCAAATTTATATAGCATCTCATTA 1859
32L07 viridis TATTATTCACTATCAGCTACCCAAGTATGCAAATTAATAGCATCTCATTA 1064


32L07 vesca TCTTGTTTCCTCTAGCTATTCTACTCAATGCATATCAACAACCTGACCCAGTTCTCCTAT 1919
32L07 viridis TCTTGTTTTC TCTACTCAATGCATATCAACAACCTGACCCAGTTCTCCTAT 1124


32L07 vesca AATTGCTGGCAGATAGTAATACCAATTACTCCAGAATCTTCACACCCAGAACTTGAAATT 1979
32L07 viridis AATTGCTGGCAGATAGTAATACCAATTACTCCAGAATCTTCACACCCAGAACTTGAAATT 1184


32L07 vesca ACACGACCTCAATACTCCAAACAGTAC ---------AAAAAAAGATGATCAAAACA 2030
32L07 viridis ACACGACCTCAATACTCCAAACAGTACTGTCAGTACAAAACAACCCAGATGATCAAAACA 1244


32L07 vesca CATAACATTCTTTATTTCATCTTATTGGGAAAATCTCTATATCTATTATCTTCATTATTC 2090
32L07 viridis CTTAAAATTCTTTATTTCATCTTATTG---------CTATCTCTATCATCTTCATTATTC 1295


32L07 vesca AATTTTTCTACACTGCATGCTATACATGTTACAAAAGAGAAAGAAAAGACACTAGTCCAT 2150
32L07 viridis AATTTTTCTACACTGCATGCTATACATGTTACAAAAGAGAACAAAAGACACTAGCCCAT 1355


32L07 vesca ATCACATAGGCCATGTCCTTCCCAATTCTAACCCAACAATTCAAGGACCACACCCATGAG 2210
32L07 viridis ATCACATAGGCCATGTCCTTCCCAATTCTAACCAACAATTCAAGGACCACACCCATGA- 1414


32L07 vesca TAGTGGCACTGAATCACTGAATCGTCGCCTTCACAACTACACTACCTATCCAACCCAGAC 2270
32L07 viridis --GTGGCACTGAATCACTGAATCGTCACCTTCACAACCACACTACCTATCCAACCCAGAC 1472



32L07 vesca TCAACACAGATGAAAATTCACAGCAGCTAAGAATATAGTACTAGTTTTGCTCTATCTTTT 2330
32L07 viridis -----ACAGATGAAATTCACAGCAGCTAAGAATATAGTACCAATTTTGCTCTATCTTTC 1527


32L07 vesca TTCTTTACCAAAACAAAAAAAACCCTGTAGTAACCAATATAACCGCTAACAGCTTTTCCC 2390
32L07 viridis TTCTTTACCAAAACAAAAAAGATCCTGTAGTAACTAATATAACAGCTAACAGCTTTTCCC 1587



32L07 vesca ATCCTGCCCATAACAGCTTTTCCCCTGCAGTATGGGAAACCCTTATCTAAAACCCCCCGA 2450
32L07 viridis ATCCTGCCCATAACAGCTTTTCCCCTGCAGTATGGGAAACCCTGATCTAAA-TCCCCCGA 1646


32L07 vesca TTTATAGTAACAAAAAAATAAATAAAATAATTTACTTTCCTCATTTACCATTTTACCCTC 2510
32L07 viridis TTTATAGTAACAAAAAAATAAATAAAATAATTTGCTTTCCTCATTTACCATTTTACCCTC 1706



32L07 vesca ATCTTCTCCTTCATTGCCACTTGAACCCCCACTCTCCATGCTCCTTGAACCTTCTCAACA 2570
32L07 viridis ATCTTCTCCTTCATTGCCACTTGAACCCCCACTCTCCATGCTCCTTGAACCTTCTCAACA 1766



32L07 vesca CCCTTTCTAGGGCAATGTCAAAAGCGTCTTTTACCGTCTCCAACCCCTCCTGCGGTTTCG 2630
32L07 viridis CCCTTTCTAGGGCAATGTCAAAAGCGTCTTTTACCGTCTCCAACCCCTCCTGCGGTTTCG 1826


32L07 vesca CGTACAGAAAATTCGGTATGTAATCGATAACTTTCTCCCGCATTTTCCT 2679
32L07 viridis CGTACAGAAAATTCGGTATGTAATCGATAACTTTCTCCCGCATTTTCCT 1875
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 49
- - - - - - - - - 6-- - - - - -


























72E18


mandshl
nilger
viridi
iinumas
ananas



vesca
mandshl
nilger
viridi
iinumag
ananas



vesca
mandshl
nilger
viridi


mandshl
nilger
viridi
iinumas
ananas;



vesca
mandshl
nilger
viridi


mandsh
nilger
viridi









34D20









DNA began to be used to generate transformed Arabidopsis lines with mutant phenotypes to

identify and clone important plant genes, such as genes involved in the control of meristem

identity and hormone perception (Feldmann, 1991), (Feldmann and Marks, 1987). A second

method to clone plant genes was devised upon the isolation of the Ac and Ds transposable

elements (Fedoroff et al., 1983).

The beginning of the sequencing era can be attributed to the determination of a

bacteriophage RNA gene sequence in 1972 (Min Jou et al., 1972). The first whole-genome

sequencing was also from a virus, Haemophilus influenza, completed in 1995 (Fleischmann et

al., 1995), whereas a draft of the Human Genome was released in 2001 (Venter JC, 2001). The

first plant genome sequenced was Arabidopsis thaliana, completed in 2000 (The Arabidopsis

Genome Initiative, 2000). In 2007, approximately 2300 sequencing projects are being carried out

or completed, of which about 130 are plant genomes, according to the Genomes Online database

(Liolios et al., 2006). Our collaborators in this Fragaria genomics project have successfully

completed 1% of the F. vesca genome. Recently, Malus (apple) was selected for full sequencing

by an Italian sequencing effort. Peach also will be sequenced through a US Department of

Energy initiative. Although these genomes are much larger than the strawberry genome, their

completion will have important ramifications to Fragaria, as annotation will provide a list of

components that are similar to those in strawberry. The work presented here is a complementary

effort to those in other rosaceous crops, providing an initial glimpse into the genome of one of

the world's most prized horticultural crops.









DNA Extraction from Plants

Pioneer methods to isolate genetic material of plants used DNA-rich matter such as germ

tissue (Lipshitz and Chargaff, 1956; Shapiro and Chargaff, 1960). Early attempts to extract DNA

from leaves resulted in degraded product due to the extreme pHs used by the procedure for

removal of RNA (Thomas and Sherratt, 1956). The currently most used protocol for plant DNA

isolation, developed by Murray and Thompson (Murray and Thompson, 1980), takes advantage

of the selective precipitation of DNA by cetyltrimethylammonium bromide (CTAB), a

phenomenon observed by Jones during DNA isolation from bacteria (Jones, 1953). CTAB is a

cationic detergent that, in high ionic strength solutions (e.g. >0.7M NaC1), complexes with

proteins and non-acidic polysaccharides, whereas at low ionic strength it precipitates nucleic

acids and acidic polysaccharides, leaving proteins and neutral sugars in solution (Sambrook and

Russell, 2001). Multiple variations of Murray and Thompson's protocol have been used by

researchers to adapt the original process to different plant species. A protocol designed by Doyle

and Doyle (Doyle and Doyle, 1987) is also frequently used for plant DNA extraction and is

ultimately a variation of the Murray and Thompson procedure. Doyle and Doyle's protocol uses

fresh tissue in place of lyophilized material and a higher concentration of CTAB and salt to

compensate for the greater water content of fresh tissue.

Although CTAB is the reagent of choice to purify DNA from organisms that produce

many polysaccharides (Sambrook and Russell, 2001), even high quantities of the cationic

detergent seem insufficient to free DNA preparations from sugar contamination. In attempt to

circumvent this problem, boric acid is added to the extraction buffer. Boric acid forms complexes

with polyphenols at pH 7.5 (King, 1971) and with carbohydrates (Gauch and Dugger Jr., 1953),

making these complexes more soluble. An additional approach to avoid co-purification of









alleles of the octoploid were detected by EcoRI digestion. The following polymorphisms were

identified in the 2.8kb analyzed: short indels of 4-12bp (9 bp insertion in F. vesca; 12 bp deletion

in F. iinumae, shown in figure 4-4), 180 SNPs, of which 125 are ambiguous (may be sequencing

or polymerase errors) and 55 likely true SNPs, because the base change occurs in more than a

single clone. Most of the likely SNPs delineate the octoploid clones from the diploid ones. It is

interesting to note that the octoploid alleles are grouped separately from diploid alleles not only

for their SNPs, but also for small indels. Two SSR motifs were identified (AAG and AT), with 4

repeats each, for every clone. Therefore no polymorphism in the number of repeats was detected.

GPH23

Atlg23740 (oxidoreductase, zinc-binding dehydrogenase family protein) and Atlg23750

(DNA-binding protein) were similar to F. xananassa with E values of 3x10-64 and 2x1057,

respectively.

Only F. mandshurica, F. iinumae, and F. xananassa were amplified by the primers

designed for this region. Larger deletions than those observed for other loci investigated, and

different alleles from the diploids were observed for GPH23. Figure 4-5 illustrates the

polymorphisms detected. After preliminary sequence alignment, the putative SNPs were verified

by observation of unambiguous peaks in the chromatograms. Therefore, for this locus, a SNP is

only an artifact if it was introduced during amplification by the polymerase. (CTC)4 SSRs were

detected and occurred in equal number of repeats for every clone, in the same position when

aligned. The implications of the polymorphisms are discussed below.

GPH10

Primers GPH10A and GPH10C were utilized to amplify a 4.4kb fragment from the

octoploid 'Strawberry Festival'. Four categories of polymorphic clones were detected by EcoRI










Predicted o Putative Gene Distr (kb
Number of Genes EST Fosmid between genes)
Fosmid a Insert Size
ab Similari Protein Hit Hits n Similarity-
Protein Hit (bp) ab initio
initio ty O (gb no.) based
8 X
27F10 11 8 37,110 3.4 4.6
L. DY675
1 kinase .
883.1
2A CX6613
A hypothetical CX661
86.1
DV438
3 unknown
706.1
4 integrase
5 integrase
6 integrase
7A unknown +
8 X
Not C03787
predicted 00.1
9 unknown
10 X
11 X
29G10 10 4 31,681 3.2 7.9
1 L transposase
21 X
3 flavin-binding + DY673
monooxygenase-like 408.1
4 X
5 X
6 X
7A X
8 phenylacetaldehyde
synthase
9 unknown +
10 t X
30124 7 5 37,599 5.4 7.5
1 X

kinase
3 X ( E value=le-10)
arabidopsis response
regulator 12
+ CX6615
4 chitinase 2
29.1
5 arabidopsis response + DY671
regulator 12 913.1
6 A transferase
7 PICKLE chromating
remodeling factor












>34D20 vesca
GCAGAAAGAAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTCAGGGGATAAAGGAAGTA
TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC
GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT
TATGTGAATC CATTGGGCATATATAATCATTCAGAACTACAAGGAAAGATTATCGGC
GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG
ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA
ATGGATTTAGTACTTCCATTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGAACTGC
AGGTGAACCGTATGTCTATATATGTCGTATGTTAGAT ACATAGTATGTGGGTGTGGATGAACTATACG
TAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGTATGAATCTCCCTCCCGGCCACACTAGAC
CACACTTTTGAACTGGCGGATTCCATCCGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCT
CCTAGCCATAGTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATATGATGAATTT
GGAATGAATACAAAAACTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATT
CTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGT
ACTTGTATAGTTGTATTGATCTGATATAACATAAATTTAATGAATCTAATAGACATTTTTCCTAGTTAACAGAGGAT
AGGTCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACACAAGACTGGTCTATATA
AGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCTCTTTATCTGTTTCATACTTTCATTGATCATATT
GTCTAGTACTGGAAGAGCTATATTTATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGA
AGGTCCTTAGTTACAATGGTGTTGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTTGTTACTTAACCGAT
AGCATCATTTGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTC
TGATCTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGA
AAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG
TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTGGAAGCTAGGAAAATGTA
TACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCACCAATAACTCCAGCTGGGACTAGTAGTTTGGCTA
CCAACAACAGTTCTACAATGAGACACGAGTGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATG
CTTGGGGTTTGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCATCC
TTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTAATCATGGAGATGATGATGATGTTAATGAACCTTGGT
ATAGAGAAGAGACTCTTATGCCTCAGCAGACGAATTTTTACGACTGCG


>34D20 iinumae
NNNNNNNNNAACTGATGTGCTTTCCGGAGGGACTGACAGTAGAAAAGGACAGTGCAGTTCAGGGGATAAAGGAAGTA
TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC
GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT
TATGTGAATCATCTGCATATTATCTATATCTCATTGGGCATATTATCTATAATCATTCAGAACTACAAGGAAAGATTATCGGC
GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG
ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA
ATGGATTTAGTACTTCCATTAACTAGTAACTGAGGAATCCAATTGCACTCATGCACAGGAGAACTGCA
GGTGAACTATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGTGATGAACTATAAGT
AGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGCATGAGTCTCCCTCCCGGCCACACTAGACC
ACCTAGATTTTGTGCCGACTATCACAATAGTGAAAGTTGGTCCTCCTAGCTATAGTTTCTAGTACTATTCTACTGAT
ATCATGTTTCGTCTCAGCTTTTGACAATGGAATATGATGAATATGGAATGAACAAAACCTGCTTTGTCCATCTATTA
GCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTT
CATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCACTACTTGTGTAGTTGTATTGATCTAAATTTTTCCTAG
TTAACAGAGGATAGGTCTCCGGCTGACGTTATCCTACAAGGAAACAGAAACGTACAATTAACGGATTCACAAGACTG
GTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCTAGTACTGGAAGAGCTATATTTATCT
GATAACAGAAAGTGCTTACTTGCTGGTTCCTACTCATTATGGATCCGAAGGTCCTTAGTTACAAAGGTGTTGACCTG
AGCATGAGCGACTAGGATATTCTTAGAGGACCTTATTACTTAACCGATAGCATCATTCGATTCTATTTCACTTATCT
TACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTCTGATCTCCTGGTAAATTCTCCGGATCCCG
AGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATCTTCGCAGTGAATGATAAC
AAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTGTATTTCAGAAAATCAAACGCATTCGTACA
TTACGACAGCTTGGGGGGTAACAATAGTTTGGAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTC
CAGCAACACAAGCACCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGA AG
TGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTTTGGGGTTTTGTTGTCAACTA
CATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAA
TCACCAATAGTAATCATGGA GATGATG ATGAACCTTGGTATAGAGAAGAGACTCTTTGCCTCAGCAGA
CGAATTTACGACTNNN




























































OCTTAAGG
OCTTAAGG
OCTTAAGG
OCTTAAGG









* Resuspend pellet in 500ul to 1 ml (depending on the amount required to dissolve the pellet)
of deionized water or TE pH 8.0.









due to the higher number cells that contained in freeze-dried samples in comparison to the same

weight of fresh tissue. While yield from T58 was not different from that of T59, increases of 73

and 50% were observed in T13-T16.

There was concern that the lyophilization process might compromise DNA quality. This

was addressed by running uncut genomic DNA on agarose gel, and the integrity of all

lyophilized samples (T13, T14, T23, and T57) appeared preserved. Therefore, lyophilization may

be a good solution for storing material that does not require immediate DNA extraction, but it is

not indispensable.

Incubation temperature and duration

Utilizing fresh 'Strawberry Festival' leaf tissue, the effects of temperature and duration of

incubation of tissue in extraction buffer were investigated. The treatment that relinquished the

most DNA was incubation at 65C for 1 hour (figure 2-2), which is the treatment specified in

most plant DNA extraction protocols. However, the resultant preparation at this temperature is

atypically viscous, complicating mechanical and enzymatic downstream manipulations.

Tissue-to-buffer ratio

Tissue-to-buffer ratios were tested for four protocols (2, 5, 14, 23; ratios and yields shown

in table 2-1), and yielded inconsistent results. For protocols 2 and 14, the lower the ratio, the

higher the yield, whereas for protocols 5 and 23, the opposite was true. Since all of the ratios

(10-200 mg/ml) tested did not use the same protocol, a last DNA extraction experiment was

conducted using leaf tissue of 'Strawberry Festival'. Volumes of extraction buffer were kept

constant at 5 ml, whereas the treatments were 50, 200, 500, or 1000 mg of fresh tissue. Each

treatment included two replicates, and incubation was carried out at 40C for 5 min. Samples were

treated with RNAse A, DNA was precipitated by isopropanol and resuspended in deionized










Predicted o Putative Gene Distr (kb
Number of Genes EST Fosmid between genes)
Fosmid a Insert Size
ab Similari Protein Hit Hits n Similarity-
Protein Hit (bp) ab initio
initio ty 0 (gb no.) based
32A10 15 4 33,577 2.2 8.4
DY667
1 catalytic/ hydrolase 800.1
800.1
2AA x
3 X
4 L X
5 X
6 X
7 copper ion binding +
8AA X
9 MADS-box
10 X
11 X
12 X
13 pathogenesis-related
14 X
15 X
32L07 6 4 32,951 5.5 8.2
Not x DY668
predicted 002.1
1 hypothetical
2 SMC2
DY666
3 disease resistance 6
677.1
4 X
CX6620
5 exostosin-like .
49.1
6 X
34D20 8 6 30,034 3.8 5.0
1 / RNA recognition +
AA motif
cysteine-type +
peptidase
3 X
4 transposase +
5 anthocyanin 5-
aromatic
X( E value = 8e-14) +
6 anthocyanin
malonyltransferase
FGENESH missed
EST
7 NAC domain NAM
Not DV438
predicted 498.1
8 X



























iridi
ilger


11D02









Material and Methods

Thirty-three DNA extraction protocols, totaling 103 treatments, were tested using either

lyophilized or liquid nitrogen-frozen leaf tissues. A broad range of genotypes were tested,

including tissue from F. nubicola, F. vesca cultivars Yellow Wonder, Alexandria, and Hawaii-4,

F. chiloensis CA 1367, F. virginiana NC 96-35-2, F. x ananassa cultivars Sweet Charlie,

Tristar, Camarosa, Quinault, Diamante, Strawberry Festival, and the laboratory transformation

genotype LF9 (Folta et al., 2006). The detailed protocols can be found in Appendix A, whereas

further below is a summary of the approaches adopted. When at least 15kg of DNA were

obtained, digestion of 5[ g of DNA with at least 2 separate restriction enzymes were carried out.

The uncut and enzyme treated samples were loaded on 1% agarose gel for assessment of DNA

quality (integrity and amenability to use of restriction enzymes), and correlation to

spectrophotometric readings. Phenols are known to absorb at 260nm as does DNA, and high

readings may be attributed to the presence of phenols, particularly when the DNA pellet has

brown coloration, caused by oxidation of phenolic compounds (phenylpropanoid and flavonoids)

to quinones (Loomis, 1974). To further test the quality of the DNA preparations, PCR was

carried out using primers for F. x aanassa 18S ribosomal DNA. The primers (forward: 5' TAT

GGG TGG TGG TGC ATG GC 3'; reverse: 5' TTG TTA CGA CTT CTC CTT CC 3') were

designed utilizing as sequence source the accession gi 184481emblX15590.1|FA18S. The

fragment to be amplified by this primer pair is not large (510bp from cDNA, -Ikb from

genomic) and should be easily amplified, since many copies of ribosomal DNA are present in the

genome. If a product was observed, a second set of primers (forward: 5' CAC TGC CAA GGA

GCG TGG TG 3'; reverse: 5' TCA GTA GGG CAG CTG ATG 3') targeting a single-copy

region, the Leafy gene, was used to provide a more challenging test. This second primer pair was

designed utilizing F. vesca 'Pawtuckaway' sequence provided by our collaborator, Dr. Thomas












CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGGTCCTACCAACCTCATCATACATGGGCAAGAAATC
ATTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTATATGCCGGTTTTGAATTATTAGAGTCTATAAAGGGA
CTAGTTACAACTGTTTTTTTCCACTTTTTTTTTTTTTTTTTTTTTTTTGAGAATGTATGCTTTTCACTTATATGGCC
TGAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGCACACAGGAACCGTTGA
GGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAGTTAGAGCAATGGGTGAGATGAACTGGAGACAAT
TTGCTGCTGAGGAGGTTACAGAGATGAGGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCAAAGTC
ACATCCCTTCCTGGATGTGAGAGTTTCCCCGATGCAGGAGGAAATATAACCGGTTCTTTCCTTGGCATTCAAGAAAA
TTTGACAATTTGATCACCAGTTCAGTTTTATTGAAGAACTCGGTACAGTTTTGAAACGTTTTTTTGTTGTATTTAGC
AAACGCATAGGAGGATAGGGTTTTCTTTTATTCAACAGGGATATAGGCGCTTTTAGGGTTTCTTTTCCTATTGAATT
TCGTTCTTTGGTATACCAAGTCCCTTCTTTGGCATTCAAAGAAACCTAAGCATTTGATCTGCCTGTCATCACGTCTA
GAGTTGCAGATTGTTTAGAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAACTTCTGGTGTTCAACAACG
CATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGATATGCAGGTAACATCTCTGACTATGCATCTTTGCTTT
TTCTTCTGTTTTTGAGAACAAGGCATCTTGTTTATTTGTGGCCAACTTGAAGCACTGTATTTAAATAATGCTAAGAC
CGTGTCAATTTTGTTACAAAAGTCTAGGCAACAATGAACTTGAACTTGATAAGAAAATAGATCCTAGAGATGGTCTA
TCTACTACCCTTGACTACACAAGTTGCTCATTCTTTACATGTGAAATGGCTATCCAGAGCAGTCGTAAATTTCATGA
GATATTAACAAGCTTTGGACGTCCAGTTGCCAGGTTCATGTCTACTCGGAGACCAAGTGAGCAAGGGCAGGCACATC
AACAGACCCCTAAA


>63F17 mandshurica
CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGGTCCTACCAACCTCATCATACATGGGCAAGAAATC
ATTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTAGATGCAGGTTTTGAATTATTAGAGTCTATAAAGGGA
CATAGTTACAACTGTTTGTATGCTTTTCCATTTTTTTATTTTTTTATTTTTTGAGAATGTATGCTTTTTCACTTATA
TGGCCTGAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGCACACAGGAACC
GTTGAGGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAGTTAGAGCAATGGGTGAGATGAACTGGAA
ACAATTTGCTGCTGAGGAGGTTACAGAGATGAGGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCA
AAGTCACATCCCTTCCTGGATGTGAGAGTTTCCCGATGCAGGAGGAAGTATAACCGGTTCTTTCCTTGGCATTCAA
GAAAATTTGACAATTTGATCACCAGTTCAATTTTATAGAAGAACTCAGTTAGTACAGTATTGAAACGTTTTTTCGTT
GTATTTAGCAAACCCATAGGAGGATAGGGTTTTCTTTTATTCAACAGGGATATAGGCGCTTTTAGGGTTTCTTCTCC
TATTCAATTTCATTCTTTGGTAGACCAAGTCGCTTCTTTGGCATTCAAGGAAACCTGAGCATTTGATCTGCCTGTCA
TCACATCCAGAGTTGCAGATTGTTTAGAGAAGAATTCCAATTCCTTTTGTACAGTTTGGTTAACTTTTGGTGTTCAA
CAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGATATGCAGGTAACATCTCTGACTATGCATCTTT
GCTTTTTCTTCTATTTTTGAGAACAAGGCATCTTGTTTATGTGTGGCCAACTTGAAGCACTGTATTTAAATAATGCT
AAAACAGTGTTAATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGATAAGAAAATAGATCCTA
GAGATGGTCTATCTACTACTCTTGACTACACAAGTTACTCATTCTTTACATGTGAAATGGCTATCCAGAGCAGTCGT
AAATTTCATGAGATATTAACAAGCTTTGGACGTCCAGTTGCCACGTTCATCTCTACTCGGAGGCCAAGTCGAGCAAG
GGCAGGCACATCAACAGACCC


>63F17Rrc ananassa 2
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNTTCAATTTCATTCTGGGATAGACCAAGTCACTTTTTTGGCGTTCAAGGAAACCTGAGCATTTGATCTGCCGG
TCATCACATCCAGAGTTGCAGATTGTTTATAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAACTTTTGG
TGTTCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGGATATGCAGGTAACATCTCTGACTATG
CATCTTTGCTTTTTCTTCTTTTTTTGAGAATAAGGCATCTTGTTTATGTGTAGCCAACTTGAAGCACTGTATTTAAA
TAATGCTAAAACAGTGTTAATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGAACTTGATAAG
AAAATAGATCCCAGAGATGGTCAATCTACTACCCTTGACTACACAAGTTACTCATTCTTTACATGTGAAATGGCTAT
CCAGAGCAGTCGTATTTCATGAGATATTAACAAGCTTTGGACGTCCAGTTGCCAGGTTCATCTCTACTCGGAGGCCA
AGTCGAGCAAGGGCAGGCACATCAACAGACCCTCAA
















































ca AGGTGAGCAGGTTAGCTAGAAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 420




inassa clone2 ATAGATATATGCTGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 476
aassa clone7 ATAGATATATGTTGAA ---CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 476
-idis ATAGATATATGTTGAA ---CTGTAAGAGACATATTTCAAGCTC--TGGTGTTCAAAGTT 474
tumae ATAGATATATGTTGAA ---CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 476
gerrensis ATAGATATATGTTGAA ---CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTC 476
idshurica ATAGATATATGTTGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 475


FACATI
FACACI
FACAC'
FACACI
FACACI




'TAATE
'TAATF
TAATF

'TAAT
TAATF
TAATF

TAATF

TAAT54









154









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andsh
esca
iridi
ilgei


tandsh
esca
iridi
ilger


landsh
esca
iridi
ilger


andst
esca
iridi
ilgei
manas
manas
manas
manas
inume





bicc
andsh
esca
iridi
ilgei















GPH23mandshuricalone3 --CAACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 1939
GPH23 ananassa clone4 ---CAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 2026
GPH23 ananassaclone3 ---CAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 1996


GPH23 iinumaeclone2 CTCTGAGAGTGTCTCTCTAGAGTGTCTGTAATTACAGCTGCCCCTGCCACGGCTAGGCC 2060
GPH23 iinumae clone5 CTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCC 2062
GPH23_mandshurica clone3 CTCTGAGAGTGTCTGCTAAATACAGCTTCCCCTGCCACAGCTGAGGCC 1989
GPH23 ananassa clone4 CTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCC 2076
GPH23 ananassa clone3 CTCTGAGAGTGTCTGCTAATTACGAGCTGCCCCTGCCACGGCTGAGGCC 2046


GPH23 iinumae clone2 TCCACGGTGCCGTTGGAGATGAAGGCGTGGGTGTA 2095
GPH23 iinumae clone5 TCCACGGTGCCGTTGGAGATGAAGGCGTGGGTGTA 2097
GPH23_mandshurica clone3 TCCACGGTGCCGTCGGAGATGAAGGCGTGGGTGTA 2024
GPH23 ananassa clone4 TCCACGGTGCCGTCGGAGATGAAGGCGTGGGTGTA 2111
GPH23 ananassa clone3 TCCACAGTGCCGTCGGAGATGAAGGCGTGGGTGTA 2081








Gene Pairs Detected Through Prediction from Genomic Sequence


GPH10


GPH10 ananassa clone GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50
GPH10 ananassa clone20 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50
GPH10 ananassa clonel8 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50
GPH10 ananassa clonel9 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50


GPH10 ananassa clone2 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100
GPH10 ananassa clone20 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100
GPH10 ananassa clonel8 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100
GPH10 ananassa clonel9 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100
**************************************************

GPH10 ananassa clone TCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGT 150
GPH10 ananassa clone20 TCCTTATCGGAGACCA CGAAACGGCGTCGCTTTAGGCGAGAGT 150
GPH10 ananassa clonel8 TCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGT 150
GPH10 ananassa clonel9 TCCTTATCGGAGACCG AACAGCCAACGGCGTCGCTTTAGGCGAGAGT 150
**************************************************


GPH10 ananassa clone GAATAGCGAACTGAGTAGTTTGGATTTAGAGAGAGGATGTAATTGGTAAC 200
GPH10 ananassa clone20 GAATAGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAAC 200
GPH10 ananassa clonel8 GAATAGCGAACTGAGTGGTTTGGATTTAGAGAGAGGATGAAATTGGTAAC 200
GPH10 ananassa clonel9 GAATAGCGAACTGAGTGGTTTGGATTTGAGAAGAGGATGAAATTGGTAAC 200


GPH10 ananassa clone GGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCATCTTTGAAGTG 250
GPH10 ananassa clone20 GGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAAGTG 250
GPH10 ananassa clonel8 GGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTAAGTG 250
GPH10 ananassa clonel9 GGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTTAGCTTTGAAGTG 250


GPH10 ananassa clone GAGTGTAGGATATAACAAACTCGTTATC--------------------- 279
GPH10 ananassa clone20 GAGTGTAGGATAATAACAAACTCGTATAAAAGACAGGATTAATGTCAG 300
GPH10 ananassa clonel8 GAGTGTAGGATAATAACAAACTCGTTATC--------------------- 279
GPH10 ananassa clonel9 GAGTGTAGGATAATAACAAACTCGTTATC-------------------- 279









In addition to the amplification of unknown regions, sequences were gathered by sample

sequencing genomic DNA. Both random and targeted sequences were studied in a F. vesca

fosmid library. The annotation scheme is described in Chapter 3 of this dissertation. Forty

combinations of PCR primer pairs were tested to amplify 18 loci, since different primer

combinations were required to amplify some of the loci. The primer pairs generated for the

putative intergenic regions are listed in table 5-1 of Chapter 5.

Following determination of location and design of PCR primer pairs, PCR was carried out

to amplify 28 loci, of which 10 gene pairs (listed in table 4-1) were inferred by the F. x

aananassa/Arabidopsis micro-colinearity approach and 18 gene pairs (listed in table 5-1) were

inferred from gene prediction from F. vesca 'Pawtuckaway' genomic sequence. The

optimizations of PCR conditions were carried out utilizing as template DNA from the species for

which primers had been designed: F. x ananssa and F. vesca 'Pawtuckaway' for micro-

colinearity- and genomic-DNA-based approaches, respectively. Once optimum conditions were

determined, the reaction was carried out for seven Fragaria species, which included the

respective control species: F. x ananssa 'Strawberry Festival', F. vesca 'Pawtuckaway',

FRA341 F. viridis, FRA377 F. iinumae, FRA520 F. nubicola, FRA1318 F. nilgerrensis, and

FME F. mandshurica.

The PCR products were cloned using the plasmid cloning vectors pJET1 (GeneJetTM PCR

cloning kit by Fermentas Life Sciences) or pCR2.1-TOPO (Invitrogen Life TechnologiesTM).

The ligation reaction was carried out according to manufacturer's directions and 1 tl of the

ligation reaction was used to transform 50tl of competent cells. The chemically competent

Escherichia coli bacterial cells (Invitrogen One Shot' TOP10) were purchased with the TOPO

cloning kit whereas XL1-Blue competent cells (Bullock et al., 1987) were prepared in the









where a = number of distinct alleles. For an octoploid containing 8 different alleles for a single

locus, the number of different combinations would be 2,451. However, this estimate is artificial,

since most polyploid plants are considered to be alloploids, and therefore display a degree of

fixed, non-segregating heterozygosity (Soltis and Soltis, 2000).

The F. x ananassa genome structure is not well understood. The first proposed genome

structures were derived from cytological analyses of meiotic pairing chromosomes. The genome

composition was first described as AABBBBCC (Fedorova, 1946), whilst the model

AAA'A'BBB'B'(Bringhurst, 1990) is currently the accepted one. More evidence gathered

through the use of molecular markers (Arulsekar et al., 1981; Haymes et al., 1997; Viruel et al.,

2002; Ashley et al., 2003) supports the fully diploidized model. In a single study using molecular

markers (Lerceteau-Kohler et al., 2003), the authors have observed some polysomic inheritance

in a Fl octoploid population. However, the deviations from disomic ratios observed may not be

due to polysomic inheritance, as segregation distortions have also been observed in diploid

segregant populations (Davis and Yu, 1997; Sargent et al., 2004; Sargent et al., 2006).

The identification of genome-specific polymorphisms may permit the monitoring of

segregation of each genome in the complex polyploid background. The "Gene-Pair Haplotype"

(GPH) is a tool developed to fingerprint the alleles present in the contributing genomes in the

octoploid strawberry. It is defined as a suite of intergenic polymorphisms-Simple Sequence

Repeats (SSRs), Single Nucleotide Polymorphisms (SNPs), insertion or deletions (InDels), and

changes in restriction sites (RFLP) that present a complex genetic marker for a given locus

within the diploid genomes. The types of polymorphisms likely to be detected in a GPH locus

and their respective expected location in the genome (within versus between genomes) are

summarized in figure 4-1.









variability that may be induced because of the leaves of various ages, leaves were cut with
a hole puncher, mixed, and split into 4 portions, one for each temperature treatment.
Enough plant tissue was ground per temperature treatment so that 2 experimental replicates
for each time treatment were derived from a single test tube (see figure 2-1).

In addition to variations of the CTAB protocol, other approaches adopted included use of

the chaotropes 8M urea, 4M guanidine thiocyanate (alone or in combination with 2% CTAB,

simultaneously or sequentially); DNA isolation using kits: QIAGEN DNeasy Plant Mini Kit

(charged resin-based), Molecular Research Center DNAzol Extra Strength (guanidine

thiocyanate-based), Epicentre MasterPureTM Plant Leaf DNA Purification, MoBio PowerPlantTM

DNA Isolation Kit; 0.5% SDS, Tris-borate extraction buffer; and crude and fine isolations of

nuclei prior to DNA extraction. Five DNA extraction procedures, QIAGEN DNeasy kit, 2%

CTAB, 2% SDS, 4M guanidine thiocyanate/l% sarkosyl, and 5% SDS/1%TIPS, were tested on

Percoll gradient-isolated nuclei. Refer to table 2-1 for all the treatments.

The amount of tissue necessary to obtain the highest DNA extraction efficient was

determined by keeping the volume of buffer constant at 5ml and varying the tissue weights at 50,

200, 500, and 1,000mg. Once the best tissue-to-buffer ratio was determined, an attempt to extract

DNA from 10 species within the genus Fragaria was made to test the universality of the method.

Each treatment had 2 replicates for both experiments. Expanded leaf tissue was ground in liquid

nitrogen, added to the buffer, and the mixture was incubated at 40C for 5 minutes. An equal

volume (5ml) of 24:1 chloroform:octanol were added to the tubes after incubation, agitated, and

centrifuged at 4,000rpm for 5 minutes. The aqueous phase was transferred to a new tube, and

nucleic acids precipitated by 1/10 volume of 5M NaCl and 7/10 volume of isopropanol. After a

second centrifugation, the supernatant was decanted, the pellet air-dried, and resuspended in

500tl water. RNAse was added to final concentration of 50ig/ml. The solution was transferred

to 1.5-ml tubes and DNA was precipitated as described above. The dry DNA pellet was




Full Text

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1 STRUCTURAL GENOMICS OF Fragaria WILD AND CULTIVATED STRAWBERRIES By DENISE CRISTINA MANFRIM TOMBOLATO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 by Denise Cristina Manfrim Tombolato

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3 To: my father Vadir Tombolato, who has taught me the importance of moral integrity; my mother, Marlene Tombolato, who has, by example, taught me persistence; my professor, Kevin Folta, who permitted and encouraged me to exercise those virtues. Yes, you will say, but the plank is very long. That is true, and so if you do not have a sure foot and a steady eye, and are afraid of stumbling, do not venture down the path. Jean de Lry, in "History of a Voyage to the Land of Brazil, Otherwise Called America", 1578

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4 ACKNOWLEDGMENTS I thank my parents Vadir and Marlene, and my brothers Eduardo and Ricardo, for their teachings, advice, support, and, above all, for their unconditional love. Though not content with my departure from Brazil, my family always supported my decisi ons. I appreciate their confidence in my choices and me, for it reaffi rmed my personal mission in moments of doubt. I am grateful to my professor Dr. Kevin M. Folta, who accepted me as his student in an altruistic gesture, and who has been a lato sensu adviser since. I tha nk the members of my committee for the enjoyable discussions about my project and about scien ce in general: drs. A. Mark Settles, Natlia A. R. Peres, and Craig K. Chandler. I also wish to thank my laboratory colleagues and friends drs. Philip J. Stewart and Amit Dhingra, Thelma F. Madzima, Stefanie A. Maruhnich, Jeremy Ramdial, Dawn Bies, and Maur een Clancy, as well as project collaborators drs. Thomas M. Davis and Daniel J. Sargent, for DNA sequences and plant material from the genetic linkage mapping population. Many people made special the almost-9 years I spent in Gainesville, while I pursued part of my undergraduate training and two advanced de grees. I convey my gratitude to all those who facilitated not only my adaptation to a new country and language, but also the discovery of who I am and of matters I learned to be truly meaningful. I rec ognize Welch McNair Bostick III (McNair), whose short life was vastly fruitful. Mc Nair caused positive impact into the lives of whomever surrounded him: his wife and my fr iend Carmen Valero, his neighbors (including myself), and his colleagues. I thank him for havi ng shown to me the importance of treasuring the time shared with loved ones, expressing honest opinions and making a difference in society. I express my appreciation for the time and assistance granted to me by professors and technicians with whom I worked since my arriva l to the University of Florida: Richard D.

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5 Berger, Terry A. Davoli, D. Pete We ingartner, Jeffrey A. Rollins, Ulla Benny, Valerie Jones, Jeffrey B. Jones, and Jerry Minsavage. I thank these individuals for the attention they have dedicated to me: Bala Terzic', Sylvia Morais de Sousa, Gisele, Jens, and Gabriel Sc hene, Mark D. Skowronski, Luciana C. B. Manfrim Bchir, Gustavo Ramirez, Juliana a nd Gustavo Astua, Aaron Hert, Botond Balogh, Abby Guerra, Ahu Demir, Petrnio Pinheiro, Il ka V. Arajo, Maggie Kellogg, Maria Beatriz Pdua, Melissa Webb, Bruno Maciel, Camila A. Brito C. Paula, Luiz Augusto de Castro e Paula, Hazar Dib, Marlise Klein, Marcus Martin, Michelle Bolton, Sonja I. Parisek, Penny E. Robinson, Anne Visscher, Ricardo da Costa Mattos, Claudi a Riegel, Valerie Rodrig uez-Garcia Schweigert, Lisa Olsen, Jared Greenberg, We ndy Gonzalez, and David Adato. Every one of them made my life in Gainesville a more enjoyable experience.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 STRAWBERRY AND TH E GENOMICS ERA....................................................................12 Introduction................................................................................................................... ..........12 Molecular Markers for Strawberry.........................................................................................13 The Genomics Era............................................................................................................... ...14 2 DNA EXTRACTION FROM RECALCITRANT SPECIES.................................................16 Introduction................................................................................................................... ..........16 The DNA Extraction Procedure......................................................................................16 DNA Extraction from Plants...........................................................................................19 Material and Methods........................................................................................................... ..21 Results........................................................................................................................ .............24 Components of the Strawberry Protocol......................................................................26 Optimization of the CTAB Protocol................................................................................27 Leaf tissue state........................................................................................................27 Incubation temperature and duration........................................................................28 Tissue-to-buffer ratio................................................................................................28 Tissue maceration method........................................................................................30 Discussion..................................................................................................................... ..........31 3 PRIMARY ANALYSES OF Fragaria GENE distribution...................................................42 Introduction................................................................................................................... ..........42 Materials and Methods.......................................................................................................... .45 Results........................................................................................................................ .............48 Expressed Sequence Tags (ESTs)...................................................................................49 Simple Sequence Repeats (SSRs)...................................................................................49 Discussion..................................................................................................................... ..........49 4 GENE-PAIR HAPLOTYPES: NOVEL MOLECULAR MARKERS FOR INVESTIGATION OF THE Fragaria ananassa OCTOPLOID GENOME......................55 Introduction................................................................................................................... ..........55

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7 Materials and Methods.......................................................................................................... .58 Results........................................................................................................................ .............62 GPH5........................................................................................................................... ....63 GPH23.......................................................................................................................... ...64 GPH10.......................................................................................................................... ...64 72E18.......................................................................................................................... .....65 Discussion..................................................................................................................... ..........66 5 GENE-PAIR HAPLOTYPES: FUNCTIONAL AND TRANSFERABLE MARKERS AS NOVEL ADDITIONS TO THE DIPLOID Fragaria GENETIC LINKAGE REFERENCE MAP................................................................................................................82 Introduction................................................................................................................... ..........82 Materials and Methods.......................................................................................................... .85 Results........................................................................................................................ .............88 Discussion..................................................................................................................... ..........89 Conclusions.................................................................................................................... .........91 APPENDIX A DNA EXTRACTION PROTOCOLS.....................................................................................98 DNA Extraction from Leaves.................................................................................................98 DNA Extraction from Isolated Nuclei..................................................................................101 Modifications of Murray and T hompson DNA Isolation Protocol......................................102 B In silico ANNOTATION AND DISTRIBUTION OF Fragaria vesca GENES..................106 C PCR PRIMERS USED TO AMPLIFY AND SEQUENCE GENE-PAIR HAPLOTYPES.....................................................................................................................115 D SEQUENCES GENERATED DURING CH ARACTERIZATION OF GENENPAIR HAPLOTYPES...................................................................................................................117 E GENE-PAIR HAPLOTYPE INDIVIDUAL LOCI ALIGNMENTS...................................153 Gene Pairs Detected by Microcolinearity.............................................................................153 Gene Pairs Detected Through Pred iction from Genomic Sequence.....................................166 LIST OF REFERENCES.............................................................................................................205 BIOGRAPHICAL SKETCH.......................................................................................................220

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8 LIST OF TABLES Table page 2-1 Nucleic acid yields from isolation protocols.....................................................................34 2-2 Ranking of 4 best nucleic acid extraction protocols..........................................................36 2-3 DNA yields (g DNA) from ten strawberry genotypes.....................................................38 2-4 Impact of interactions between macer ation methods and incubation temperatures on DNA yield and purity.........................................................................................................38 3-1 Number of simple sequence repeats (w ith a minimum of 5 repeats) observed in Fragaria vesca genomic sequence.....................................................................................54 3-2 Different types of dinucleotide and trinucleotide repeats observed in Fragaria vesca genomic sequence..............................................................................................................54 4-1 PCR primers designed for amplificati on of micro-colinearity-inferred putative intergenic fragments...........................................................................................................72 4-2 PCR primers that allowed amplicon generation................................................................73 4-3 Overview of insertions and deletions detected through alignment of all sequenced clones......................................................................................................................... ........80 5-1 PCR primer pairs and amplifica tion conditions used in this study....................................94 5-2 Fragment sizes of parental amplic ons digested with restriction enzymes.........................95

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9 LIST OF FIGURES Figure page 2-1 Design of incubation temperat ures and durations experiment...........................................33 2-2 Effect of incubation temp erature and time on DNA yields...............................................37 2-3 Effect of tissue-tobuffer ratios on DNA yields.................................................................37 2-4 Relationships between DNA yield, tissue-to -buffer ratios, and sample amenability to amplification by PCR.........................................................................................................39 2-5 DNA contamination by carbohydrate (estimat ed by the ratio between absorbance at 260nm and 230nm) and its influence on PCR outcome....................................................40 2-6 Effect of interactions between maceration method and incubation temperature in the absorbance at 220-340nm..................................................................................................41 2-7 The effect of Polytron homoge nization on nucleic acid recovery.....................................41 3-1 Flowchart of genomic D NA sequence annotation scheme................................................52 3-2 Diagram of two fosmid inserts of variab le length, with their putative proteins and Simple Sequence Repeats (SSRs)......................................................................................53 3-3 EST classes identified by homo logy searches between large genomic F. vesca sequence and Rosaceae ESTs............................................................................................54 4-1 An idealized GPH locus.................................................................................................... .70 4-2 Fragaria species and their geogra phical locations............................................................70 4-3 GPH design upon comparison between strawberry ESTs and Arabidopsis database.......71 4-4 Subset of the alignment of GP H5 octoploid and diploid clones........................................76 4-5 Diagrammatic representation of alignment of full GPH23 clones.....................................76 4-6 EcoRI Restriction patterns observed for GPH10 clones from the octoploid Strawberry Festival, indicating four different allele classes...........................................77 4-7 GPH10 clones, 4 alleles from the octoploid Fragaria ananassa ...................................77 4-8 Subset of GPH72E18 alignment displaying SSR polymorphisms....................................78 4-9 Cladograms of F. ananassa and diploid alleles for six independent GPH loci..............79

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10 5-1 Fosmid 40M11 with primers designed on exons of FGENESH-predicted genic regions........................................................................................................................ ........94 5-2 Amplicon restriction patterns for GPHs 34D20 and 72E18..............................................96 5-3 Gene-Pair Haplotypes assigned to linkage groups of the reference Fragaria map...........97

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STRUCTURAL GENOMICS OF Fragaria WILD AND CULTIVATED STRAWBERRIES By Denise Cristina Manfrim Tombolato August 2007 Chair: Kevin M. Folta Major: Horticultural Science The extensive phenotypic variability and co mplex genetic makeup of the cultivated strawberry Fragaria ananassa permits advances in plant improvement, a factor breeders have exploited to great benefit. Howeve r, the introgression of specific ch aracters is complicated due to the cumbersome genetics and limited knowledge of genome structure an d function of genes relevant to traits of interest The present study represents the first genomics-level insight into strawberry genome structure and explores the hy pothesis that a new type of molecular marker, the Gene-Pair Haplotype represents a transferable marker that may hasten linkage mapping in the diploid and octoploid strawberry. My research presents the findings of four relate d research activities. First, an efficient and unified method for genomic DNA isolation was derived from over 100 experimental tests and conditions. Next, 1% of the Fragaria genome was sequenced and functionally annotated, using a bioinformatics approach and computational tool s. Over 120 kb of intergenic regions were sequenced using the Gene-Pair-Haplotype approach, allowing for some initial relationships to be formulated concerning the diploid subgenome co ntribution to octoploi d strawberry. Finally, Gene-Pair Haplotypes were used to a dd a suite of allele s to the growing Fragaria linkage map. These findings provide a starting point for fu rther analyses of th e strawberry genome.

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12 CHAPTER 1 STRAWBERRY AND THE GENOMICS ERA Introduction The cultivated strawberry, Fragaria ananassa Duch belongs to the family Rosaceae as do the also economically important crops rose, ap ple, pear, peach, cherry, plum, raspberry, and almond. Linnaeus named the genus Fragaria due to its fragrant properties, whereas the odor, taste and berry shape was thought to be similar to pineapple, or ananas, in Latin (Darrow, 1966). In 1765, the F. ananassa parentage was proposed by An toine Nicolas Duchesne, whose father worked at the Court of Louis XV (Darrow, 1966). F. ananassa was first observed in several countries in Europe si nce the 1750s and it originated from a spontaneous hybridization between F. virginiana and F. chiloensis, both from the American continent. F. virginiana is thought to have been imported to Europe by tw o routes (Wilhelm and Sagen, 1974): to France by the explorer Jacques Cartier during his first e xpedition to the Quebec Ca nadian Province in 1534; and to England, by Thomas Hariot, who vis ited the New Found Land of Virginia in 1588. Later, in 1714, F. chiloensis was taken to France by the engineer Amde Franois Frzier. During his mission to study the defense fortificat ions of Chile and Peru, Frzier noticed the large-fruited berries at Concepcin, Chile, and collected several plants to take back to his country (Darrow, 1966). The result of the accidental cross between the two Fragaria species was the basis for the creation of the fruit cultivate d and appreciated thr oughout the world today. Profitable strawberry production is challenged by several factor s: diseases, pests, market competition, and, arguably most importantly, by the phase-out of methyl bromide. This fumigant is considered essential for the production of ma ny crops, including strawberry (Rosskopf et al., 2005), but because methyl bromide has great strato spheric ozone depletion ability, the Montreal Protocol mandates that its use be reduced (Anonymous, 1998). Although traditional plant

PAGE 13

13 breeding has been used to remedy several of th e above-mentioned challenges, the knowledge of the Fragaria genome structure may streamline the vari ety improvement process, potentially permit discovery of gene function, and ultimatel y lead to more diverse and hypothesis-based solutions to traditional and contemporary problems not only for the strawberry but also for other Rosaceous crops. Molecular Markers for Strawberry The cultivated strawberry has a complex ( 2n=8x=56) (Ichijima, 1926), (Fedorova, 1946) and poorly understood genome. Despite strawberrys commercial value of 1.4 billion dollars as a fruit crop (Folta et al., 2 005), substantial knowledge of Fragaria structural genomics before this project was virtually nonexistent. Sequence information facilitates the development of molecular markers that can be used for marker-assisted selection (Haymes et al ., 1997), (Van de Weg, 1997), (Albani et al., 2004), (Sugimoto et al., 2005) (Haymes et al., 2000), (Lerceteau-Khler et al., 2002), clone characterization in germplasm ba nks (Harrison et al., 1997), (James et al., 2003), identification of cultivar proprietary (Aru lsekar et al., 1981), (B ringhurst et al., 1981), (Gidoni et al., 1994), (Nehra et al., 1991), (B ell and Simpson, 1994), (H ancock et al., 1994), (Levi et al., 1994), (Parent and Page, 1995), (L andry et al., 1997), (Degani et al., 1998), population genetics studies (Deg ani et al., 2001), (Harrison et al., 1997), (Graham et al., 1996), (Arnau et al., 2003), (Hadonou et al., 2004), and construction of genetic linkage maps (Williamson et al., 1995), (Yu and Davis, 1995), (Davis and Yu, 1997), (Deng and Davis, 2001), (Lerceteau-Khler et al., 2003), (Sargent et al., 2003), (Sargent et al., 2004). Pioneer molecular markers were based on polym orphisms observed on punctual loci or the whole genome: isozymes and intron length po lymorphism; Randomly Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Poly morphism (RFLP), and Amplified Fragment Length Polymorphism (AFLP). More recently, Si mple Sequence Repeats (SSRs) have been

PAGE 14

14 employed to address the challenge of marker tran sferability (Monfort et al., 2005), (Nourse et al., 2002), (Ashley et al., 2003). The present work disc usses the creation of a novel marker type that, in addition to responding to the transferability necessity of modern markers, also attaches functional information to markers generated. The Genomics Era Genomics has been defined as the study of all nucleotide sequences, including structural genes, regulatory sequences, and nonc oding DNA segments, in the chromosomes of an organism. (The American Heritage, 2006) The complexity of plant genomes began to be investigated in the mi dto late-1970s using quantitative DNA reassociation ki netics (i.e. Cot curves) (Gol dberg, 2001). It was determined that plant genomes had families of repetitive se quences and that these repeats varied in copy number and arrangement in the genome (Flavell et al., 1974), (Goldberg, 1978). By the end of the 1970s, with the ability to construct cDNA clones, there was the surprising finding that the coding regions of e ukaryotic genes were interrupted by introns (Gilbert, 1978 ), what led to i nvestigation of posttranscriptiona l splicing mechanisms (Jeffreys and Flavell, 1977). The first plant gene was cloned in 1979 (Bedbr ook et al., 1980), demonstrating that plant DNA was not different from the DNA of other organisms and th erefore could be manipulated using the same enzymes, cells, and vector system s. The result was the construction of both plant genomic and cDNA libraries of many plants and organs (Goldberg, 2001). The demonstration that Agrobacterium tumefaciens tumor DNA (T-DNA) integrates into the chromosomes of plant cells (Chilton et al ., 1977) created the opportunity to generate transgenic plants, the first one being sunflower cells expressi ng bean phaseolin seed storage protein gene (Murai and Sutton DW, 1983). In add ition to being a vector to foreign genes, T-

PAGE 15

15 DNA began to be used to generate transformed Arabidopsis lines with mutant phenotypes to identify and clone important plant genes, such as genes involved in th e control of meristem identity and hormone perception (Feldma nn, 1991), (Feldmann and Marks, 1987). A second method to clone plant genes was devised upon the isolation of the Ac and Ds transposable elements (Fedoroff et al., 1983). The beginning of the sequencing era can be attributed to the determination of a bacteriophage RNA gene sequence in 1972 (Min Jou et al., 1972). The first whole-genome sequencing was also from a virus, Haemophilus influenza completed in 1 995 (Fleischmann et al., 1995), whereas a draft of the Human Genome was released in 2001 (Venter JC, 2001). The first plant genome sequenced was Arabidopsis thaliana completed in 2000 (The Arabidopsis Genome Initiative, 2000). In 2007, approximately 2300 sequencing projects are being carried out or completed, of which about 130 are plant geno mes, according to the Genomes Online database (Liolios et al., 2006). Our collaborators in this Fragaria genomics project have successfully completed 1% of the F. vesca genome. Recently, Malus (apple) was selected for full sequencing by an Italian sequencing effort. Peach also wi ll be sequenced through a US Department of Energy initiative. Although these genomes are much larger than the strawberry genome, their completion will have important ramifications to Fragaria as annotation will provide a list of components that are similar to those in strawber ry. The work presented here is a complementary effort to those in other rosaceous crops, providi ng an initial glimpse into the genome of one of the worlds most prized horticultural crops.

PAGE 16

16 CHAPTER 2 DNA EXTRACTION FROM RECALCITRANT SPECIES Introduction Strawberry ( Fragaria ananassa ) is an important crop worldwide, and it supports many regional economies in the United States. However, relatively little is known about the genes that govern agriculturally important traits or their expression. Contemporary genomics tools have the potential to accelerate study of st rawberry and bring additional resolution to strawberry gene form and function. Strawberry belongs to the genus Fragaria a genus that includes a number of species of varying ploidy with a small haploid genome size. These facets make strawberry an excellent candidate for genomic studies repres enting the Rosaceae family. Because it is easily transformable, it is particularly well suited for translational-genomics studies. Any genomics effort, whether tran slational, structural or func tional, is generally dependent on a reproducible and effective means to isolat e quality genetic mate rial. Although protocols have been streamlined over the last several decad es, it is challenging to isolate large amounts of quality DNA from strawberry (Manning, 1991; Po rebski et al., 1997). A similar problem has been encountered in other species Plants like cotton (Katterman and Shattuck, 1983; Dabo et al., 1993; Chaudhry et al., 1999; Li et al., 2001), sugarcane (Aljanabi et al., 1999), conifers (Crowley et al., 2003), tomato (Peterson et al., 1997), gr ape (Collins and Symons 1992; Lodhi et al., 1994), and the rosaceous chestnut rose (Xu et al., 2004) have been reported to be recalcitrant to DNA extraction. The high content of polysacch arides and polyphenols either limit DNA isolation or inhibit downstream enzymatic reactions. The DNA Extraction Procedure A typical DNA extraction is accomplished by three basic steps: lysis of the cell, removal of proteins, and separation of nucle ic acids from other cellular compounds. Cell lysis is easily

PAGE 17

17 achieved by removal of membrane li pids with detergents such as sodium dodecyl sulfate (SDS), triisopropylnaphthalenesulfonic acid (TIPS) (Bies and Folta, 2004), and N-laurylsarcosine (sarkosyl) when extracting DNA from bacterial or animal cells; how ever, because plants have a solid cell wall in addition to the cellular me mbrane, solvents alone are not enough to expose organelles, and mechanical force must be applie d. Samples can be sonicated but generally are either treated with ethyl ether (Watson a nd Thompson, 1986; Peterson et al., 1997; Folta and Kaufman, 2000; Peterson et al., 2000), lyophilized or frozen in liquid nitrogen to make the material more friable prior to manual grinding. Additional homogenization is performed with a Polytron or comparable tissue disruptor. Cell lysis is carried out either as a single step, breaking open all cellular compartments simultaneously, or in a stepwise fashion, first rupturing outer membranes to expose the nucleus, then solubilizing the nuclear envelope to free nuc leic acids. The first membrane lysis is induced by osmotic pressure generated by 0.35M sorbitol (Fulton et al., 1995; Ha nania et al., 2004) 0.35M glucose (Chaudhry et al., 1999) or Tr iton X-100 (which lyses chloroplasts and mitochondria, but does not solubilize nuclear DNA) (Watson and Thompson, 1986; Peterson et al., 1997), while the second lysis is performed by detergents and ethylen ediaminetetraacetate (EDTA). During this perturbation of the cell, DNA-degrading enzymes must be inhibited, which is accomplished by manipulating pH and removing divalent cations. Since DNAses act at pH 7.0, Tris is added to raise the pH to between 7.5 and 8.0. The chelation of divalent cations (Ca2+, Mg2+) by EDTA prevents the activity of metal-dependent enzymes. Cellular and histone proteins can be di ssociated by SDS (Kay and Dounce, 1953), proteases, chaotropic agents, chloroform (Sevag et al., 1938 ), and phenol. Because phenol solubilizes proteins (Cohn and C onant, 1926), it has been used to deproteinize preparations of

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18 carbohydrates (Westphal et al., 1952; Westphal and Jann, 1965) and nucleic acids (Kirby, 1956). Chaotropic agents denature protei ns by increasing the solubility of nonpolar substances in water (Voet et al., 1998). Hofmeister (Hofmeister, 1888) de fined the series of an ions and cations with increasing protein destabilizing prop erties when he measured the concentration of various salts needed to precipitate proteins from whole egg white (translated by (Kunz et al., 2004)). According to the Hofmeister series, urea, gua nidinium, thiocyanate (Sawyer and Puckridge, 1973) and perchlorate (Wilcockson, 1973) are ex tremely chaotropic agents. Thus, high concentrations of urea (Settles et al., 2004), guanidine hydrochloride (Logemann et al., 1987), and guanidine thiocyanate have been used in isolation of RNA (Cox, 1968; Chomczynski and Sacchi, 1987) and DNA (Cho mczynski et al., 1997). Chemical or physical means such as preci pitation by isopropanol, et hanol, butoxyethanol (Manning, 1991), acetone (Vogelstein and Gillesp ie, 1979), adsorption to silica (Vogelstein and Gillespie, 1979), paramagnetic particles (Anony mous, 1980, 2001; Koller and al., 2001), and ion exchange resin (QIAGEN Anion-Exchange Resin ma nual) can be utilized to retrieve DNA from solution. The resin is coated with diethylethanolamine (DEAE) and DNA recovery is due to interaction between negatively charged phosphate s of the DNA backbone and positively charged DEAE groups. In the case of silica columns, DNA is recovered from solutions because it tends to adsorb to silica in the presence of chaotropic salts, su ch as sodium iodide (NaI) (Vogelstein and Gillespie, 1979), guanidine thiocyanate, and guanidine hydrochloride. The binding capacity depends on the solutions ionic strength and pH, being higher in concentr ated solutions and at pH<7.5 (GeneClean Manual). Silic a columns have been used to eliminate polysaccharide contaminants, and the ratio A260/230 increases as polysaccharides are removed (Abdulova et al., 2002).

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19 DNA Extraction from Plants Pioneer methods to isolate genetic material of plants used DNA-rich matter such as germ tissue (Lipshitz and Chargaff, 1956; Shapiro and Chargaff, 1960). Early attempts to extract DNA from leaves resulted in degraded product due to the extreme pHs used by the procedure for removal of RNA (Thomas and Sherratt, 1956). Th e currently most used protocol for plant DNA isolation, developed by Murray and Thompson (Murray and Thompson, 1980), takes advantage of the selective precipitation of DNA by cetyltrimethylammonium bromide (CTAB), a phenomenon observed by Jones during DNA isolati on from bacteria (Jones, 1953). CTAB is a cationic detergent that, in high ionic strength solutions (e.g. >0.7M NaCl), complexes with proteins and non-acidic polysaccharides, whereas at low ionic strength it precipitates nucleic acids and acidic polysaccharides, leaving proteins and neutral sugars in solution (Sambrook and Russell, 2001). Multiple variations of Murray and Thompsons protocol have been used by researchers to adapt the original process to di fferent plant species. A pr otocol designed by Doyle and Doyle (Doyle and Doyle, 1987) is also fre quently used for plant DNA extraction and is ultimately a variation of the Murray and Thomps on procedure. Doyle and Doyles protocol uses fresh tissue in place of lyophilized material a nd a higher concentration of CTAB and salt to compensate for the greater wa ter content of fresh tissue. Although CTAB is the reagent of choice to purify DNA from organisms that produce many polysaccharides (Sambrook and Russell, 2001), even high quantities of the cationic detergent seem insufficient to free DNA preparat ions from sugar contamination. In attempt to circumvent this problem, boric acid is added to the extraction buffer. Boric acid forms complexes with polyphenols at pH 7.5 (King, 1971) and wi th carbohydrates (Gauch and Dugger Jr., 1953), making these complexes more soluble. An add itional approach to avoi d co-purification of

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20 polysaccharides during DNA isolation is to differentially precipit ate the sugars by manipulating the 2-butoxyethanol conc entration (Manning, 1991). Cytoplasmic compounds come into contact with nuclei contents when cells are disrupted and the oxidized polyphenols covalently link to DNA (Loomis, 1974), restraining subsequent DNA manipulation (Katterman and Shattuck, 198 3). Reducing agents like -mercaptoethanol, dithiothreitol, ascorbic acid, sodium bisulfite, and diethylcarbamic acid can be added to the extraction buffer to inhibit the oxidation proce ss and protect DNA from quinones, disulfutes, peroxidases, and polyphenoloxydases. Polyvinyl pyrrolidone (PVP) and its insoluble, crosslinked form, PVPP (Gegenheimer, 1990), also protect DNA from phenolics and alkaloids by sequestering them. Additional approaches to avoid problems caused by phenolics like freezing tissue prior to homogenization (Katterman a nd Shattuck, 1983; Leutwiler et al., 1984), purification by cesium chloride gradient (T ravaglini and Meloni, 1962; Williamson, 1969; Murray and Thompson, 1980), and extraction of DNA from isolated nuclei (Hamilton et al., 1972; Katterman and Shattuck, 1983; Watson and Thompson, 1986; Peterson et al., 1997) have been used. As genomics tools become more common in stra wberry research, it is imperative to devise a standard protocol that is effective across cu ltivars and species of different ploidy levels. Examination of the literature on strawberry ( Fragaria spp.) indicates that the many published DNA isolation methods are not univ ersally transferable between cu ltivars or species. An optimal protocol should use readily available plant materi al (such as mature leaves), be inexpensive, rapid, reproducible, and have high yields of high molecula r weight DNA, amenable to downstream manipulation. Of all these traits, quality is most important, yield second in importance, followed by cost and ease of protocol.

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21 Material and Methods Thirty-three DNA extraction protoc ols, totaling 103 treatments, were tested using either lyophilized or liquid nitrogen-fr ozen leaf tissues. A broad ra nge of genotypes were tested, including tissue from F. nubicola F. vesca cultivars Yellow Wonder, Alexandria, and Hawaii-4, F. chiloensis CA 1367 F. virginiana NC 96-35-2 F. ananassa cultivars Sweet Charlie, Tristar, Camarosa, Quinault, Diamante, Strawberry Festival, and the laboratory transformation genotype LF9 (Folta et al., 2006). The detailed pr otocols can be found in Appendix A, whereas further below is a summary of the approaches adopted. When at least 15g of DNA were obtained, digestion of 5g of DNA with at least 2 separate restri ction enzymes were carried out. The uncut and enzyme treated samples were lo aded on 1% agarose gel for assessment of DNA quality (integrity and amenab ility to use of restriction enzymes), and correlation to spectrophotometric readings. Phenols are know n to absorb at 260nm as does DNA, and high readings may be attributed to the presence of phenols, particularly when the DNA pellet has brown coloration, caused by oxidation of pheno lic compounds (phenylpropanoid and flavonoids) to quinones (Loomis, 1974). To further test th e quality of the DNA preparations, PCR was carried out using primers for F. ananassa 18S ribosomal DNA. The primers (forward: 5 TAT GGG TGG TGG TGC ATG GC 3; reverse: 5 TTG TTA CGA CTT CTC CTT CC 3) were designed utilizing as sequence source the accession gi|18448|emb|X15590.1|FA18S. The fragment to be amplified by this primer pa ir is not large (510bp from cDNA, ~1kb from genomic) and should be easily amplified, since ma ny copies of ribosomal DNA are present in the genome. If a product was observed, a second set of primers (forward: 5 CAC TGC CAA GGA GCG TGG TG 3; reverse: 5 TCA GTA G GG CAG CTG ATG 3) targeting a single-copy region, the Leafy gene, was used to provide a more challenging test. This second primer pair was designed utilizing F. vesca Pawtuckaway sequence provided by our collaborator, Dr. Thomas

PAGE 22

22 M. Davis, and encompassed a 770-nucleotide regi on. Both PCR reactions were carried out for 35 cycles, with 55C as annealing temperat ure, and 1min as extension at 72C. The original CTAB protocol designed by Murray and Thompson (Murray and Thompson, 1980) is extremely laborious, requi ring a long centrifugation period in a cesium chloride (CsCl) gradient. Since the aim of this project was to develop a rapid, practical method to extract DNA, the CsCl step was omitted from all DNA extraction attempts. Further modifications of the protocol were tested systematica lly to pyramid the beneficial asp ects of each preparation into a unified and effective means to generate highquality DNA for downstream analysis as described bellow: CTAB was tested at 1, 2, 6, and 20% Inclusion of one or combinations of the following reagents to prevent DNA oxidation: 0.01% -1% sodium (bi)sulfite, 5mM ascorbic acid, 1-4% PVP EDTA concentration from 10mM (as pr oposed by Murray and Thompson) to 200mM Tris concentration ranged from 50mM (as in original protocol) to 200mM. The pH was adjusted to 8.0 by addition of HCl. In cases wh ere boric acid was used to adjust the pH, the Tris-borate solution was brought to pH 7.6 because at that pH, boric acid forms complexes with polyphenols The original protocol removes protei ns by treating the solution with 24:1 chloroform:octanol. Alternative deprot eination methods tested were: 25:24:1 phenol:chloroform:isoamyl alcohol, 1M sodium perchlorate, and 150 g/ml proteinase K DNA was recovered by either adsorption to silica, or precipitation by ethanol, isopropanol, 2-butoxyethanol, or 5M potassium acetate. In Murray and Thompsons original protocol, DNA is precipitated by decr easing salt concentration Attempts to remove water-soluble contaminan ts by adsorption to silica column (QIAGEN DNeasy kit) and by dialyses of DNA solution into TE pH 7.0 at 4C Instead of adding buffer subsequent to grindi ng the plant tissue, an additional tissue/buffer homogenization step was performed. An aliquot of the final buffer was used to either produce a tissue/buffer paste in the mort ar and pestle or Polytron homogenizer In place of the standard incubation in buffe r at 50-60C for 20-30 minutes, incubation was carried out at 4, 20, 42, and 65C for 0, 5, 30, and 60 minutes. In order to eliminate

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23 variability that may be induced because of the leaves of various ages, leaves were cut with a hole puncher, mixed, and split into 4 porti ons, one for each temperature treatment. Enough plant tissue was ground per temperature treat ment so that 2 experimental replicates for each time treatment were derived from a single test tube (see figure 2-1). In addition to variations of the CTAB protoc ol, other approaches adopted included use of the chaotropes 8M urea, 4M gua nidine thiocyanate (alone or in combination with 2% CTAB, simultaneously or sequentially); DNA isolati on using kits: QIAGEN DN easy Plant Mini Kit (charged resin-based), Molecular Research Center DNAzol Extra St rength (guanidine thiocyanate-based), Epicentre MasterPureTM Plant Leaf DNA Purificat ion, MoBio PowerPlantTM DNA Isolation Kit; 0.5% SDS, Tris-borate extracti on buffer; and crude and fine isolations of nuclei prior to DNA extraction. Five DNA extr action procedures, QIAGEN DNeasy kit, 2% CTAB, 2% SDS, 4M guanidine thiocyanate/1% sarkosyl, and 5% SDS/1% TIPS, were tested on Percoll gradient-isolated nuclei. Refer to table 2-1 for all the treatments. The amount of tissue necessary to obtain the highest DNA extraction efficienty was determined by keeping the volume of buffer consta nt at 5ml and varying the tissue weights at 50, 200, 500, and 1,000mg. Once the best tissue-to-buffer ra tio was determined, an attempt to extract DNA from 10 species within the genus Fragaria was made to test the universality of the method. Each treatment had 2 replicates for both experime nts. Expanded leaf tissue was ground in liquid nitrogen, added to the buffer, and the mixture was incubated at 4C for 5 minutes. An equal volume (5ml) of 24:1 chloroform:octanol were ad ded to the tubes after incubation, agitated, and centrifuged at 4,000rpm for 5 minutes. The aqueou s phase was transferred to a new tube, and nucleic acids precipitated by 1/10 volume of 5M NaCl and 7/10 volume of isopropanol. After a second centrifugation, the supernatant was decante d, the pellet air-dried, and resuspended in 500l water. RNAse was added to final concentr ation of 50g/ml. The solution was transferred to 1.5-ml tubes and DNA was precipitated as described above. The dry DNA pellet was

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24 resuspended in 200l water and DNA quantit ies were estimated by a NanoDrop ND-1000 spectrophotometer. Nucleic acids were extracted from 96 individuals that be long to a diploid Fragaria mapping population. Minimal quanti ties of lyophilized tissue were processed, ranging from 3 to 14mg (average = 6.44mg, standard deviation =1.98). Because the buffer volume was kept constant, there was an opportunity to furthe r study tissue-to-buffer ratios, under different conditions from those tested above. This time, tis sue was macerated in buffer after having been ground in liquid nitrogen and incubated at 65C for 1hour. The absorbance values at 230, 260, and 280nm were determined by a NanoDrop to ma ke inferences about nucleic acid purity. Absorbance ratios A260/A230 and A260/A280 are m easures of contamination by polyphenols or carbohydrates (Craigie and McLachlan, 1964; Lo gemann et al., 1987), cited by (Manning, 1991) and protein, respectively. The ultimate usefulne ss of each sample was determined by PCR with two primer pairs in separate reactions leafy primers amplify a short fragment of 770 nucleotides; 72E18 challenged amplification, for it is a relatively long fragment of 2622 nucleotides. Like primers for leafy primers 72E18 (Fb: GCT AGG GAA AAC AGC TCG TG; Rb: TGG GTT TGG TTT TGG GA T AA) were designed for F. vesca cv. Pawtuckaway and are transferable to F. nubicola Results The majority of the protocols tested either failed to render appr eciable amounts of DNA from mature plant leaf tissue, or yielded plenty of material th at was not amenable to further manipulations, such as restriction digestion or PCR (data not shown). However, a variable previously considered minor had an unexpectedly gr eat impact in the retrieval of nucleic acids: further maceration of tissue in extraction buffer. Most of these preparations do not separate DNA

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25 from RNA, so quantification is generally a comb ination of nucleic acids. This is important for two reasons. First, the RNA isola tion protocols for strawberry ar e principally revisions of DNA extraction methods. Those that yield high amount s of RNA also contai n proportionate amounts of DNA, and RNA is removed with selective Li Cl precipitation. In these preparations RNA and DNA recovery is generally parallel and so quantification of both as nucleic acids provides a general measure of DNA recovery. Also, in an a ttempt to identify an efficacious method, the step of removing RNA, and verifying its remova l would limit the number of protocols and experimental conditions that could be tested. Table 2-1 lists yields from th e different DNA isolation protoc ols described in the Appendix A. Different numbers of treatment replications and amounts of plant tissue were used in the DNA extraction attempts. Therefore, to allow compar ison between treatments, values for yield shown in the table are averages of replications, st andardized using 1 g of plant tissue as the denominator. Table 2-2 ranks the four methods th at had highest nucleic acid returns per g of tissue. Control samples were excluded from the calculation of averages. For example, T85 was a control in protocol 30tissue was not macerated in buffer. Because the factor in question was the formation of slurry due to maceration, T85 was excluded from the calculation of the average for slurry protocols. Although the strawberry protocol permitted ex traction of nucleic acids 10 times greater than CTAB-based methods, DNA obtained through the former protocol cannot be digested by restriction enzymes or PCR-amplified by primers for the 18S ribosomal DNA. The DNA remains intractable even after treatment with proteinase K and subsequent dialysis. Similar situations occurred with DNA extracted by CTAB/Tris-borat e or guanidine thiocyanate. Only after purifying the guanidine thiocyan ate prep utilizing the DNeasy Plant Mini kit, did the DNA

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26 become PCR-amplifiable. It is interesting to note that the differe nce in spectrophotometer readings before and after the pur ification was minor (treatments 8 versus 10), suggesting that the kit may be a viable alternative to oher me thods used to purify PCR-recalcitrant DNA. The 4th highest ranked protocol type in table 2-2 is in fact th e only one of the four listed that resulted in tractable DNA. Of the many CTAB protocols th at were investigated, the ones that required maceration of plant tissue in buffer cluster together at the top in terms of g of nucleic acid obtained per gram of tissue (presented later in Figure 2-6). Components of the Strawberry Protocol Because the strawberry protocol had such high yield relative to the other methods tested, attempts to determine the reason for its superior ity were made. The objective was to discover the variable responsible and incor porate it into a protocol that would yield DNA amenable to enzymatic reactions. The factors tested were: i, nucleic acid pr ecipitation by 2-butoxyethanol; ii, boric acid (rather than HCl) used to adjust th e pH of Tris for the extraction buffer; iii, second round of extraction from plant ti ssue after chloroform treatment; iv, dilution of upper phase with Na+ solution before DNA precipitation. Treatments T30-T37 (comparing precipitati ons by isopropanol against 2-butoxyethanol) verified that the latter has a detrimental effect on DNA pr ecipitation. Considering all 4 experimental variables, 65 to 200% more nucleic acids were r ecovered by isopropanol rather than by 2-butoxyethanol precipitation. The absolute importance of boric acid to nuc leic acid isolation has not been tested, though borate appears to contribute to hi gher in yields when in combination with other factors. In the extractions using guanidine thiocyanate, bor ate-containing buffer (T36) had on average 10x higher yield than HCl-containing bu ffer (T8, T9). However, this incr ease may be attributed to the different tissue-to-buffer rati os among treatments. A second comparison, this time between

PAGE 27

27 CTAB buffers, strengthens the argument for the contribution of borate: T30 (Tris-borate) versus T82 (Tris-HCl), where T30 had a tissue-to-buffer ratio = 16mg/ml and T82 had the ratio that was determined to be optimum for DNA extraction (i llustrated in figure 2-2) Perhaps borate was at least partially responsible for T30s 25x greater yield than with T82. When used in substitution to Tris, though, boric acid alone was not able to increase the retr ieval of nucleic acids. T38 (1M boric acid, no Tris) was a similar treatm ent to T30, but the yield was 60x lower. A second round of extraction from plant ti ssue increased approximately 50% the DNA recovery relative to a single incubation in ex traction buffer. T34 and 35 yielded 60 and 45% of single-extraction treatments T32 and T33, resp ectively. Although this may be a considerable increase, it is not the sole factor responsible for the dramatic advantage of the strawberry protocol (3 times higher yield than the 2nd highest ranked protocol). The dilution of the aqueous phase also plays an important role in th e recovery of nucleic acids. Observing the results for treatments T24, T27: no dilution; T25, T28: dilution by 2.5 volumes of Na+ solution (detailed in Appendix A); T 26, T29: dilution by 4 volumes, it became apparent that the 2.5 volumes were superior to the other two, in a ratio of 50:125:1 (no dilution : 2.5vol : 4vol). Optimization of the CTAB Protocol Protocols containing CTAB in the extraction buffer produced th e highest yield of tractable DNA. Therefore, an optimum protocol was devised to further investigate the following factors: leaf tissue state, incubation temperature and duration, tissueto-buffer ratio, leaf tissue maceration. Leaf tissue state DNA was extracted from the same mass of fr esh and lyophilized tissues. As expected, yield per gram of sample was generally higher fr om lyophilized samples. However, this likely is

PAGE 28

28 due to the higher number cells that contained in freeze-dried samples in comparison to the same weight of fresh tissue. While yi eld from T58 was not different fr om that of T59, increases of 73 and 50% were observed in T13-T16. There was concern that the lyophilization pr ocess might compromise DNA quality. This was addressed by running uncut genomic DNA on agarose gel, and the integrity of all lyophilized samples (T13, T14, T23, and T57) app eared preserved. Therefore, lyophilization may be a good solution for storing material that doe s not require immediate DNA extraction, but it is not indispensable Incubation temperature and duration Utilizing fresh Strawberry Festival leaf tissue, the effects of temperature and duration of incubation of tissue in extracti on buffer were investigated. The treatment that relinquished the most DNA was incubation at 65C for 1 hour (figur e 2-2), which is the treatment specified in most plant DNA extraction protocols. However, the resultant prepar ation at this temperature is atypically viscous, complicating mechanical and enzymatic downstream manipulations. Tissue-to-buffer ratio Tissue-to-buffer ratios were tested for four pr otocols (2, 5, 14, 23; ra tios and yields shown in table 2-1), and yielded inc onsistent results. For protocols 2 and 14, the lower the ratio, the higher the yield, whereas for protocols 5 and 23, the opposite was true. Since all of the ratios (10-200 mg/ml) tested did not use the same pr otocol, a last DNA extraction experiment was conducted using leaf tissue of Strawberry Festiv al. Volumes of extraction buffer were kept constant at 5 ml, whereas the treatments we re 50, 200, 500, or 1000 mg of fresh tissue. Each treatment included two replicates, and incubation was carried out at 4C for 5 min. Samples were treated with RNAse A, DNA was precipitated by isopropanol and resuspended in deionized

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29 water. Figure 2-3 illustrates the result of the optimization of th e tissue-to-buffer ratio, where the optimum observed was at 40 mg of fresh tissue per milliliter of buffer. Using the optimum tissue-to-buffer ratio determ ined in the experiment above (40 mg/ml), the procedure of extracting DNA w ith incubation at 4C for 5 min was tested on ten strawberry cultivars, 2 replicates each. S trawberry Festival was included as a control. DNA recovery was dependent of plant species and cultivars (table 2-3). Plants with rigid leaves, such as F. chiloensis and the more F. chiloensis -like F. ananassa Diamante had negligible yields. Perhaps solely grinding leaves in liquid nitroge n is not sufficient to break down the cells and expose contents to the ex traction buffer solvents. An attempt to determine the optimum tissue-to -buffer ratio for lyophilized tissue was made utilizing material from a Fragaria diploid mapping population. Tissue weights varied from 3 to 14 mg, with average of 6.8 mg and standard de viation of 2 mg. Tissue was macerated in liquid nitrogen and, subsequently, in extraction buffe r for approximately 30 s. Grinding in buffer was conducted until the material was the consistency of paste. Incubation was performed at 65C for 1 hour. No correlation between amount of tissu e processed and DNA rec overed was apparent (figure 2-4). PCR was performed using 1l of the extracted DNA at variable nucleic acid concentrations (40ng/l to 4.5g/l) and the pr imer pairs designed for the Leafy gene: FvLFYintron2F (5 CAC TGC CAA GGA GCG TGG TG 3) and FvLeafy3' (5 TCA GTA GGG CAG CTG ATG 3). Due to inability to PCR-amplif y 50% of the diploid mapping population samples, an effort was made to monitor for correlations between PCR outc omes and i, nucleic acid concentration in the sample (figure 2-4); ii, tissue-to-buffer ra tio during DNA extraction (figure 2-4); and iii, A260/A230 ratios (figure 2-5) that could be indicative of carbohydrat e contamination. The

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30 absorbance ratios at 260nm and 230nm wavelengths were grouped into seven categories, and the number of samples in each categor y is indicated in figure 2-5. No conclusive correlation be tween success of amplification reaction and any of the three variables cited above could be determined. Although not statistically analyzed, subjective evaluation indicated no need to apply statistical techniques. Su rprisingly, there was no pattern suggesting a relationship between template concentration and P CR amplification. This outcome indicates that other factors are c ontributing to inhibition of the pro cess. In an attempt to dilute a possible polymerase inhibitor, lower tissue-to-b uffer ratios were tested. However, no correlation between ratios and PCR outcome was apparent since all permutations were detected: amplification was observed for both low and high tissue-to-buffer ratios; lack of amplification was also observed for both low and high ratios. Regarding the A260/230, according to Manning (Manning, 1991), the ratio 1.8 indica tes the purest nucleic acid sa mple. From the samples that were classified in this category (47 samples out of 91), 2/3 of them were amenable to amplification. Amplification was also obser ved for both extreme A260/230 ratios: 0.6 and 6.2. Therefore, the ratio either is a poor estimator of polysaccharide inhibition, or the polymerase inhibition was caused by polyphenols or other indeterminate factors. These trials indicate that there is no simple measure that serves as an indicator of a samples potential to be used successfully in downstream applications. Tissue maceration method The processes of breaking leaf tissu e down solely in liquid nitrogen versus preliminary pulverization in liquid nitrogen w ith subsequent grinding in buffe r were compared. Formation of slurry by maceration of tissue in buffer not only increased the yield by many fold (table 2-4 A), but also permitted the extraction of allegedly purer DNA, indicated by the lower absorbance at 230nm (figure 2-6). The most pr ominent absorbance peak at 2 60nm was observed for samples

PAGE 31

31 that were processed at 60C and ground in buffer (figure 2-6). Samples macerated this manner and incubated at 4C appear to contain many polys accharide contaminants, as a peak is seen at 230nm. The desired A260:A230 and A260:A280 rati os are equal to 1.80. Samples that were ground in liquid nitrogen only and in cubated at 4C absorbed more at 230nm than 260nm (ratio = 0.61, table 2-4), indicating that they proba bly had low content of nucleic acids. Due to the extraordinary increase in DNA c ontent by the maceration procedure, several treatments combining speed (1/2, full ) and duration (5, 15, 30, 60, 120 seconds) of homogenization with a Polytron were invest igated. Incubations pos t-homogenization were carried out at 65C for 1hour. The more aggressive the trea tment, the higher the amount of DNA obtained (figure 2-7). None of the samples a ppeared degraded on 1% agarose gel, DNA was digestible by restriction enzymes and amenable to PCR amplification with Leafy primers. Discussion The profound effect on nucleic acid yield by the aggressive maceration method suggests that the cell wall plays a major role in preventing DNA is olation. This hypothesis is further substantiated by the lower DNA yields observed for genotypes that contain harder leaves with a glossy, conspicuous cuticle, such as F. chiloensis and Diamante (table 2-3). However, when the cell wall was removed prior to DNA extraction, DNA extraction from isolated nuclei did not present appreciable yields. It is possible that th e isolated nuclei were no t pure and therefore the number of nuclei used for DNA extraction was overestimated, explaining the low yield observed. Guanidine thiocyanate has been used in nucleic acid isolation for a variety of plants. The compound is known to act as protein denaur ant by breaking intramolecular hydrogen bonds (Kauzmann, 1954) and, therefore, it causes inhi bition of enzyme activity. We hypothesized that the lack of amplification by PCR and digesti on by restriction enzymes occurred due to the presence of this chaotropic sa lt in the DNA preparation. To test this hypothesis, two approaches

PAGE 32

32 were adopted to purify the DNA from the guani dine thiocyanate: DNA adsorption to a silica column and dialysis of the DNA preparat ion. DNA purified by the first method rendered tractable DNA, whereas DNA remained unsuited fo r enzymatic reaction after dialysis. When isolated by the strawberry protocol proposed by Manning, DNA was also in tractable even after treatment with proteinase K and dialysis. Therefor e, it is possible that the co-purified guanidine thiocyanate or other inhibitors are retained in the dialysis tube. A m odification of DNA during the extraction procedure was considered as a po ssible explanation to enzyme activity inhibition, but the fact that previously in tractable DNA purified by a silica column permits amplification by PCR refutes this idea. The disappearance of an absorbance peak at 230nm when incubation was carried out at higher temperatures (figure 2-6) may be explained by the solubilization of sugars. At lower temperatures, the sugars are present and are not so lubilized by the extraction buffer, therefore are carried throughout the remaining steps of the DNA extraction protocol. Th eir solubilization in the early phase favors produc tion of a purer product. When considered together it is clear that many variables have no effect on yield. Whereas many protocols alter CTAB concentration, Na co ncentration, method of precipitation, additional organic extraction and use of affinity matrices, it is clear that concurrent physical and chemical disruption of cells is the most critical parame ter in the generation of pure genomic DNA suitable for downstream manipulations.

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33 Figure 2-1. Design of incubation te mperatures and durations experiment. The scheme illustrated above was followed for each of the incuba tion temperatures of 4, 20, 42, and 65C. Samples for a specific temperature were ground and homogenized together to decrease random variation between time points.

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34 Table 2-1. Nucleic acid yields from isolation protoc ols. P: Protocol number as listed in Appendix A; T: Treatment number; Status: condition of leaves prior to DNA isolation. F: fresh, L: lyophilized; T/B: tissue-tobuffer ratio (mg of tissue per ml of buffer). n/a: not aplicable; Yield: g of nucleic acids obtai ned if 1 g of tissue had been used for DNA isolation P T Status T/B Yield Brief Description mgtissue gnucl ac /mlbuffer /gtissue 1 1 F 100 0 Nuclei crude isolation 2 F 200 0 Nuclei crude isolation 3 F 400 0 Nuclei crude isolation 2 4 F 100 3 PEG 5 F 100 1 PEG 6 F 10 235 PEG 7 F 10 232 PEG 3 8 F 200 112 Guanidine thiocyanate, newly expanded leaf 9 F 200 774 Guanidine thiocyanate, unexpanded leaf 10 F 200 96 T8 cleaned by QIAGEN kit 11 F 200 11 T8 cleaned by dialysis 4 12 F 1000 35 Guanidine thiocyanate, CTAB consecutively 5 13 L 20 450 Guanidine thiocyanate, CTAB simultaneously 14 L 200 750 Guanidine thiocyanate, CTAB simultaneously 15 F 20 260 Guanidine thiocyanate, CTAB simultaneously 16 F 200 500 Guanidine thiocyanate, CTAB simultaneously 6 17 L 66 0 DNAzol kit by Molecular Research Center, Inc 18 L 333 0 DNAzol kit by Molecular Research Center, Inc 19 F 66 0 DNAzol kit by Molecular Research Center, Inc 20 F 333 0 DNAzol kit by Molecular Research Center, Inc 7 21 F 70 15 Pine tree minus lithium chloride 8 22 F 400 30 Urea 23 L 50 580 Urea + antioxidants 9 24 F 15 5000 No dilution 25 F 15 15000 2.5vol dilution 26 F 15 120 4vol dilution 27 F 30 8200 No dilution 28 F 30 18800 2.5vol dilution (not amenable to restriction digestion, even after treatment with proteinase K and dialysis) 29 F 30 150 4vol dilution 10 30 F 16 5700 Tris-borate, isopropanol 31 F 16 3130 Tris-borate, 2-butoxyethanol 11 32 F 25 3515 1st extraction, isopropanol 33 F 25 1190 1st extraction, 2-butoxyethanol 34 F 25 2135 2nd extraction, isopropanol 35 F 25 545 2nd extraction, 2-butoxyethanol 12 36 F 16 4300 Guanidine thiocyanate/Tr is-borate, isopropanol 37 F 16 2600 Guanidine thiocyanate/Tris -borate, 2-butoxyethanol 13 38 F 20 90 1M Boric acid, no Tris 14 39 F 33 50 Epicentre kit

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35 Table 2-1. continued P T Status T/B Yield Brief Description mgtissue gnucl ac /mlbuffer /gtissue 40 F 100 15 Epicentre kit 41 F 333 5 Epicentre kit 15 42 F 635 0 Mo Bio kit 16 43 F 125 8.5 Qiagen DNeasy kit 17 44 F 2.5 150 Silica 45 F 25 40 Silica 18 46 F n/a 18 Isolated nuclei, Qiagen DNeasy kit 47 F n/a 5 Isolated nuclei, Qiagen DNeasy kit 19 48 F n/a 12 Isolated nuclei, CTAB 49 F n/a 14 Isolated nuclei, CTAB 20 50 F n/a 3 Isolated nuclei, SDS 51 F n/a 1 Isolated nuclei, SDS 21 52 F n/a 0 Isolated nuclei, guanidine thiocyanate 22 53 F n/a 0 Isolated nuclei, SDS, TIPS 23 54 F 14 0 Murray and Thompson + solid CTAB, ppt by low ionic strength 55 F 70 25 Murray and Thompson + solid CTAB, ppt by low ionic strength 56 L 14 0 Murray and Thompson + solid CTAB, ppt by low ionic strength 57 L 70 1300 Murray and Thompson + solid CTAB, ppt by low ionic strength 24 58 F 66 45 Murray and Thompson, 6% CTAB, ppt by low ionic strength 59 L 66 50 Murray and Thompson, 6% CTAB, ppt by low ionic strength 25 60 L 1.6 1250 Murray and Thompson, precipitation by ethanol 61 L 8 60 Murray and Thompson, precipitation by ethanol 62 L 16 100 Murray and Thompson, precipitation by ethanol 26 63 F 250 0 Murray and Thompson, 5% CTAB, ppt by isopropanol 64 F 250 1 Murray and Thompson, 5% CTAB, ppt by isopropanol 27 65 F 100 0 CTAB + SDS 66 F 100 0 CTAB + SDS 28 67 F 40 16 4C, 0min 68 F 40 80 4C, 5min 69 F 40 98 4C, 30min 70 F 40 69 4C, 60min 71 F 40 34 20C, 0min 72 F 40 28 20C, 5min 73 F 40 95 20C, 30min 74 F 40 142 20C, 60min 75 F 40 28 42C, 0min 76 F 40 41 42C, 5min 77 F 40 34 42C, 30min 78 F 40 57 42C, 60min 79 F 40 41 65C, 0min 80 F 40 25 65C, 5min 81 F 40 76 65C, 30min 82 F 40 211 65C, 60min 29 83 F 75 387 Unexpanded leaf 84 F 75 28 Expanded leaf

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36 Table 2-1. continued P T Status T/B Yield Brief Description mgtissue gnucl ac /mlbuffer /gtissue 30 85 F 100 92 Powder 86 F 100 400 Slurry 31 87 F 50 2476 Slurry, 4C 88 F 50 300 Powder, 4C 89 F 50 3048 Slurry, 60C 90 F 50 700 Powder, 60C 32 91 F 40 1400 2% CTAB 92 F 40 1450 6% CTAB 93 F 40 665 20% CTAB 33 94 F 40 660 No polytron 95 F 40 1000 speed, 5sec 96 F 40 940 speed, 15sec 97 F 40 1155 speed, 30sec 98 F 40 1605 speed, 60sec 99 F 40 955 Full speed, 5sec 100 F 40 975 Full speed, 15sec 101 F 40 1335 Full speed, 30sec 102 F 40 1455 Full speed, 60sec 103 F 40 2245 Full speed, 120sec Table 2-2. Ranking of 4 best nuc leic acid extraction protocols Average g nucleic acid/g tissue Treatments included in average calculation Protocol # Protocol type 11,750 T24, T25, T27, T28 9 Strawberry 4,415 T30, T31 10 CTAB with tris/borate 3,450 T36, T37 12 Guanidine thiocyanate 1,232 T83, T84, T86, T87, T89, T91-T103 29-33 CTAB with slurry

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37 0 100 200 300 400 500 600 700 4204265Temperature (C) 0 min 5 min 30 min 60 min Figure 2-2. Effect of incubati on temperature and time on DNA yi elds. The standard plant DNA extraction procedure of carrying out inc ubation at 65C for 1 hour displayed, as expected, superior yields to other in cubation time lengths and temperatures. 0 50 100 150 200 250 300 350 400 450 500 050100150200250 Tissue-to-buffer ratio (mg tissue/ml buffer) Figure 2-3. Effect of tissue-tobuffer ratios on DNA yields. The optimum ratio for DNA isolation was 40 mg of leaf tissue per ml of extrac tion buffer. The yield declined rapidly as more tissue was processed by the same volume of buffer.

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38 Table 2-3. DNA yields (g DNA) from ten strawb erry genotypes. Plant tissue incubation with the extraction buffer was carried out at 4C for 5min. Aver ages of 2 replicates, 200mg tissue each, extracted by 5ml buffer. Genotype g DNA/200mg tissue F. vesca cv Yellow Wonder 127 F. vesca cv Alexandria 59 F. virginiana 54 F. chiloensis 0.85 F. ananassa cv Diamante 0.65 F. ananassa cv Strawberry Festival 50 F. ananassa Laboratory Festival #9 52 F. ananassa cv Camarosa 100 F. ananassa cv Sweet Charlie 64 F. ananassa cv Quinault 55 Table 2-4. Impact of interacti ons between maceration methods and incubation temperatures on DNA yield and purity. The ratio between absorbance at 260nm and 230nm (A260/230) estimate contamination by polysaccharides, whereas the ratio A260/280 estimate contamination by proteins. Pure samples have both ratios equal to 1.80. Yield g DNA/50mg tissue A260/230 A260/280 4C 60C 4C 60C 4C 60C slurry 31 38 1.02 1.78 1.71 1.91 no slurry 3.8 8.8 0.61 1.46 1.67 1.95

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39 0 200 400 600 800 1000 1200 0246810121416 Tissue-to-buffer ratio (mg tissue per ml buffer) Figure 2-4. Relationships between DNA yield, tissueto-buffer ratios, and sa mple amenability to amplification by PCR. DNA was extracted utilizing lyophilized tissue from 94 F2 individuals from a Fragaria diploid mapping population. Th e range of tissue weights was 3-14mg, with average of 6.7mg and standard deviation of 2mg. Because the volume of extraction buffer was kept constant at 1ml, the tissue-to-buffer ratios also represent the amount of tissu e (in mg) processed per sa mple. Correlations between amount of tissue processed, tissue-to-bu ffer ratio, DNA yield, and PCR outcomes were not apparent. no amplification amplification

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40 01020304050 0.5-0.6 1.1-1.4 1.5-1.6 1.7-1.9 2.0-2.3 2.3-4.0 4.1-6.2Absorbance at 260nm / Absorbance at 230 nmNumber of samples observed No amplification Amplification Figure 2-5. DNA contamination by carbohydrate (estim ated by the ratio between absorbance at 260nm and 230nm) and its influence on P CR outcome. Absorbance at 230nm and 260nm wavelengths were observed for 94 sa mples from a genetic linkage mapping population. The A260/230 ratio was calculated for each sample and the ratio data were grouped into 7 categories, varying fr om 0.5 to 6.2. Most samples presented ratio in the 1.7-1.9 range (1.8 is the optim um for DNA purity from carbohydrates). However, even within the purest DNA category, amplification by PCR was not observed for 1/3 of the samples. Theref ore, contamination by carbohydrates may not be considered the sole responsib le for the polymerase inhibition.

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41 Figure 2-6. Effect of interacti ons between maceration method and incubation temperature in the absorbance at 220-340nm. The most de sirable product from a DNA isolation procedure has a peak at 260nm. A peak at 230nm indicates contamination by polysaccharides. The more aggressive m aceration method, combined with higher temperatures, appears to be the best combination of treatments. Figure 2-7. The effect of Polytron homogenizat ion on nucleic acid recovery. Leaf tissue was ground in liquid nitrogen and further blended with buffer by utilization of a Polytron. The full uniformization promoted by higher speeds and prolonged durations yielded the best results on DNA isolation. Full speed, 2min speed, 60sec Full speed, 60sec Full speed, 30sec speed, 30sec speed, 5sec Full speed, 15sec Full speed, 5sec speed, 15sec No Polytron slurry no slurry 60C 4C 4C 60C

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42 CHAPTER 3 PRIMARY ANALYSES OF Fragaria GENE DISTRIBUTION Introduction Although the cultivated strawberry genom e is complex and polyploid, its monoploid genome is particularly small and tractable (a pproximately 200 Mb (Folta and Davis, 2006)). When compared to other rosaceous species, the strawberry genome is exceptionally well suited for rapid elucidation of its sequence, leading to meaningful descriptions of gene distribution and content. Here, small portions of the genome may be sampled and annotated to describe the basis of the Fragaria genome. These studies may then be ex tended to other rosa ceous species or utilized in comparative genomics efforts. The goal of the research described in this chapter is to provide a basic description of the first expanses of the Fragaria genome. The sequences obtained originate from a fosmid library constructed by Dr. Thomas M. Davis. Individual fosmids were selected by hybridization to genes of interest, and some were randomly selected. These studies provide a primary characterization of the Fragaria genome, revealing an understanding of gene content and placement as well as other f eatures of the genome of strawberry. Genome annotation has been defined as t he process of taking the raw DNA sequence produced by the genome-sequencing projects and adding the layers of analysis and interpretation necessary to extract its biological significance and place it into the context of our understanding of biological processes. (Ste in, 2001) The first challenge to annotate any genomic sequence information is to discriminate between tw o types of sequences: coding (DNA sequences encoding a protein) and non-coding (DNA is not transcribed into R NA or it is transcribed but not translated into a protein). Regul atory sequences such as promot ers and enhancers are examples of non-coding DNA sequences. Other non-coding DNAs are transfer RNA, ribosomal RNA,

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43 small RNAs (snoRNAs, microRNAs, siRNAs, piRNAs ), and long RNAs (Xist, Evf, Air, CTN, PINK). The second challenge for annotation is to as certain or predict gene function, how gene products might interact, and how they are re gulated (Salamov and Solovyev, 2000). Gene finding can be accomplished by similarity-based or ab initio gene prediction software. Similarity is defined by the NCBI glossary as the extent to which nucleotide or protein sequences are related. Similarity-based algorithms provide informa tion on alternative transcription (Li et al., 2006), translation start sites, and s licing and are more specific than ab initio. However, the latter is more sensitive than the former because it does not bias findings based on prior descriptions (Birney et al., 2004). Similarity-based algorithms like GeneWise (B irney et al., 2004) predict genes by testing putative translation products fo r similarity to known proteins A nucleotide comparison against cDNA, to an expressed sequence tag (EST), or a protein database using the Basic Local Alignment Search Tool (BLAST) are also simila rity-based gene predic tions Non-coding rRNAs are also identified using this approa ch (Stein, 2001). In contrast, the ab initio approach attempts to predict genes from sequence data without pr ior information on gene characterization. Most gene predictors attempt to define a gene us ing neural networks (modeled according to the learning process in cognitive systems), rule-based systems (algorithms that use an explicit set of rules to make decisions), or hidden Markov mo dels (HMMs). HMMs are statistical algorithms typically utilized in natural language processing. In gene prediction, they are trained with known gene structures (Stein, 2001; Yandell and Majo ros, 2002). A Markov model is a statistical model in which the system being modeled is assumed to be a Markov process, i.e., a stochastic

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44 (random) process in which the condi tional probability distribution of future states of the process depends on previous states. While in the Markov model one or more states can be directly observed, in the hidden Markov model, they cannot. H MMs are popular because they are relatively simple, and efficient methods that exis t for training and testing HMMs, these being the Baum-Welch and the Viterbi algorithms respectively (Mark D. Skowronski, personal communication ). For a review on HMMs, refer to Rabi ner, 1989 (Rabiner, 1989). Examples of ab initio HMM gene prediction software are GenS can (Burge and Karlin, 1997), GeneMark (Besemer and Borodovsky, 1999), and FGENESH (S alamov and Solovyev, 2000). When used to annotate the rice genome, FGENESH was more sens itive and more specific than GeneMark and GenScan (Yu et al., 2002). Plant genomic annotation mechanisms gained favor in the year 2000, shortly after the completion of sequencing of Arabidopsis thaliana a widely used genetic, developmental and physiological model for plants (The Arabidopsis Genome Initiative, 2000), followed by rice in 2002 (Yu et al., 2002). The in itial annotation of the Arabidopsis genome was submitted by numerous centers, each of them utilizing their own annotation method and terminology. The genome has been re-annotated and classified us ing Gene Ontology terms as a solution to the cumbersome handling of the information that ha d resulted from non-centr alized annotation (Haas et al., 2005). Since the completion of the first draft of th e rice genome, sequencing of many plants has progressed: high-quality finishing of rice and deep draft coverage of maize, alfalfa ( Medicago truncatula the model legume), tomato ( Lycopersicon esculentum ) (National Plant Genomics Initiative, 2002), and black cottonwood ( Populus trichocarpa ) (Tuskan and Difazio S, 2006). Despite the high commercial value of strawberri es, there is extensive more nucleotide sequence

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45 information for the above-mentioned species than for Fragaria The availability of strawberry nucleotide sequences was so scarce in 2004 that, if one searched for Fragaria in public databases, only 58 gene sequences were retrie ved (Folta and Davis, 2006). In 2007, this number jumped to over 20,000 sequences, of which 50% are Expressed Sequence Tag (EST) sequences. Collaborative work between the laboratories of Drs. Thomas M. Davis (University of New Hampshire), Kevin M. Folta (University of Fl orida), Jeffrey L. Bennetzen (University of Georgia), and Phillip SanMigue l (Purdue University) have added an additional 50 genomic DNA sequences, constituting slightly less than 2 mega bases of genomic information. The sequences are derived from a Fragaria vesca Pawtuckaway genomic libr ary and represent 1% of F. vesca s 200Mbp haploid genome (Folta and Davis, 2006). Due to its minute genome size and to the facts that F. vesca is the most geographica lly predominant diploid Fragaria species (Folta and Davis, 2006) and it is a plausible ancestor of the cultivated, octoploid strawberry (Ichijima, 1926; Davis and DiMeglio, 2004), this diploid serves as a valuable model for development of molecular markers and comparisons amongst several Fragaria species, as well as other genera of the Rosaceae family. This study aimed to annotate the newly sequenced parcels of the F. vesca genome. This represents the first opportunity to explore th e gene distribution and the composition of the Fragaria genome, which, at 200 Mbp, is comparable to the 157 Mbp (Bennett et al., 2003) genome size of the model plant A. thaliana Materials and Methods Dr. Thomas M. Davis at the University of New Hampshire used fosmids (CopyControlTM pCC1FOSTM from Epicentre) as vectors to produce a F. vesca genomic library with 8x coverage. The theory is that if the genome was dige sted into 35kb fragments, approximately 45,000 colonies would be necessary to represent th e 200Mbp haploid genome 8 times. Fosmid vectors

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46 were developed by Kim et al. (Kim et al., 1992) to address undesirable recombination during cloning in multicopy cosmid vectors. Due to the single-copy F-factor replicon, DNA inserted into fosmid vectors underwent a lower rate of rearrangements a nd deletions than did fragments inserted into cosmids. In order to annotate the newly available F. vesca sequence, a complement of ab initio and similarity-based approaches was utilized. Prelim inary screening for putative genes was executed by using the gene prediction so ftware FGENESH (accessible at http://www.softberry.com) for each of 26 fosmid insert sequences, using Medicago as the gene model. Subsequently, a series of different types of sequence similarity sear ches were performed using BLAST algorithms (http://www.ncbi.nlm.nih.gov/BLAST/), as illustrated in figure 3-1. The amino acid sequences from each gene predicted by FGENESH were used as query sequences against the non-redundant protein sequences database for all organisms using the BLASTP algorithm. Significant similarities be tween a query sequence and a sequence in the database, termed hits, were indicated by an expectation value (E value) lower than 10-15. (The lower the E value, the more significant is the score because the E value ultimately represents how likely two sequences are of being sim ilar by chance alone.) The threshold of 10-15 was defined based on thresholds used in the Arabidopsis genome annotation (The Arabidopsis Genome Initiative, 2000), where BLASTP E values < 10-20 and 10-10 were adopted to identify protein families and functional roles betw een different organisms, respectively. The BLASTP results that produced significant hits were used to guide the subsequent BLAST interrogations because they determined which nucleotide fragments should be further analyzed. Though the entire 30-45kb sequence could c onceivably be analyzed at once, it is more convenient to do the analysis in sequence par cels. The response to a BLAST submission of

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47 sequences larger than 12kb may require protracted time frames and the process may get aborted before the result is retrieved (T. M. Davis, personal communication ). A second reason to perform searches in parcels is that if two genes are c ontained in the large query nucleotide sequence and one of them has very high similarity to mo re than 100 hits, this condition may mask the similarity results to the second gene, appearing as if the seco nd gene was non-coding sequence. Similarity searches with BLASTX were pe rformed using sequence segments for which BLASTP detected amino acid matches. The translated nucleotide query was delimited to sequence fragments of 8kb whereas the non-redunda nt (nr) amino acid database against which the F. vesca sequences were compared was confined to green plants (green algae and embroyphytes) Viridiplantae. BLASTX was ca rried out to determine coding sequence orientation, to assign tentative gene identificati on and function to the query sequence, and to note the accession and locus tag numbers for the best Arabidopsis thaliana orthologs. The Arabidopsis loci are sequentially tagged according to their physical position in the genome. Therefore, the tag numbers could be us ed to assess microcolinearity between Arabidopsis and F. vesca. The BLASTN algorithm was utilized in separa te searches against the EST and the nr nucleotide collection databases. The query seque nces originated from fragments for which a gene had been predicted by FGENESH. EST databa ses searched were delimited to the Rosaceae, in an attempt to detect homologs (sequences that display similarity due to their shared ancestry) and the best Fragaria, Malus, Prunus, Rubus, and Rosa hits were noted. When no identities were detected within this botanical family, the search was expanded to the Viridiplantae database to detect ESTs that would facilitate detection of genes in the genomic sequence. BLASTN against the nr database was executed to detect repe titive elements and nontranslated sequences

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48 features such as rRNA, tRNA, and was also usef ul to detect duplications within the query sequence. If two different regions from a single query were similar to single subject from the database, that indicated a dup lication in the fosmid insert sequence under investigation. To address the sensitivity aspect of the FGENESH gene predictor software, a second search utilizing the BLASTN algorithm was carri ed out. This time, the query sequences were 812kb fragments of genomic sequence (regardless of whether or not genes were predicted in that segment), compared against Rosaceae ES Ts. The objective was to determine if Medicago suffices as a gene model for gene prediction in Fragaria A survey of the simple sequence repeats (SSRs) present in the newly accessible F. vesca sequences was carried out and their location, composition and predominance were noted. The tool used, SSRIT (Temnykh et al., 2001), is avai lable online at the Gram ene website: http:// www.gramene.org/ db/searches/ssrtool. Results The average fosmid insert fragment si ze was 35kb and FGENESH predicted 235 genes from the 26 fosmid insert sequences. Of the total number of nucleotides, 42% were predicted to belong to genic sequences. A list of the numbe red predicted genes a nd their corresponding BLASTX results is available in Appendix B. The software specificity wa s 55%, since out of the 235 genes predicted, 129 had hits in the ami no acid database having as threshold E < 10-15. Enzymes related to mobile elements lik e transposase, integrase, polyprotein, retrotransposon polyprotein, transcriptase, and reverse transcriptase were putatively present ubiquitously: 14 out of 26 fosmids contained at le ast one of those types of enzymes. In some cases, several of these enzymes were present in tandem, as depicted for fosmid 18A19 in figure

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49 3-2. The second fosmid diagrammed in figure 3-2, fosmid 34D20, contained putative proteinencoding sequences, including inverted repeat s of a gene next to a transposase. Expressed Sequence Tags (ESTs) ESTs facilitate genome annotation (The Ar abidopsis Genome Initiative, 2000) because they are strong evidence th at a sequence is transc ribed. In the case of Fragaria, only a small percentage of protein hits was supported by EST h its (32 of 129), exacerba ting the need for more rosaceous ESTs. Three classes of ESTs were id entified (figure 3-3): i, those that displayed identity to predicted, putative protein-enc oding genomic sequence; ii, those that were FGENESH-predicted genes, but fo r which there was no protein hit; and, more interestingly, iii, those that were identifie d spanning DNA sequences for which no ORF was predicted. Simple Sequence Repeats (SSRs) Due to their widespread presence, SSRs have been used to construct a linkage map in diploid strawberry (Sargent et al., 2004). Here, SSRs were iden tified in all fosmid insert sequences, except three: 11D02, 15B13, and 32L07. It is interesting to note that these fosmids putatively contain plastid and R NA genes and belong to the 50% cl ass that did not contain any putative transposon-related enzymes. A total of 195 SSRs containing at least 5 motif repetitions were identified. Of the nearly 4,000 nucleotides contained in the SSRs, 71% occurr ed in regions that were predicted to be intergenic. The great majority (92%) of the repe ats were dinucleotides (t able 3-1). The numbers of times a specific motif was observed are listed in table 3-2. Discussion Amplification of repetitive elements, together with polyploidy, are the mechanisms responsible for genome expansion (Bennetzen and Kellogg, 1997). Evolutionary mechanisms for genome downsizing also exist, though they are less well characterized. Bennetzen et al.

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50 (Bennetzen et al., 2005) proposed that retrotransposon removal as well as small deletions caused by unequal homologous recombination and ille gitimate recombination, lead to genome shrinkage. Grasses like rice, maize, sorghum (Be nnetzen et al., 1998), and wheat (Li et al., 2004) are known to have large gene-empty regions and abundant transposons in the intergenic sequences of gene clusters (Barakat et al., 1998). Fosmid insert 38H05 appe ared to be one such gene-empty space, since the only similarity de tected between its 32kb sequence and the protein database was to polyprotein, which comprised on ly a small portion of the fosmid sequence. The pattern of gene distri bution was more similar to Arabidopsis than to grasses. Arabidopsis has been determined to have 15 to 32 Open Reading Frames (ORFs) per 100 kb (Barakat et al., 1998), or 1 ge ne per 3-6.6kb, whereas rice has one gene per 6.46 kb (Yu et al., 2002) and barley has one gene per 1520 kb (Keller and Feuillet, 2000). The Fragaria average gene distribution was calculated as 1 gene /4kb or 1 gene/9kb, depending on the prediction method used: ab initio gene prediction software FGENES H or BLASTX similarity-based approach at E<10-15, respectively. In either case, strawb erry ranks among the more gene-dense species. Since a portion of the fosmid sequen ces analyzed arose from non-random, gene of interest selections, it is possibl e that the sample was biased to ward genic regions, and that the number of kb containing one ge ne will increase as more random expanses of the genome are sequenced. The number of putative genes per fosmid ranged from 6 to 15 (identified by ab initio ) or from 1 to 11 (according to homology to protein database). The discrepancy between the numbers from the two methods may be attributed to the fo llowing possibilities: i, the gene structure used for prediction was from Medicago not Fragaria. There is a possibility that the gene structures between these two organisms are distinct e nough that a sequence encoding a protein in Medicago

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51 is not coding in Fragaria ; ii, the gene prediction is correct, but the putative amino acid sequence is not represented in the protein database because the transcript is not translated (RNA genes in fosmid 15B13, for example), or because the protei n has not yet been desc ribed; iii, the amino acid sequence is indeed represented in the database, but it is not conserved with Fragaria so the E value threshold chosen as a threshold is too st ringent. If a less stringent threshold is used (E value 10-10, rather than 10-15), the number of BLASTX hits increases from 129 to 166 and, therefore, software specifi city rises from 55 to 70%. Half of the ESTs that were identified in ge nomic regions for which no gene was predicted (figure 3-3) were detected in fosmids that e ither contained sequence si milar to chloroplast DNA (11D02 and 32L07) or to ribosomal RNA (26S in fosmid 15B13). One of the ESTs displayed identity starting in the first nucleotide of the fo smid insert. Perhaps the gene predictor failed to perceive this ORF because the query sequence di d not contain transcription initiation signals. The other half of the ESTs that were identi fied but not predicted was similar to genomic sequences from other species, and the reason why the gene predicti on software failed to predict them is not clear. This may suggest some facet of Fragaria gene structure that is not recognized by other conditioned algorithms. The detecti on of putative genes through homology-based similarity search reveals the n eed to utilize various homology s earch methods in combination to ab initio gene prediction for the opt imum genome annotation. Th is finding is exceedingly important as the genomes of peach and appl e will soon be sequenced. Accurate genome annotation will depend on the capac ity to adapt current gene prediction methods to these genomes.

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52 Figure 3-1. Flowchart of geno mic DNA sequence annotation sc heme. The software FGENESH was used with Medicago as the gene model to predict possible gene positions in the genomic sequence. BLASTP algorithm was utilized as preliminary validation FGENESH prediction, whereas BLASTX wa s used to determine coding sequence orientation and assign tentat ive gene function. Putative homologs within Rosaceae, conservation amongst various taxonomical fam ilies, as well as sequence repeats and duplications were detected by different homology searches utilizing BLASTN. Finally, putative genes that had not been predicted by FGENESH were identified by searching similarities between large fragments of genomic sequence (containing or not FGENESH-predicted genes) and Rosaceae EST.

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53 Figure 3-2. Diagram of two fosmid inserts of vari able length, with their putative proteins and Simple Sequence Repeats (SSRs). Fosmid 34D20 contained an inverted repeat of an anthocyanin gene next to a transposase, in addition to other protein-encoding genes. Fosmid 18A19 contained mostly trans poson-related enzymes, integrase and transferase. SSRs were identified bo th in genic and inergenic spaces.

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54 Figure 3-3. EST classes identified by ho mology searches between large genomic F. vesca sequence and Rosaceae ESTs. BLASTX similar ity searches were carried out between genomic sequence and the Viridiplantae pr otein database. A fraction (25%) of the protein matches was validated by BLASTN-d etected similarities to the Rosaceae EST database. Other 19 ESTs present no functional information, since no similar amino acid was identified. Of these, a set of 8 ESTs belong to genomic sequence for which there were no genes predicted by FGENESH utilizing Medicago as the gene model. Table 3-1. Number of simple sequence repeats (with a minimum of 5 repeats) observed in Fragaria vesca genomic sequence Motif length Number of Repeats Frequency 2 bp 1864 92.1% 3 bp 149 7.4% 4 bp 10 0.5% Table 3-2. Different types of dinucleotid e and trinucleotide repeats observed in Fragaria vesca genomic sequence Motif Number of Repeats Frequency AG/GA/CT/TC 1105 54.9 AT/TA 658 32.7 AC/CA/TG/GT 91 4.5 AGA/GAA/CTT/TCT 79 3.9 CAC/GGT/GTG/TGG 21 1.0 AAC/ACA/GTT/TGT 19 0.9 AAT/TTA 15 0.7 GC/CG 10 0.5 ATG/CAT/TGA//TCA 10 0.5 AGG/CCT 5 0.2

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55 CHAPTER 4 GENE-PAIR HAPLOTYPES: NOVEL MOLE CULAR MARKERS FOR INVESTIGATION OF THE Fragaria ananassa OCTOPLOID GENOME Introduction The cultivated strawberry, Fragaria ananassa contains 8 copies of a set of 7 chromosomes (2n=8x=56). The amount of DNA c ontained in a single co mplete strawberry chromosome set is approximately 200 million ba ses (Nehra et al., 1991; Akiyama et al., 2001; Folta and Davis, 2006), a very small genome size re latively to other angios perms. There is some controversy as to which angiosperm contains the lowest C-value (or C x -value, terminology proposed by Greilhuber (Greil huber et al., 2005) to spec ify the monoploid genome of polyploids), due to different size standards used among vari ous flow cytometry studies. However, likely candidates to the smallest genome are Arabidopsis thaliana with 157Mb (Bennett et al., 2003), and perh aps the green strawberry Fragaria viridis with 0.108pg (Antonius and Ahokas, 1996). If the formula proposed by Dol eel (Doleel et al., 2003) (where 1pg = 978 Mb) is applied, the estimate for F. viridis genome size is 105Mb. However, according to the correction proposed by Bennett (Bennett et al., 2003), F. viridis current C-value estimate is 206 Mb (Folta and Davis, 2006). Considering that angiosperm C-value varies approximately 1000fold between species (Bennett and Leitch, 2005), the difference in monoploid genome sizes between F. ananassa and A. thaliana is negligible. Although strawberrys small basic genome size makes Fragaria species attractive organisms for genomic studies, the process of sor ting out segregation in an octoploid background is an extremely complex task, posing a formidable barrier to development of molecular markers and genetic linkage mapping. In a polyploi d where reassortment amongst all homologous chromosomes occurs, the number of possibl e genotypes for a single locus would be 1 1 2 a 2 aC 2,

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56 where a = number of distinct alleles. For an oc toploid containing 8 different alleles for a single locus, the number of different combinations woul d be 2,451. However, this es timate is artificial, since most polyploid plants are c onsidered to be alloploids, a nd therefore display a degree of fixed, non-segregating heterozygosity (Soltis and Soltis, 2000). The F. ananassa genome structure is not well unde rstood. The first proposed genome structures were derived from cytological analys es of meiotic pairing chromosomes. The genome composition was first described as AABB BBCC (Fedorova, 1946), whilst the model AAAABBBB(Bringhurst, 1990) is currently the accepted one. More evidence gathered through the use of molecular markers (Arulsekar et al., 1981; Haymes et al., 1997; Viruel et al., 2002; Ashley et al., 2003) supports the fully dipl oidized model. In a single study using molecular markers (Lerceteau-Khler et al., 2003), the auth ors have observed some polysomic inheritance in a F1 octoploid population. However, the devia tions from disomic ratios observed may not be due to polysomic inheritance, as segregation di stortions have also been observed in diploid segregant populations (Davis and Yu, 1997; Sargent et al., 2004; Sargent et al., 2006). The identification of genome-specific pol ymorphisms may permit the monitoring of segregation of each genome in the complex pol yploid background. The Gene-Pair Haplotype (GPH) is a tool developed to fingerprint the a lleles present in the cont ributing genomes in the octoploid strawberry. It is de fined as a suite of intergenic polymorphismsSimple Sequence Repeats (SSRs), Single Nucleotide Polymorphisms (SNPs), insertion or deletions (InDels), and changes in restriction sites (R FLP) that present a complex genetic marker for a given locus within the diploid genomes. The types of polymor phisms likely to be detected in a GPH locus and their respective expected location in the genome (within versus between genomes) are summarized in figure 4-1.

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57 GPH markers were also used to in vestigate polymorphisms in diploid Fragaria species, in an attempt to identify genome contributor s and trace the diploid ancestors. The genus Fragaria contains 23 species of different ploidy numbers. Most of the Fragaria species are represented in figure 4-2, with locations based on maps and descriptions publis hed elsewhere (Hancock et al., 2004) (Darrow, 1966) (Staudt, 1973) (Hummer et al., 2005) (Staudt, 200 3) (Staudt, 2005). F. ananassa is not included in the figure, as cu ltivated strawberry is ubiquitous. According to T. M. Davis, Lake Baikal marks a major geographical boundary for strawberry distribution. F. vesca and F. viridis are not found in India, Tibet, China, Japan, or southeast Asia. Likewise, no Asian species grow to the west of Lake Baikal. (http://www. strawberrygenes.com/map.html) Fragaria species have been cultivated for a l ong time. The French started transplanting fraise des bois, or the wood strawberry F. vesca (vesca means little, in Latin (Fay, 1903)) from the wilderness to gardens in the 1300s, whereas the hexaploid F. moschata, the musky strawberry, was common in gardens in the 1700 s (Darrow, 1966). The modern cultivated strawberry has a very well documented history. It was first cited by Philip Miller in the 1759 edition of the Gardeners Dictionary (Darrow, 1966) and it received the name of F. ananassa due to its resemblance to pineapple in odor, tast e, and berry shape. In 1765, Duchesne correctly proposed that the new species parents were F. virginiana and F. chiloensis. Although both parents are native to America, the spontan eous hybridization occurred in Europe. F. virginiana with its rather small fruits was transported overseas in the 1600s. Because of its relatively large fruits, F. chiloensis was collected by the Frenchman Am de Franois Frzier, during a reconnaissance mission to the Spanish West I ndies ordered in 1714 by the king Louis XIV. Disappointingly, no fruits were obs erved during the first years, pr obably because, in an attempt

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58 to collect only the largest-fruited plants, Fr ezier imported only female plants. About 50 years later, the product of the pollination of F. chiloensis by F. virginiana was observed in Germany, Switzerland, Holland, and the Trianon gardens in France (Darrow, 1966). F. ananassa s nuclear genomic content can be traced to fifty-three founding clones (Sjulin and Dale, 1987), whereas as few as seventeen cytoplas m donors are represented in the cultivated strawberry (Dale and Sjulin, 1990). Wild accessions from the octoploid parents have been used relatively recently in strawberry breeding programs for in trogression of various characteristics (Hancock, 1999), including day neutrali ty into California cult ivars (Ahmadi et al., 1990). Athough the identities of the direct ancestor of F. ananassa are known, their genome constitutions and evolution are not. The presen t research investigated polymorphisms in the intergenic regions of diploid sp ecies, as well as the cultivated octoploid to attempt to trace ancestry and make inferences about the octoploid genome mode of inheritance. Materials and Methods Before the commencement of this study in the year 2004, virtually no Fragaria genomic sequence was available. Therefore, it was nece ssary to develop a means to capture useful sequences for analysis. Two different approaches were adopted: i, inference of gene adjacency by putative micro-colinearity between F. ananassa and Arabidopsis thaliana ; ii, construction and annotation of a F. vesca genomic library (discussed in Chapter 3). Potential micro-colinearity was detected usi ng the approach described in figure 4-3. This approach was possible be cause the genome of Arabidopsis has been completely sequenced and the genes were numbered in such fashion that th eir locus tags indicate their position on the chromosomes. The hypothesis was that if two genes were adjacent in Arabidopsis they would also be adjacent in Fragaria. Similarity between F. ananassa ESTs and A. thaliana transcripts

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59 was tested using the FASTA software available at The Arabidopsis Information Resource (TAIR)s website (http://arabidopsis.org/cgi-bin /fasta/nph-TAIRfasta.pl) and the best match for Arabidopsis was recorded. The sequences of each of the Arabidopsis genes adjacent to the Arabidopsis matches were retrieved from the Salk Institute Genomic Analysis Laboratory (SIGnaAL) T-DNA Express Arabidopsis Gene Mapping Tool websit e (http://signal.salk.edu/ cgi-bin/tdnaexpress). The ne xt step was to detect Fragaria sequences that were similar to each of the Arabidopsis gene sequences retrieved. The Basic Local Alignment Search Tool (BLAST) was used to search the Fragaria translated nucleotide database using the Arabidopsis translated nucleotide query, since TBLASTX is the most sensit ive algorithm to detect sequence similarities. Results with an E-value < 10-4 were considered positive hits and primers were designed for the putative gene pair to amplify the presumably intergenic sequence flanked by the conserved Fragaria and Arabidopsis primers. In addition, forward and reverse primers (table 4-1) were designed to amplify at least 100 bp of the EST. This allowed validation that the amplification sequenced was specific to the target regions. A second approach was adopted to increase th e micro-colinearity detection level. Twohundred and fifty F. ananassa EST sequences were randomly sel ected for similarity searches against Arabidopsis utilizing FASTA and all (rather than only the best match) of the Arabidopsis sequences that had a si milarity E-value < 10-4 were considered for furthe r analysis. The loci tags were recorded on two separate tables, one table keeping the correspondence between F. ananassa and Arabidopsis similar sequences, and the other table had the Arabidopsis loci tag sorted in crescent order. When th e difference between two consecutive Arabidopsis loci tag numbers was equal to or lower than 10, a putative gene pair was detected and the F. ananassa EST sequences were retrieved from the non-sorted table.

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60 In addition to the amplification of unknown re gions, sequences were gathered by sample sequencing genomic DNA. Both random and targeted sequences were studied in a F. vesca fosmid library. The annotation scheme is descri bed in Chapter 3 of this dissertation. Forty combinations of PCR primer pairs were tested to amplify 18 loci, si nce different primer combinations were required to amplify some of the loci. The primer pairs generated for the putative intergenic regions are li sted in table 5-1 of Chapter 5. Following determination of location and design of PCR primer pairs, PCR was carried out to amplify 28 loci, of which 10 gene pairs (listed in table 4-1) were inferred by the F. ananassa / Arabidopsis micro-colinearity approach and 18 gene pairs (listed in table 5-1) were inferred from gene prediction from F. vesca Pawtuckaway genomic sequence. The optimizations of PCR conditions were carried out utilizing as template DNA from the species for which primers had been designed: F. ananassa and F. vesca Pawtuckaway for microcolinearityand genomic-DNA-based approaches respectively. Once optimum conditions were determined, the reaction wa s carried out for seven Fragaria species, which included the respective control species: F. ananassa Strawberry Festival, F. vesca Pawtuckaway, FRA341 F. viridis FRA377 F. iinumae, FRA520 F. nubicola FRA1318 F. nilgerrensis and FME F. mandshurica The PCR products were cloned using the pl asmid cloning vectors pJET1 (GeneJet PCR cloning kit by Fermentas Life Sciences) or pCR2.1-TOPO (Invitrogen Life Technologies). The ligation reaction was carried out according to manufacturers directions and 1l of the ligation reaction was used to transform 50l of competent cells. The chemically competent Escherichia coli bacterial cells (Invitrogen One Shot TOP10) were purchased with the TOPO cloning kit whereas XL1-Blue competent cells (B ullock et al., 1987) were prepared in the

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61 laboratory using and the rubidi um chloride method (Hanahan, 1985). The putative recombinant plasmids and competent cells were gently mixed, iced for 30min, heat-shocked at 42C for 2min, and immediately iced again for at least 5min. XL1-Blue cells w ith no vector were included in each transformation round as a negative control. When large fragments we re cloned, a separate treatment with a smaller fragment for which tr ansformation had been successful before was included as a positive control. Two-hundred l of Luria-Bertan i (LB) broth (10g tryptone, 5g yeast extract, 10g NaCl, per litter of deionized water) were added to the transformed cell and were incubated in a shaker fo r 1 hour at 37C, with agitation of 220rpm, after which 100l of cells were spread onto LB-agar plates contai ning 50g ampicillin/ml medium. The TOPO vector has the -galactosidase reporter ge ne. Therefore, when this vector was used, an overlay of 50l of the chromogenic substrate 5-bromo-4-chloro -3-indolyl--D-galactoside (X-gal) at 20mg/ml dissolved in N-N'-dimethyl-formamide and 10l of the filter-sterilized inducer isopropylthiogalactoside (IPTG) at 1M were added to the LB-agar plates before the transformed cells were plated. IPTG and X-Ga l were not added to the LB plat es when the pJET1 vector was used. This vector contains a ge ne for a restriction endonuclease in the cloning site. If disrupted by an insert, the lethal endonucleas e is not expressed and the transf ormants are able to propagate. After the cells were plated, they were incubate d at 37C overnight and single colonies were selected for screening for transformantswhite colonies for TOPO a nd, supposedly, any colony for pJET1. The screening procedure was carried out by setting up individual PCR reactions for each colony using annealing temper ature of 55C and primers specifi c for the vector (pJET1F: 5 GCC TGA ACA CCA TAT CCA TCC 3, pJET 1R: 5 GCA GCT G AG AAT ATT GTA GGA GAT C 3; TOPO, M13F: 5 GTA AAA CGA C GG CCA GTG AAT TGT A 3; M13R: 5 CAG GAA ACA GCT ATG ACC ATG ATT AC 3). Appr oximately 10 colonies were initially

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62 selected from each plate with transformants containing PCR products fr om diploids. Because several different alleles were s ought for the octoploid, 30 colonies were selected from the plates that had transformants with inserts amplified fr om Strawberry Festival. The tested colonies were streaked on a separate LB/ampicillin plate during the set up of the colony PCR reactions. The PCR products were resolved in 0.8% agar ose gel with 1x TAE buffer. PCR-confirmed transformants were grown in 3ml LB broth co ntaining 50g amplicillin/m l for approximately 4 hours at 37C, with agitation at 220 rpm. Plasmi ds were extracted from 1.5ml culture by the alkaline lysis method, followed by 24:1 chloro form extraction. Isolated plasmids were resuspended in 50l of deionized water and 5 l were digested with 1 unit of restriction enzymes: EcoRI or XbaI/XhoI for amplicons ligated to TOPO or pJET1, respectively. Transformants carrying distinct alleles were detected by diges tion with EcoRI. The digested bands were resolved in 2% Metaphor/1xTBE or 2% agarose/1xTAE. Clones with similar restriction patterns were grouped in to different classes and a repres entative clone of each class was sent to DNA sequencing facili ties. A list of primers generated for sequencing reactions can be found in Appendix C, whereas the sequen ces generated are included in Appendix D. Sequences obtained were analyzed for conser vation between diploid a nd octoploid alleles. Alignments were performed using the global alignm ent tool ClustalW available at the European Bioinformatics Institutes webs ite at http://www.ebi.ac.uk/clust alw/. Except for the penalty for gap extension, which was set at 0.05 instead of th e default 6.66, all other penalty settings were the default ones: gap open: 15; end gap: -1; gap distance: 4. Results Considering the number of Fragaria ESTs available at the time this study was initiated (approximately 1,500 ESTs), and the estimated 26,000 genes in the Arabidopsis genome (Sterck et al., 2007), if micro-co linearity indeed existed, the chance that two adjacent genes would be

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63 detected in the pool of 1,500 was a pproximately 10% as calculated by 1,500 1 25,999 1 25,998 Therefore, the amplification of 2 lo ci out of the 10 investigated (table 4-2) may be regarded as fairly successful. A th ird set of primers (GPH4) permitted amplification, though after fragment cloning and sequencing, amplification was determined to be unspecific. There was no similarity between sequences obt ained and the 770bp from the template sequences for primer design. The gene prediction from F. vesca genomic sequence enabled de tection of 18 potential gene pairs. Of those, primers designed for 11 loci rendered amplif ication of at least the positive control DNA template of F. vesca Table 4-2 summarizes the results of PCR am plification using primers designed through both gene-pair detection approaches, as well as results on cloning amplicons and sequencing inserts. The clone # in the table is in mo st cases the PCR reaction number, followed by the colony that was determined to be a transforma nt by PCR and/or restriction enzyme digestion. The hypothesis that a fingerprint for each alle le belonging to the octoploid Strawberry Festival would correspond to al leles from different diploids could be tested by GPHs 5, 23, 10, 27F10, 34D20, and 72E18. The full alignments for ever y GPH characterized in this dissertation can be found in appendix E. Data of Individual Loci GPH5 GPH5 was detected by the micro-colinearity search approach. The adjacent genes in Arabidopsis were At3g07320 (E value of 9x10-21, encoding a glycosyl hydrolase family 17 protein) and At3g07330 (E value of 2x10-60, encoding a glycosyl transferase family 2 protein). GPH5 is a particularly interesting locus, since amplification was observed for all diploids and 2

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64 alleles of the octoploid were detected by EcoRI digestion. The following polymorphisms were identified in the 2.8kb analyzed: short indels of 4-12bp (9 bp insertion in F. vesca ; 12 bp deletion in F. iinumae shown in figure 4-4), 180 SNPs, of wh ich 125 are ambiguous (may be sequencing or polymerase errors) and 55 likely true SNPs, because the base change occurs in more than a single clone. Most of the likely SN Ps delineate the octoploid clones from the diploid ones. It is interesting to note that the octoploid alleles ar e grouped separately from diploid alleles not only for their SNPs, but also for small indels. Two SSR motifs were identifie d (AAG and AT), with 4 repeats each, for every clone. Therefore no polymorphism in the number of repeats was detected. GPH23 At1g23740 (oxidoreductase, zinc-binding dehydr ogenase family pr otein) and At1g23750 (DNA-binding protein) were similar to F. ananassa with E values of 3x10-64 and 2x10-57, respectively. Only F. mandshurica F. iinumae and F. ananassa were amplified by the primers designed for this region. Larger deletions than those observed for other loci investigated, and different alleles from the diploids were obs erved for GPH23. Figur e 4-5 illustrates the polymorphisms detected. After preliminary sequence alignment, the putative SNPs were verified by observation of unambiguous peaks in the chromatograms. Therefor e, for this locus, a SNP is only an artifact if it was introduced during amp lification by the polymerase. (CTC)4 SSRs were detected and occurred in equal number of repe ats for every clone, in the same position when aligned. The implications of the pol ymorphisms are discussed below. GPH10 Primers GPH10A and GPH10C were utilized to amplify a 4.4kb fragment from the octoploid Strawberry Festival. Four categor ies of polymorphic clon es were detected by EcoRI

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65 restriction digestion (figure 4-6) and sequence was obtained for th e full clones (figure 4-7). The primers flaking the most polymorphic region (10 PPR1 and 10AB#22) were utilized to amplify that region from all six diploids included in this study. A cla dogram based on this polymorphic region is shown in figure 4-9. 72E18 72E18 was the only GPH sequenced that presente d polymorphism in the number of repeats in SSRs. Estimations of Relatedness from Sequence Variation Cladograms are branching diagrams assumed to be an estimate of a phylogeny where the branches are of equal length. Therefore, cl adograms show common ancestry, but do not indicate the amount of evolutionary "time" separating ta xa (information from the http://www.ebi.ac.uk/ website). In this study the use of cladograms generated from multiple sequence alignments provide an outstanding means to gauge the re latedness between strawberry genomes. When compared against each other, th e use of cladograms depicts the relative divergence between similar sequences, and thus is a useful estima te of SNP frequency between the alleles in F. ananassa and the putative diploid subgenome donors. The following cladograms derive from all GPH that contained at least one allele repres enting the octoploid strawb erry compared to all cases where products could be amplified from di ploids. The results in dicate that octoploid alleles cluster together, as do dipl oid alleles. The most related diploid to octoploid alleles is consistently F. iinumae, and surprisingly, alleles closely matching F. vesca were not detected for any of these GPH loci. Relatedness may also be assessed by studying the order of insertion-deletions and SSRs. Presumably, a set of similar indels or SSRs ma y be conserved between the diploid subgenome donors and the modern cultivated octoploid. The pr esence and order of these features provides

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66 evidence of relatedness. Table 4-3 represents the length and position of indels and SSRs identified in the sequenced clones. In this tabl e, indels and SSRs are presented as variable features in genomic sequence as it is parsed from 5 to 3. With this method the size and position can be best described, pres enting evidence of relatedness. In this table the variable features present in all genomes are revealed. When two or more values in the same column are shown, this represents indels pr esent in the same region of a given locus, as the corresponding genotype deviates from a consensus sequence co mpiled from multiple sequence alignment of all sequences. A blank box indicates agreement with consensus in a given region. The corresponding genotype does not deviate from cons ensus. The sequence of the clones listed in the table is conserved with the sequence as they appear in the cladog rams of figure 4-9 to facilitate the perception of relatedness. Discussion Synapsis between F. vesca and F. virginiana chromosomes has been shown to occur (Ichijima, 1926). This is regarded as the first evidence that F. vesca is a likely genome donor to F. ananassa since F. virginiana is the pollinating parent to F. ananassa Another study published a year later showed that the crosses between F. vesca F. chiloensis and F. vesca F. virginiana produced sterile hybrids (M angelsdorf and East, 1927). The occurrence of natural hybrids between F. chiloensis and F. vesca (Bringhurst and Gill, 1970), the geographical predominance of F. vesca and a recent study on chloro plast DNA showing that the F. vesca is closely related to F. ananassa (Potter et al., 2000) support the hypothesis that F. vesca is a contributor to the genome of oct oploid strawberries. In this study large intergenic regions were sequenced from a series of oct oploid and diploid alleles to as sess the relatedness between the cultivated strawberry and potenti al subgenome donors. Two central methods were used to detect

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67 relatedness, both based on multiple sequence alignments. The first used cladograms to display consolidation of single nucleotide polymorphi sms (Table 4-2). The second method was as assessment of multinucleotide polymo rphisms, detected as indels or SSRs that varied between accessions and a consensus sequence. The use of these complementary methods provides two levels of resolution that descri be relatedness between alleles. Contrary to the expected, however, data from five characterized loci and the inability to PCR-amplify F. vesca using primers designed for F. ananassa (GPH23) display F. vesca Pawtuckaway as the most unrelated sequence to any of the sequenced octoploid alleles. From the few loci studied, it does not appear that F. vesca is a more likely A-genome donor than any of the other diploids studied. Th is surprising finding contrasts di rectly with cytological evidence and suggests that F. vesca may not be a contributor to at leas t the Strawberry Festival cultivar. F. iinumae on the other hand, was confirmed as one of the most distinct diploid. Table 4-3 shows that F. viridis and F. iinumae had the most dramatic changes in relationship to the other four diploids concerning size of their indels. F. viridis displays large indels: 44, 500, and 800bp in loci 11D02, 27F10, and 32L07, respectively. None of the deletions, however, corresponded to any of the F. ananassa alleles sequenced. In the case of 32L07, no octoploid allele was sequenced because PCR amplification could not be detected for any of the following octoploids: Strawberry Festival, Carmine, Diama nte, Rosa Linda, and Sweet Charlie. F. iinumae has a deletion greater than 500bp in the fragment 10PPR1AB22 of GPH10, five indels of approximately 30 bp (t hree in 11D02, and two in 34D20), and one of approximately 50 bp in 72E18. The indels in 34D20 and 72E18 from F. iinumae coincided with F. ananassa suggesting that F. iinumae is indeed a genome donor to the diploid. The cladograms from figure 4-9 suggest that in every locus studied, F. iinumae was the most similar diploid haplotype to the

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68 octoploid alleles. Phylogenetic analysis of th e intron-containing region of the Adh gene of 20 Fragaria species identified two major clades, and pointed to F. iinumae as the best B clade candidate for Adh allele donor to oct oploids (Davis and DiMeglio, 2004). The data identified here provide furthe r evidence to support the hypothesis that F. iinumae is a subgenome donor to the modern octoploid. In all comparisons herein where octoploid sequence was obtained, the octoploi d related more closely to the F. iinumae haplotype. Thus, one conclusion that can be made is that F. ananassa cv. Strawberry Festival contains clear evidence of the F. iinumae characters within its subgenome composition. But what about the A genome? The B genome donor has been disputed, but almost 100 years of evidence implicates F. vesca as an A-genome donor. In this data set, little evidence of the A-genome exists. There are several ways to reconcile this discrepanc y, although all of them ar e speculative. The one important caveat is that Strawberry Festival is only one octoploid accession and was used almost exclusively as the octopl oid representative. Strawberry Festival has a broad east-coast, west-coast lineage, so in many ways it is an excellent representative for this study. It is possible, albeit unlikely, that the allelic content of Str awberry Festival is skewed to the B-genome F. iinumae components and somehow the A-genome is not being detected. This is surprising because the primers that detect the B genome va riants were derived from the A genome donor. One alternative explanation is that perhaps the A genome underwent extensive modification, such as expansion, therefore preventing amp lification of octoploid sequences by PCR. Alternatively, these regions could have been dele ted from the octoploid genome, as the octoploid genome is smaller than four diploid genomes, i ndicating a loss of genetic material (Folta and Davis, 2006). A final explanation is that not all diploid sp ecies, including many F. vesca accessions, were tested, so the A genome may be represented by a genotype not tested in this

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69 study. There is no simple answer, and this finding may indicate that some higher-order mechanism is at work to limit the presence of subgenome sequence in the polyploid. Polysomic inheritance has been documented (Lerceteau-Khler et al., 2003). If polysomic inheritance led to a trait of interest early on, it may have been selected as beneficial in bree ding populations and fixed in subsequent lines. Another unlikely explanation is that changes in F. iinumae paralled those in F. ananassa in two separate and unrelated instances. Probabil ity suggests that this ca nnot be the case, yet it remains a formal possibility, especially if the cha nges induced result in re gulatory alterations that affect gene expression, biological function and po ssibly selection. It is also possible that cultivation and selection have important conseque nces in skewing subgenome representation. It has been demonstrated that F. iinumae is a robust plant, with more vigorous growth than F. vesca (Sargent et al., 2004). These characters may ha ve lent themselves to the wild octoploids and were attractive to potential early breeders. These alleles may dominate certain selections, like Strawberry Festival. Other cultivars need to be tested to assess allelic composition to further query this hypothesis.

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70 Figure 4-1. An idealized GPH locus. Arrows represent primers designed to amplify the intergenic spaces of a GPH. The combina tion of polymorphisms within (SSR, SNP) and between subgenomes (InDels, change in restriction sites) de fine each haplotype. Figure 4-2. Fragaria species and their geogra phical locations

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71 Figure 4-3. GPH design upon comparison between strawberry ESTs and Arabidopsis database. When the quest for homologies culminates in the detection of potentially neighboring genes, primers are designed in the stra wberry EST and the intergenic region is amplified if the adjacency is true, the ge ne space is smaller than 4kb, and the gene orientations are conserved.

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72 Table 4-1. PCR primers designed for amplifica tion of micro-colineari ty-inferred putative intergenic fragments Primer name Fragaria EST Arabidopsis gene Intergenic fragment size in Arabidopsis (bp) Primer sequence GPH4a FA_SEa0007-G07 At2g20120 acgagggcttggaagaaagg GPH4b FA_SEa0015-B08 At2g20140 3,415 gcccaacaacagaaagacc GPH5a#2 FA_SEa0002C03r At3g07320 caatgccatggtctccggtc GPH5b#2 FA_SEa0018E10r At3g07330 2,196 tgccgttgcacacaccttcc GPH20 FA_SEa0012D10r At5g13440 gagggtaacgctcatggtt GPH20 FA_SEa0012E08r At5g13450 1,043 gtctccttcaattctttctcctc GPH21a AY679587 At5g06750 tgacatcccataagccatca GPH21b DQ011163 At5g06760 gggaggactacggcacataac GPH21c DQ011163 At5g06760 1,509 atcagatgtcggcactgc GPH22 FaSCH6rgene gi 48249442 At5g11250 tttcagctcagcaagcaagg GPH22 FaHy5 FA_SEa0004E09r At5g11260 1,632 gctcccaggaccaaacca GPH23F FA_SEa0013H07r At1g23750 cttgagggccatcagcac GPH23R V01014C10_558132 At1g23740 982 tacacccacgccttcatctc GPH27F FA_SEa0014E11r At1g74260 tgccgctgccatttctct GPH27R FA_SEa0011H07 At1g74280 2,300 ccatgctcttgataggccaaat GPH31F FA_SEa0014B05r At2g30100 aatggagctgatggtttcgat GPH31R FA_SEa0016G12r At2g30110 724 aaggatgatgacacgaactatca GPH51F CX662192 At3g176600 ggacacatggctcccaga GPH51R AY961594 At3g17670 1,867 caagacagcgggagcagt GPH56F AB211167 At4g38970 ccagggacgatgttttgctc GPH56R AJ414709 At4g38990 ggtggattacattttgggtgaca GPH56R2 AJ414709 At4g38990 3,017 ttcaagctttggacaactaacg

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73 Table 4-2. PCR primers that allowed amplicon ge neration. A minus sign in the amplification and clone # columns signify, respectivel y, no amplification and no transformants were observed. Primer name Template PCR product size (kb) Clone # Vector E. coli strain Sequence obtained from forward end (bp) Sequence obtained from reverse end (bp) GPH4 13 TOPO TOP10 1,109 Strawberry Festival 2.0, 1.0, 0.5 15 TOPO TOP10 638 GPH5 F. vesca 2.8 21 TOPO TOP10 1,268 1,299 F. viridis 2.8 5 TOPO TOP10 1,249 1,287 F. iinumae 2.8 5 TOPO TOP10 1,256 1,298 F. nubicola 2.8 7 TOPO TOP10 1,261 1,263 F. nilgerrensis 2.8 19 TOPO TOP10 1,262 1,295 F. mandshurica 2.8 1 TOPO TOP10 Full clone 2 TOPO TOP10 1,257 1,306 6 TOPO TOP10 749 755 Strawberry Festival 2.8 7 TOPO TOP10 Full clone GPH23 F. vesca F. viridis 2 TOPO TOP10 Full clone F. iinumae 2.0 5 TOPO TOP10 Full clone F. nubicola F. nilgerrensis F. mandshurica 2.0 3 TOPO TOP10 Full clone (2,024) 3 TOPO TOP10 Full clone (2,081) Strawberry Festival 2.0 4 TOPO TOP10 Full clone (2,111) GPH10 4.4 2 TOPO TOP10 Full clone 4.4 7 TOPO TOP10 Full clone 4.4 18 TOPO TOP10 Full clone 4.4 19 TOPO TOP10 Full clone Strawberry Festival 4.4 20 TOPO TOP10 Full clone F. vesca 0.728 TOPO TOP10 Full clone F. viridis 0.726 TOPO TOP10 Full clone F. iinumae 0.266 TOPO TOP10 Full clone F. nubicola 0.722 TOPO TOP10 Full clone F. nilgerrensis 0.652 TOPO TOP10 Full clone F. mandshurica 0.724 TOPO TOP10 Full clone 528 2 Subset of GPH10 sequence 644 7 Subset of GPH10 sequence 584 18 Subset of GPH10 sequence 584 19 Subset of GPH10 sequence 10PPR 1/10A B22 Strawberry Festival 643 20 Subset of GPH10 sequence 11D02 F. vesca 1.6 library F. viridis 1.6 2031-1 pJET1 XL1-Blue 1,403 F. iinumae 1.6 2032-1 pJET1 XL1-Blue Full clone F. nubicola 1.6 2033-1 pJET1 XL1-Blue 1,299 F. nilgerrensis 1.6 F. mandshurica 1.6 Strawberry Festival 1.6 10PPR1/10AB22 is a locus within GPH10

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74 Table 4-2. continued Primer name Template PCR product size (kb) Clone # Vector E. coli strain Sequence obtained from forward end (bp) Sequence obtained from reverse end (bp) 17O22 F. vesca 1.4 library F. viridis 1.4 678 pJET1 XL1-Blue Full clone F. iinumae 1.4 & 1.0 668 pJET1 XL1-Blue Full clone F. nubicola 1.4 653 pJET1 XL1-Blue Full clone F. nilgerrensis F. mandshurica 1.4 655 pJET1 XL1-Blue Full clone Strawberry Festival 1.5 & 1.4 27F10 F. vesca 1.0 library F. viridis 1.5 2039-1 pJET1 XL1-Blue 537 582 F. iinumae 1.0 2040-1 pJET1 XL1-Blue Full clone F. nubicola 1.0 2041-1 pJET1 XL1-Blue Full clone F. nilgerrensis 1.8 F. mandshurica 1.0 2043-1 pJET1 XL1-Blue Full clone 1.0 2046-1 pJET1 XL1-Blue Strawberry Festival 2046-2 pJET1 XL1-Blue Full clone 29G10 F. vesca library F. viridis F. iinumae F. nubicola 0.7 2049-1 pJET1 XL1-Blue Full clone F. nilgerrensis 0.7 2050-1 pJET1 XL1-Blue Full clone F. mandshurica 0.7 2051-1 pJET1 XL1-Blue Full clone Strawberry Festival 32L07 F. vesca 2.7 library 640 pJET1 XL1-Blue Full clone F. viridis 1.9 1090-11 TOPO TOP10 F. iinumae F. nubicola 2.7 647 pJET1 XL1-Blue No seq F. nilgerrensis 2.7 993 TOPO TOP10 No seq F. mandshurica 2.7 1000 TOPO TOP10 No seq Strawberry Festival I attempted to amplify fragment from the octoploids Carmine, Diamante, Rosa Linda, and Sweet Charlie, but amplification was not observed for any of them 34D20 F. vesca 2.0 1826-3 pJET1 XL1-Blue library F. viridis 2.0 1827-3 pJET1 XL1-Blue Full clone F. iinumae 2.0 1828-4 pJET1 XL1-Blue Full clone F. nubicola 2.0 1829-1 pJET1 XL1-Blue Full clone F. nilgerrensis 2.0 1830-3 pJET1 XL1-Blue Full clone F. mandshurica 2.0 1831-5 pJET1 XL1-Blue Full clone Strawberry Festival 2.0 1832-2 pJET1 XL1-Blue Full clone 40M11 F. vesca 3.1 library F. viridis 3.1 F. iinumae 2.9 F. nubicola 3.1 F. nilgerrensis 3.1 F. mandshurica 3.1 1088-1 TOPO TOP10 884 2.9 Strawberry Festival

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75 Table 4-2. continued Primer name Template PCR product size (kb) Clone # Vector E. coli strain Sequence obtained from forward end (bp) Sequence obtained from reverse end (bp) 63F17 F. vesca 1.2 library F. viridis 1.2 2 pJET1 XL1-Blue Full clone F. iinumae 1.2? 3 pJET1 XL1-Blue F. nubicola F. nilgerrensis F. mandshurica 1.2 3 pJET1 XL1-Blue Full clone Strawberry Festival 1.2 1 pJET1 XL1-Blue 570 72E18 F. vesca 2.6 library TOPO TOP10 F. viridis 2.6 1096-4 TOPO TOP10 1,052 1,515 F. iinumae 2.6 1097-1 TOPO TOP10 1,000 973 F. nubicola ? F. nilgerrensis 2.5 1099-1 TOPO TOP10 1,958 F. mandshurica 2.6 1100-6 TOPO TOP10 Full clone Strawberry Festival 2.5 1101-10 TOPO TOP10 1,170 1,245 73I22 F. vesca F. viridis 3.0 1834-16 pJET1 XL1-Blue No seq F. iinumae F. nubicola 3.0 1836-1 pJET1 XL1-Blue No seq F. nilgerrensis F. mandshurica 3.0 1838-5 pJET1 XL1-Blue No seq Strawberry Festival

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76 GPH5_ananassa_clone2 C AGAAGGTAATATGCATGATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAAT A 582 GPH5_ananassa_clone7 C AGAAGGTAATATGCATGATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAAT A 582 GPH5_viridis TAGAAGGTAATATGCATGATATAAATATCAAGTTAATTGTACA G TGATAT---TTGTAA C C 576 GPH5_iinumae TAGAAGGTAATAT-------------ATCAAGTTAATTGTACAAT A ATAT---TTGTAATC 566 GPH5_nilgerrensis C AGAAGGTAATATGCATGATATAAATA C CAAGTTAATTGTACAATGATAT---TTGTAATC 579 GPH5_mandshurica TAGAAGGTAATA C GCATGATATAAATATCAAGTTAATTGTACAATGATAT---TTGTAATC 578 GPH5_nubicola TAGAAGGTAATA C GCATGATATAAATATCAAGTTAATTGTACAATGATAT---TTATAATC 583 GPH5_vesca TAGAAGGTAATATGCATGATATAAATATCTAGTTAATTGTACAATGATAT---TTGTAA C C 579 ************ ************* **** ** *** GPH5_ananassa_clone2 GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_ananassa_clone7 GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_viridis GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_iinumae GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_nilgerrensis GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGA A CT 240 GPH5_mandshurica GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_nubicola GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGA A CT 240 GPH5_vesca GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 ********************************************************* ** Figure 4-4. Subset of the alignment of GPH5 oc toploid and diploid clone s. Single Nucleotide Polymophisms (SNPs) are in bold font. Same base changes that appear in a determinate position for more than one clone are likely to reflect real differences and are colored red. Hyphens signify indels whereas SSRs are magenta-colored. Figure 4-5. Diagrammatic representation of a lignment of full GPH23 clones, depicting all polymorphisms identified, such as Single Nu cleotide Polymorphisms, insertions, and deletions. Numbers in triangles i ndicate the length of indels.

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77 Figure 4-6. EcoRI Restriction patterns observed for GPH10 clones from the octoploid Strawberry Festival, indicat ing four different allele cl asses. M: molecular weight marker; V: empty vector; 2-20: polymorphic clones Figure 4-7. GPH10 clones, 4 al leles from the octoploid Fragaria ananassa detected by distinct EcoRI (green, vertical arrows) restrictio n patterns. The primers designed to amplify and sequence all 4.4kb clones are repr esented by black and blue arrows. The numbers between primers are the distan ces (in bp) between primers. The boxed region contained most of the polymor phism observed for GPH10, and it is comprehended between primers 10PPR1 and 10AB#22.

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78 72E18_vesca GAAAA-AAA GAGAGAGA --AAATTACAGATTTAAAGCGACGAACAA-TGAAAAGGAATGA 601 72E18_mandshurica GAAAA-AAA GAGAGAGA --AAATTACAGATCTAAAGCGACGAACAG-TGAGAAGGAATGA 594 72E18_nilgerrensis NNAAA-AAA GAGAGAGAGA ---TTACAGATCTAN-GCGACGAACAA-TGAGAAGGAATGA 593 72E18_viridis AGAAATAAA GAGAGAGA --AAATTACAGATCTAAAGTGACGAACAA-TGAGAAGGAATGA 604 72E18_iinumae GAAAAAAAGAA GAGAGA --AAATTACAGATCTAAAGCGACGAACAAATGAGAAGGAATGA 658 72E18_ananassa GAAAAAAAAAA GAGAGAGA AAATTACAGATCTAAAGCGACGAACAA-TGAGAAGGAATGA 638 *** ** ** **** ******** ** ******** *** ********* 72E18_vesca GAGGCAAAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGG GAGAGA 661 72E18_mandshurica GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGG GAGAGA 654 72E18_nilgerrensis GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGT-------GA 645 72E18_viridis GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGG-GAGA 662 72E18_iinumae GAGACAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGT-------GAGAGA 710 72E18_ananassa GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGA----------GTGAGG GAGAGA 688 *** ** ******************************* **** *** *** 72E18_vesca GAGAGAGAGA TCGACGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 721 72E18_mandshurica GAGAGAGAGA TCGACGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 714 72E18_nilgerrensis GAGAGAGAGA TCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 706 72E18_viridis GAGAGAGAGA TCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 722 72E18_iinumae GAGAGAGAGA TCGAAGACGAGGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 771 72E18_ananassa GAGAGAGAGA TCGAAGACGAAGCTGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 749 ************* ***** ** ************************************ Figure 4-8. Subset of GPH72E18 ali gnment displaying SSR polymorphisms.

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79 Figure 4-9. Cladograms of F. ananassa and diploid alleles for six independent GPH loci. Amplified loci were sequenced, aligned with ClustalW, and their relatedness represented through cladograms. The F. iinumae clones were the mo st related diploid to F. ananassa clones in every locus analyzed. F. vesca clones, on the other hand, were the furthest from the octoploid, cont rary to prediction based on data of other authors previous studies.

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80 Table 4-3. Overview of insertions and deletions detected through alignment of all sequenced clones. Each column represents an ali gned region within haplotypes of a specific locus. The aligned regions where an indel or SSR were identified were named with Roman numbers. No relationship between cl ones of different loci is implied by the utilization of the same Roman number, as each locus was analyzed independently from the others. The Arabic numbers signify the number of bases in the deletions or insertions ( minus or plus signs, respectively) in re lationship to the consensus observed. White boxes represent accordance to the consensus sequence for the region in focus. Clone Indels and SSRs in Polymorphic Loci 10PPR1AB22 I II III IV V VI VII VIII IX X XI XII XIII XIV nubicola -5 6 TA mandshurica -5 5 TA vesca -5 8 TA viridis -5 7 TA nilgerrensis -4 +6 +4 -44 ananassa_18 -4 +36 -15 -176 ananassa_20 -4 +19 +3 +7 +71 +6 -12 -15 -181 ananassa_19 -4 +36 -15 -181 ananassa_2 -4 +6 -20 -15 -181 iinumae +5 -4 -566 -8 11D02 I II III IV V VI viridis -4 +8 +44 nubicola +5 vesca +5 iinumae -28 +32 +27 17O22 I II III IV vesca -36 mandshurica viridis +5 -5 nubicola -5 -16 iinumae 27F10 I II III IV V VI VII VIII IX X vesca +2 -2 mandshurica +2 -2 -2 nubicola -9 -2 -3 iinumae -7 +6 +12 -5 -3 ananassa -14 +6 +12 -26 +8 +3 -3 viridis +505 29G10 I II III IV V vesca mandshurica nubicola -1 +2 -13 +7 nilgerrensis -1

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81 Table 4-3. continued Clone Indels and SSRs in Polymorphic Loci 32L07 I II III IV V VI vesca -9 viridis -792 -4 -9 -5 34D20 I II III IV V VI VII VIII IX X XI vesca +4 +38 mandshurica +4 +38 nilgerrensis -7 -13 -11 -3 +3 +15 iinumae -28 -2 -30 -3 ananassa -2 -30 -3 -3 +15 viridis -2 nubicola -2 -13 63F17 I II III IV V VI vesca -2 mandshurica -4 -6 viridis -6 +2 -4 -12 ananassa -2 72E18 I II III IV V VI VII VIII IX X XI vesca 4 GA 8 GA mandshurica -8 4 GA 8 GA nilgerrensis 5 GA 6 GA -11 -13 -18 -10 -44 viridis 4 GA 7 GA +3 iinumae +53 3 GA 8 GA ananassa -11 +45 4 GA 8 GA -96

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82 CHAPTER 5 GENE-PAIR HAPLOTYPES: FUNCTIONAL AND TRANSFERABLE MARKERS AS NOVEL ADDITIONS TO THE DIPLOID Fragaria GENETIC LINKAGE REFERENCE MAP Introduction Strawberry ( Fragaria ananassa Duch.) is an economically valuable fruit crop, with average consumption of over 7.3 pounds per capita in 2005 in the United States (FAO STAT). The demand tends to increase due to public awar eness of the potential health benefits of strawberry: small fruits have been shown to have high content of antioxidants (Wang, 2006), polyphenols and micronutrients that ma y play a role in human health. Despite of the great importance of strawbe rry, knowledge of its genetic composition is very modest. The cultivated strawberry is oc toploid, complicating development of molecular markers and construction of genetic linkage maps. Researchers have resort ed to utilizing wild diploid strawberries to generate the first linkage relationships in the hope of extending the findings to octoploid genomes. The first gene tic linkages identified showed relationships between fruit color (Williamson et al., 1995) and runnering (Yu and Davis, 1995) to the shikimate dehydrogenase and phosphoglucoisomerase loci, respectively. These associations were shown in Fragaria vesca a diploid that has been proposed to be a possible A type genome donor to the cultivated strawbe rry (Potter et al., 2000). The first indirect evidence of F. vesca as a genome contributor to the cultivated octoploi d comes from cytological studies by Ichijima in 1926, where he showed the formation of 21 biva lents and 7 univalents during the pairing between F. vesca (then called F. bracteata ) and F. virginiana the pistillate parent to F. ananassa. The first genetic linkage map developed for strawberry wa s constructed using Randomly Amplified Polymorphic DNA (RAPD) markers deve loped for an F2 population derived from a cross between two subspecies of the diploid F. vesca : ssp. vesca Baron Solemacher (red-

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83 fruited, runnerless) and ssp. americana wild accession WC6 (Davis and Yu, 1997). The map was populated with 3 isozymes and 75 RAPD marker s, of which 11 were codominant. This was possible due to a novel approach to Polymera se Chain Reactions (PCR), using mixed DNA templates for formation of heteroduplex bands (D avis et al., 1995). The locations of six genes involved in the anthocyanin path way were assigned into this map later (Deng and Davis, 2001). The second strawberry linkage map was developed for F. ananassa to increase the knowledge of the octoploid genome and to addr ess questions on inher itance patterns in strawberry (if disomic or polysomic) (Lercet eau-Khler et al., 2003) Amplified Fragment Length Polymorphism (AFLP) markers were used to generate separate maps for the male and the female parents, with 235 ma rkers in 30 linkage groups, and 280 markers in 28 linkage groups, respectively. Though the study generated very deta iled maps, with a total of 789 markers, AFLP markers are not easily transferable between species or even popul ations. The density of markers did add evidence of polysomic inheritance, sin ce genes apparently crossed between subgenomes with some frequency. A third map was constructed for strawberry (Sargent et al., 2004) addressing RAPD and AFLP transferability issues through the use of microsatellite markers or polymorphic Simple Sequence Repeats (SSRs). The map was based on a polymorphic F2 population generated from a wide inter-specific cross between the diploids F. vesca ssp. vesca f. semperflorens FDP815 (pistillate parent) and F. nubicola FDP601 (pollinating parent). Thes e diploids have been shown to be the most closely related diploid relatives to the cultivated octoploi d species (Potter et al., 2000). The creation of a reference map using a diploi d relative is an approach commonly used to map genetically complex polyploids. Examples of polyploids for which reference maps have been constructed are wheat (Kam-Morgan et al ., 1989), alfalfa (Diwan et al., 2000), and potato

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84 (Milbourne et al., 1998). The map published in 2004 had 78 markers and new microsatellite loci were added later, totaling 182 markers (Sargent et al., 2006). Strawberry belongs to the Rosaceae family, to which the horticulturally important peach, cherry, apple, raspberry, and rose also bel ong. Although SSRs are markers transferable between mapping progenies within and between species (Dirlewanger et al., 2002) (Hadonou et al., 2004), they are generally not transferable between ge nera. The challenge in developing transferable markers resides in the fact that markers are, by definition, placed on polymorphic regions of the DNA and, to be transferable, such markers are must be located on conserved regions. A recent study (Sargent et al., 2007) explored intr on length polymorphisms, having PCR primers anchored in flanking exons that were conserved across Prunus and Malus and thus generated highly transferable markers. In addition, because these markers were gene-linked, they also provided functional information. A new approach to development of transfer able and functional markers was explored by this research. The innovative mapping tool, named Gene-Pair Haplotype (GPH) consists of a stretch of intergenic space and takes advantage of its rich polym orphism for the development of markers. GPHs are PCR-amplifiable, with PCR primers anchored to exons of adjacent genes, making these makers transferable between spec ies where microcolinearity is maintained. A significant degree of conservation between Fragaria, Medicago and Arabidopsis has been demonstrated (T. M. Davis, personal communication ) suggesting that these same intervals might be easily transferable between rosaceous crops. This investigation aimed to introduce the ge ne-pair haplotype con cept as an innovative mapping tool, thereby increasing th e number of transferable and functional markers genetically linked to the existing F. vesca x F. nubicola diploid reference map.

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85 Materials and Methods The diploid mapping populat ion generated by Sargent et al. (Sargent et al., 2004) (a cross between F. vesca ssp. vesca f. semperflorens FDP815 and F. nubicola FDP601) was used in this study. Lyophilized tissue was received from the rosaceous genomics research group in East Malling Research station, in Kent, England. DNA was extracted as described in protocol #29, appendix A. Approximately 7 mg of lyophilized tissue was frozen in liquid nitrogen and ground in a mortar to a fine powder. After addition of 1ml of extraction bu ffer (2% CTAB, 1.4M NaCl, 100mM Tris-HCl pH 8.0, 20mM ED TA pH 8.0, 1% 2-mercaptoethanol), the tissue was further macerated until no defined leaf particles were observed. The volume was split into two 1.5-l tubes, samples incubated at 65C for 1h, and 1 vol of 24:1 chloroform:octanol was added to each tube. After mixing the organic solvents with th e extraction buffer and plant tissue, the samples were centrifuged at 13,000 rpm for 5 min. The upper phases were transferred to new tubes, and the nucleic acids precipitated by equal volum e of isopropanol, centr ifuged, the supernatant discarded, the pellet air-dried, and resuspe nded in 50l TE pH 8.0. The DNA concentration varied from 40ng/l to 4,543ng/l. Target regions for marker development were derived from the F. vesca Pawtuckaway sequence annotation described in Ch apter 3. Similarities between the F. vesca genomic sequence and either proteins or ESTs were sought for each of the 26 fosmid insert sequences. Within each fosmid clone, the most suitable pair of genes fo r PCR amplification was determined according to the following criteria: i, The putative intergenic space should be large enough to permit detection of polymorphisms, but not larger than 3.5 kb due to technical limitations of amplification by PCR. Putative genes in fosmids 15B13 and 22L05 were separated by > 4kb, therefore these clones were excluded from the potential GPH pool.

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86 ii, Tandem and non-tandem duplications were av oided as potential targets for PCR primer design. Target sequences should be unique to yield locus-specific am plification, since the assessment of more than one locus at a time w ould complicate data scoring. Tandem duplications were detected when adjacent F. vesca query sequences that had the same BLASTX hit, which appeared to indicate gene fa mily clusters (e.g.: putative genes in fosmids 05N03, 13I03, and 18A19). An exception was made for the chalcone synthase (CHS) gene, which was included in the study although tandemly duplicated. The intergenic region is ~2,300 bp in F. vesca 'Pawtuckaway', but varies from 2 kb to over 8 kb in different rosaceous species tested, making this marker transferable across genera (T. M. Davis, personal communication ). Non-tandem duplications required indirect evidence, since only 1% of the F. vesca genomes sequence was available for analyses. If nucleotide identity was detected through BLASTN between a F. vesca sequence and more than one locus belonging to a single organism, that was regarded as eviden ce of potential duplication in F. vesca iii, Some fosmid clones did not appear to contain gene pairs when similarity to databasedeposited protein sequence was the criterion adopted to classify a sequence as a putative gene. In those cases, a potential gene pair was inferred by two sequences di splaying similarities, one to proteins and the other to ESTs. Once apparent single copy, P CR-amplifiable putative gene pairs were identified, the software Primer3 (Rozen and Skaletsky, 2000) was us ed to facilitate design of PCR primers. For each primer pair, the forward primer was designe d on the 3 end of a putative exon sequence of a gene, whereas the reverse primer was designed in the 5 end of the putative exon sequence of the downstream gene. In some cases, more than one pr imer pair was designed to generate a single band product, polymorphic between the parentsbefo re or after restrict ion digest. In those

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87 cases, a primer with the fosmid clone name a nd orientation (F or R) had a suffix added to indicate another set. Figure 5-1 illustrates the case of pr imers designed for fosmid 40M11. The PCR amplification 50l-reaction component s and conditions for the parental DNA and for the 94 F2 samples were: 1x buffer (at 10x concentration, com position was 35mM MgCl2, 37.5g/ml BSA, 160mM KCl, 400mM TricineKOH pH 8.0), 0.2mM each dNTP, 0.2M each primer, 0.05unit Taq polymerase, 1l DNA template, at variable concentr ations (40ng/l to 4.5g/l). Initial denaturation: 94C, 2 min, followed by 35 cycles of: denaturation at 94C for 15 sec, annealing for 45 sec, and extension at 72C A last extension of 5min at 72C after the 35 cycles was executed. Table 5-1 contains functiona l information of the gene pair amplified, as well as annealing temperatures and the extensi on times dependent on the primer pair used. In general, extension was carried out for 1 minute per kb amplified, and the annealing temperature was primarily based on the primer melting temperature (Tm) calculated by Primer3 (Rozen and Skaletsky, 2000) using the formula described in (Rychlik et al., 1990) (though in many cases a range of temperatures had to be tested). Po sitive control primers FvLFYintron2F/ FvLeafy3' are anchored to the exons that flank Leaf y genes second intron (P. J. Stewart personal communication ). This intron size is variable among diploid species, being 770bp-long in F. vesca Pawtuckaway. The annealing temperature and ex tension time were variable, since the primer pair under investigation was their determinant. Following successful PCR amplification, 10l of each single-band amplicons were digested with 1unit of different restriction enzymes (table 5-2) in a total volume of 20l. Amplicon polymorphisms were resolved in 2% ag arose gel, 1x TAE buffer, 0.5g/ml ethidium bromide, at 80V, during variable times that were a function of the size of the digested fragments. The gel was exposed to 300-nm UV light for visualization of DNA fragments.

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88 In order to obtain the most precise linkage, analyses were performed against the data set presented in Sargent et al. (Sargent et al., 2006), including new information available since the last publication. The novel GPH markers were assigned into linka ge groups utilizing the software JoinMap 3.0 (Van Ooijen and Voorrips, 2001) wi th the application of the Kosambi mapping function and a minimum LOD score threshold of 3.0. The maps presented were constructed using MapChart software (Voorrips, 2002). Results Amplification was observed for all primer pair s, though not all were suitable for mapping purposes. Eight GPH primer pairs produced single -band amplicons that were scorable after restriction digest. The remaining primer pairs we re not scored for the population for a variety of reasons. Primer pairs 01L02Fb/Rb, 01L02Fb/ Rc, 22H18F/R, 22H18F/Rb, 30I24F/R, 32A10F/R, 32A10Fb/R, and 38H02F/R amplified multiple bands even at stringent annealing temperatures and restrictive extension times. The banding pattern for 01L02Fb/Rb appears to be due to a duplication, since two major bands ar e detected, one of the expected size, the other with higher molecular weight. Amplification by the other prim er pairs displayed multiple bands, similar to non-specific amplification. There were primer pair s for which amplifications were observed, but they were not polymorphic (e.g. 10B08FbRb). For others, amplicons were polymorphic, but only a few members of the F2 population were amp lifiable. This was the case of both 10B08 (GPHleafy/GPHacs, which amplified a 3.8 kb region that was polymorphic when digested with EcoRI ) and 32L07F/Rb, polymorphic after treatment with HaeIII Both parents, when amplified by primers for 34D20, produced amplicons that were the same size. Restriction digestion revealed a rather complicated banding pattern. All of the digested amplicon fragment sizes < 700bp observe d for 34D20 were expected, according to the

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89 predicted restriction pattern for F. vesca Pawtuckaway. An unexpected fragment of 1249 bp was observed for F. vesca raising a concern that the putative single locus was in fact two loci. The other possibility was that the higher molecular weight band was a different F. vesca allele from the same locus. Had that been the cas e, a heterozygote should have been observed containing the female allele (1249, 300, 251bp) and the male allele (702, 335, 308, 251 bp). Such an individual was not observed, as 1249bp band cosegregated with the 758 and 429 bp bands. The presence of the 1.25 kb band was attributed to partial restriction di gestion and the scoring was therefore carried out based sole ly on the expected 758 and 429bp bands versus the 702 and 335bp bands. Figure 5-2 shows banding pattern fo r digested amplicons of 34D20 and 72E18. GPH40M11 is a dominant marker and amplif ies a band only for the pistillate parent, F. vesca Since the PCR amplification was precluded fo r half of the F2 population for some reason, this raised a concern about wrongly scoring individuals as homozygous F. nubicola Thus, amplification patterns for all other 7 loci were comp ared, using a primer pair as positive control. Individuals for which amplification was observed in all those primer pairs but not observed for 40M11 were scored as homozygous for the F. nubicola allele. The majority of the GPHs investigated were assigned to linkage group VII, as shown in figure 5-3. Discussion Gene pair haplotypes are intergenic, multiple character signatures that define suites of variability between two genomes. The purpose for these markers is to provide a complex field of discrete variation that can be related to a specific subgenome donor with the goal of eventually mapping genes to specific subgenomes of the oct oploid strawberry. This chapter outlines the

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90 first step in this process, that is to test if intergenic variability could be used to assign GPH loci to the diploid linkage map. In all cases the GPH loci were assigned to the linkage map using a CAPS marker approach. Here amplicons were digested with a restricti on enzyme that corresponded to sequence variation in the parental lines. A mappi ng population was treated with iden tical conditions to reveal the genotype of the specific F2 plant. Analysis of segregation with isozyme, morphological and molecular markers allowed assignment of thes e GPH loci to the diploid linkage map. The assignment of these loci to the current ma p is important for two reasons. First, it demonstrates that the GPH is a viable markerin this case based on a single restriction site. Other variable characters certainly exist in th ese regions that will complement the detection noted by this restriction site. In the future, th ese GPH loci will likely serve as anchors for the octoploid linkage map, because their likely variabili ty supercedes that which is possible from a simple SSR or other marker used for diploid mapping. This study places markers on linkage groups I, VI, and VII, with several independent markers in the latter. The next step is to translate these markers to an octoploid mapping population. This will immediately bring relevance to the endeavor because GPH loci stem from or are located near genes of known function. In this study GP H 17O22 is localized near F3H whereas 73I22 associates with chalcone synthase two genes necessary for fruit color production and protective leaf pigments. A breeder with an interest in improving fr uit color or possibly increasing plant survival in high light environm ents may find such loci useful in breeding selections. The localization of the CHS gene determined by the GPH approach was different from the linkage group to which the gene was assigned when intron length was used to map it in a F.

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91 vesca ssp. bracteata DN1C x F. vesca ssp. vesca Yellow Wonder F2 population (Deng and Davis, 2001). This may be evidence of mu ltiple copies of the CHS gene in the Fragaria genome. While described as a single-copy gene in Brassicaceae (Koch et al., 2000), CHS is a multigene family in many plant species (Jin-Xia et al., 2004). The CHS gene family is comprised of at least seven members, which, at least in petunia and po plar, are mapped to diffe rent linkage groups: II and V (Koes et al., 1987), and I and III (Tsai et al., 2006), respectively. It is possible that the different localizations in the genome correlate wi th different gene functions. In common morning glory ( Ipomoea purpurea Convolvulaceae) (Durbin et al., 1995) as well as in Gerbera hybrida (Asteraceae) (Helariutta et al., 1996) different family members have shown to have functional divergence. The experimental outcomes of this chapter va lidate the use of GPH loci for mapping in the diploid strawberry and suggest great utility in application to octoploid mapping and breeding populations. Their complex characters, ease of detection, coupled to apparent disomic inheritance within octoploid subge nomes, indicate that these ma y be implemented in practical breeding scenarios. Conclusions The experimental trials outlined in this work test various aspects of strawberry structural genomics. From difficult honing of protocols to hasten DNA prepara tion from recalcitrant tissue, to computational anal yses, development and proof-ofconcept assessment of a novel molecular marker, these trials present new f acets of understanding the complicated genome of the cultivated strawberry. Recalcitrance to DNA extraction from plants is commonly attributed to their polyphenol and carbohydrate contents. Strawberry appears to be recalcitrant not only due to high sugars and phenols, but also because of strong physical barr iers that guard the DNA. The results of over 103

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92 systematic tests of various experimental c onditions indicate that the most important consideration is complete disruption of the tissu e via maceration, and that this process may be greatly enhanced by co-application of chemical lysis to disrupt tissu e. My study provides a comprehensive evaluation of all published techniques and provides a unified protocol that works to some degree in all strawberry cultivars and species tested. The importance of sequence information as a foundation for functional genomics studies in strawberry has been revealed by the discovery of enzymes associated with flavor (Wein et al., 2002) and fruit firmness (Llop-Tous et al., 1999 ) (Benitez-Burraco et al., 2003). This project represents the first efforts to examine the genome structure of F. vesca The data indicate that the small genome of F. vesca maintains a character and compostion similar to other model plant species, suggesting that this species will have ut ility in answering questio ns within the Rosaceae family. Annotation of fosmid inserts leads to the understanding of gene c ontent and distribution, and permits marker generation for linkage mapping. More importantly, this initial survey of the strawberry genome is the first opportunity to comp are strawberry to sequen ces to those of other organisms. Here relationships between the ge neral properties of the genome have been deciphered. Strawberry is a gene-d ense organism that maintains a significant content of mobile elements, and microcolinearity with other known genomes (T. M. Davis, personal communication ). Detection of gene pairs by search ing for micro-colinearity between F. ananassa and Arabidopsis is a clever approach, but it needs to be automated to increase the chances of finding adjacent genes. This approach has th e advantage that it is not based on F. vesca sequence. Therefore, amplification of hapl otypes is not biased towards F. vesca -like alleles. In addition,

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93 because sequences utilized for similarity search were from F. ananassa this method is better than the annotation of F. vesca genome method to address ques tions of diploid subgenome contributions to the octoploid. Primer pairs desi gned for gene pairs detected through this method amplified the octoploid, whereas most (8 out of 11) of the primer pairs generated through F. vesca genomic sequence did not amplify alleles fr om the cultivated strawberry. This study further supports the likelihood of F. iinumae as the B genome donor to the octoploid. The approach based on gene prediction to iden tify gene pairs, had a higher amplification success rate and it is useful to characterize in tergenic regions, serving as a tool to detect polymorphisms between diploids. Chapter 5 s howed how this approach was successfully employed to create molecular markers in the Fragaria diploid reference map. We have described the development and mapping of 8 markers, linked to at least one gene of known function. Therefore, this inve stigation proved the concept th at putative intergenic regions may be used as functional markers. In additi on, because the markers ar e designed for conserved sequences across different taxa in Viridiplantae, th ere is great potential for transferability and use on comparative mapping to appreciate Rosaceae structural genomics.

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94 Figure 5-1. Fosmid 40M11 with primers design ed on exons of FGENESH-predicted genic regions. Table 5-1. PCR primer pairs and amplific ation conditions used in this study Primer Putative Gene Function or EST gb number Sequence 5 to 3 Tannealing (C) Extension Time Control F FvLFYintron2F Leafy CACTGCCAAGGAGCGTGGTG Control R FvLeafy3' Leafy TCAGTAGGGCAGCTGATG variable variable 01L02Fb EST AY573376 GAACCGTTCAAGTTCATAATTGG 01L02Rb unknown protein AAGGGAGGACGTTCAATGTG 54-65 130230 01L02Rc unknown protein ACGGAGATCGGGGACTTGT 54-58 230 10B08F Leafy protein GGGCCAACTACATCAACAAGC 10B08R ACC synthase TGTTCTGTTGGGTGGACATGA 58-63 3-4 10B08Fb ACC synthase TGCCATCGTTTCCATCAGTA 10B08Rb ribosomal protein CGCGAAGATCATGAAGAACA 52 1 11D02F EST BQ105541 GAGCTGCTGTGTGAACCAAA 11D02R heat shock binding protein GTTCAACTCCAGATGAAGTGAGG 56-60 230 17O22F Oligopeptidase AAAATGGGTTGCACGAGTTC 17O22Rb Putative protein GGGTTTCCTCACAAACTTCG 60 2 17O22Fb Oligopeptidase GGTACCTCCAATGCAAGGAA 17O22R Putative protein TTCATCAGAGAAGGCGGACT 53-60 1 2 22H18F EST DY646954 ACCAATGCTTGGACACACAC 22H18R unknown protein GATGAAATTCCATGCTTGTGAC 52-65 230 22H18Rb unknown protein GGACTCCATGTAACACGGCTA 56-65 230 27F10F kinase CCTGCAGGGTTTTTCATCAT 27F10R hypothetical protein TGGAAATGTATTCTGGTTCTCC 59 1 29G10F phenylacetaldehyde synthase TGGCCTTGTTTCCTAAACTCTT 29G10R unknown protein AGAAGAAGGCAGCACCCAAT 59 1 30I24F transferase TTGAGAGAGGTCTCCAAGCTC 30I24R chromating remodeling factor CGGAAGATGGCAAGCTATTG 54, 59 4 32A10F CGGAGAGAACGATGGAGTTG 1 32A10Fb isomerase (E > 10-12) CCAAATGAATCAAGCTCAAGTG 32A10R pathogenesis-related protein ATTGTCGACCAGTGCAGCAA 52-62 130 32L02F GAGTTGAAAAACGGGTCGAA 32L03Fb CCTTCCAAGGTCACCTCCTT 32L02Fc SMC2 (Structural maintenance of chromosomes) TTAGCCCGGTTATGGAGTTG 32L02R GAAGGTTCAAGGAGCATGGA 32L02Rb AGGAAAATGCGGGAGAAAGT 32L02Rc Exostosin GAACGATTTCCGAGGTGTGT 53-61 2

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95 Table 5-1. continued Primer Putative Gene Function or EST gb number Sequence 5 to 3 Tannealing (C) Extension Time 34D20Fb RNA recognition motif GCAGAAAGAAACTGATGTGCTT 34D20Rc cysteine-type peptidase CGCAGTCGTAAAAATTCGTCT 60 330 38H02F serine/threonine kinase CCAGGCCTAAGCTTGTCATC 38H02R exportin AAGGCATTGAAATCATTCTACCA 53, 54, 60 4 40M11F ACACAGGTCATTGGGTCCAT 40M11Fc F-box protein TTGACCCGGATAACATGGAT 40M11R transposase (E > 10-13) GTGTTGCACAAGTCCATTCG 40M11Rc expressed protein (E > 10-9) CTGACAGCGAATCAATCTGC 40M11Fb GGCCTTCTTGACATTCCAGT 40M11Fd secretory protein SEC14 CAACATTTTGGTGGCCTTCT 40M11Rd CGGCCTATGAAACCACAGTT 60 4 40M11Rb ATPase TGGGGTTGTTGGAAAGAGAG 63F17F phospholipase D CGCTCTATGGAAGGGACAAG 63F17R unknown protein TTAAGGGGTCTGTTGATGTGC 59 1 72E18Fb actin GCTAGGGAAAACAGCTCGTG 72E18Rb elongase TGGGTTTGGTTTTGGGATAA 60 230 73I22F chalcone synthase A CAAGCCTGAGAAGTTAGAAGC 73I22R chalcone synthase B GAAAGTAGTAGTCGGGGTATGT 62 5 GPH10a unknown protein GGCTTCTTCTTGTCCGGCAGC GPH10b unknown protein GAACTCCAGGTCAGATCTTCG 230 GPH10c unknown protein CTCGCTGCAAATCAGCTACC 4 Table 5-2. Fragment sizes of parental amp licons digested with restriction enzymes Locus Restriction Enzyme Amplicon estimate fragment sizes (bp) Non-digested Digested F. vesca F. nubicola 17O22FRb RsaI 1,374 486, 413, 292, 83, 67, 28, 5 511, 414, 293, 83, 67, 28, 5 34D20FbRc AluI 2,050 1249, 758, 429, 300, 251, 107, 69, 48, 26 702, 335, 308, 251, 107, 69, 48, 45, 41, 26 40M11FdRd Dominant marker 3,100 present absent 63F17 HaeIII 1,266 992, 234, 40 840, 234, 40 72E18FbRb HhaI 2,620 1,400, 800, 300 2,300, 300 73I22 PvuII 3,000 2,200, 1,000, 600 2,200, 1,500

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96 Figure 5-2. Amplicon restriction patterns for GPHs 34D20 and 72E18. M: molecular weight marker; U: uncut amplicon; P1: female parent, F. vesca P2: male parent, F. nubicola ; H: heterozygote.

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97 Figure 5-3. Gene-Pair Haplotypes assigned to linkage groups of the reference Fragaria map.

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98 APPENDIX A DNA EXTRACTION PROTOCOLS The numbered items bellow represent different protocols, whereas numbers preceded by a T signify treatment number and correlate with the treatment numbers used in Table 2-1. In all protocols that used either 2-mercapto ethanol, sodium bisulf ite or sulfite, these reducing agents were added just prior to use of buffers. Most pr ocedures included at least one 25:24:1 phenol:chloroform:isoamyl alcohol deproteination step followed by one 24:1 chloroform:octanol extraction. When RNAse-trea ted, the enzymatic reaction was carried out at 50 g/ml. Precipitation of DNA was executed by adding 0.7 to 1 volume of isopropanol or by sodium acetate to reach final concentration of 0.3M plus two volumes of absolute ethanol, then washed with 70% ethanol, dried, and resuspended in sterile, deionized water. Except for buffers that involved guanidine thiocyanat e, which were kept at room te mperature, plant material in buffer was incubated 30-60 minutes at 65C, unless otherwise stated. When product was obtained, 5-10 g of DNA were digested with 2-4 re striction enzymes. Below is a brief description of each protocol. DNA Extraction from Leaves 1. Tomato [Fulton, 1995]: utilizes a comb ination of a DNA extraction buffer (0.35M sorbitol, 0.1M Tris-bas e, 5mM ethylenediaminetetraacetic acid, EDTA, pH 7.5) and a nuclei lysis buffer (0.2M Tris, 0.05M EDTA, 2M NaCl 2% CTAB) to make the micro prep buffer (42% extraction buffer, 42% nuclei lysis buffer, 16% sarkosyl 5%, and 0 .02% sodium bisulfite). Used 0.5g (T1), 1g (T2), and 2g (T3) of Strawberry Festival fresh mature leaf tissue, extracted by 5ml buffer. 2. Woody plants [Kobayashi, 1998], modified by A. M. Hadonou. Two extraction buffers are consecutively used, buffer 1 being used twice and the buffer 2 only once. Following centrifugation with buffer 1 (50mM Tris-HCl pH 8.0, 5mM EDTA, 0.35M sorbitol, 0.3% 2mercaptoethanol, 10% polyethele neglycol, PEG), the supernatan t is discarded before adding buffer 2 (50mM Tris-HCl pH 8.0, 5mM EDTA, 0.35M sorbitol, 0.3% 2-mercaptoethanol, 1% sarkosyl, 0.7M NaCl, 0.1% CTAB). Used 0.1g of two cultivars of F. vesca ssp. vesca f. semperflorens : Yellow Wonder (T4, T6) and Alexandria (T 5, T7); fresh expanded leaf tissue, extracted by 1ml (T4, T5) or 10ml (T6, T7) of buffer. 3. Guanidine thiocyanate [Chomczynski, 1987] : The incubation was carried out for 5-15 minutes only and at room temperature instead of 65C. Buffer composition: 4M guanidine thiocyanate, 100mM Tris-HCl, 10mM EDTA, 0.5M NaCl, 1% sa rkosyl, 1% sodium sulfite. Newly expanded (T8) and unexpanded (T9) leaves of Sweet Charlie were used for extraction from 100mg tissue in 100l buffer. Further treatments to aliquots of the product of this prep were performed, aiming removal of contaminants: adsorp tion to a column from the DNeasy Plant Mini kit (T10) or dialyses into TE pH 7.0 at 4C (T11). Dialyses was performed overnight, TE buffer replaced by fresh buffer, and dialyzed again for another day. Sample was 50 g/ml RNAseand 150 g/ml proteinase K-treated. DNA isolation was continued with phenol extraction and standard downstream steps. 4. Guanidine thiocyanate and CTAB util ized consecutively (T12): DNA isolation according to Chomczynski [Chomczynski, 1987], and resuspension of the ethanol-precipitated DNA in CTAB buffer described in Chang [Chang, 1993] for re-extraction, an attempt to rid

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99 DNA prep of polysaccharides. The incubations were carried out at room temperature and 65C with guanidine thiocyanat e and CTAB, respectively. 5. Guanidine thiocyanate and CTAB used simu ltaneously: extraction bu ffer kept at room temperature, 15 minutes: 4M guanidine thio cyanate, 100mM Tris-HCl 10mM EDTA pH 8.0, 0.5M NaCl, 1% sodium sulfite, 1% sarkosyl, 2% CTAB, 1% PVP, 2% 2-mercaptoethanol. Treatments included extraction from 10mg (T 13, T15) and 100mg (T14, T16) of lyophilized (T13, T14) or fresh (T15, T16) tissues. 6. DNAzol Extra Strength kit [Chomczynski, 1997] : incubation at room temperature, as suggested by manufacturer. Exact composition of buffers is cryptic, though it is known to contain a guanidine detergent. Tested extrac tion from 100mg (T17, T19) and 500mg (T18, T20) of lyophilized (T17, T18) or fresh (T19, T20) tissues. 7. Pine tree [Chang, 1993]: this protocol was or iginally designed fo r RNA extraction and was adapted here to DNA extraction by omitting th e lithium chloride step. Buffer: 2% CTAB, 2% polyvinyl pyrrolidone (PVP), 100mM Tris-HCl, 25mM EDTA, 2M NaCl, 0.5g/L spermidine, 2% 2-mercaptoethanol. After the ad dition of equal volume of chloroform, samples were homogenized using a Polytron for 1 minute, at 9/10 of maximum speed. Tissue: 0.5g in 7ml buffer (T21). 8. Urea [Settles, 2004]: Phenol deproteination st ep was done together with incubation with extraction buffer, at room temperature for 20 mi nutes in 8M urea, 0.4M NaCl, 60mM Tris-HCl pH 8.0, 25mM EDTA pH 8.0, 1.5% sarkosine (T22). A variant of the buffer was also experimented, which consisted of supplementati on with 1% sodium sulfite and 1% PVP to prevent oxidation of phenols (T23). 9. Strawberry (Manning, 1991): Buffer: 0.2M Tr is, pH adjusted to 7.6 using boric acid (which forms complexes with polyphenols at pH 7.5 (King, 1971) and with carbohydrates (Gauch and Dugger Jr., 1953)), 10mM Na2EDTA, 0.5% SDS, 2% 2-mercaptoethanol. After a 10-minute incubation at room temperat ure, equal volume of 25:24:1 of phenol:chloroform:isoamyl alcohol was added, mixed, and centrifuged for 10 min at 3,500rpm. Upper phase was transferred to a new tube (cal led Tube A here). T ube B contained interand lower phases from this first round of ch loroform extraction. A second volume of extraction buffer was added to Tube B and a second round of chloroform extraction took place. The new upper phase from Tube B was combined with Tu be A and split into 6 aliquots. Two aliquots (T24, T27) had polysaccharides precipitated by addition of 0.4 volume of 2-butoxyethanol, iced for 30 minutes, and centrifuged at 3,500rpm for 10 minutes. The othe r four aliquots were diluted by 2.5 (T25, T28) and 4 volumes (T26, T29) of a combination of 1M Na acetate buffer (pH adjusted to 4.5 by acetic acid) a nd water. The relative volumes of water and 1M Na acetate/acetic acid buffer were calculated to raise the Na concentr ation to 80mM. Considering that at this point each treatment had a volume of 3.3ml, the dilution by 2.5 volumes brought the volume to 8.3ml. Therefore, 664l of the 1M Na acetate/acetic ac id buffer and 4.3ml of water were required to reach the desired concentration of 80mM Na+. In the case of the dilution by 4 volumes, and still considering initial volu me as 3.3ml, the final volume was 13.2ml. The sample received 10ml of (water+ sodium buffer), of which 9.2ml were water and 800l were the 1M Na acetate/acetic acid buffer. After dilutions were made, T25, T26, T28, and T29 were precip itated as before: 2butoxyethanol, were iced, and centrifuged. The goal of the centrifugation here is to precipitate polysaccharides, not nucleic acids. The six supernat ants were transferred to new tubes and equal volumes of 2-butoxyethanol were added to precipitate nucleic aci ds. After icing for 30 minutes, the tubes were centrifuged for 10 min at 3,500rpm, the supernat ant discarded, and the pellet

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100 washed with a 1:1 solution of 0.2M boric acid/Tris, 10mM Na2EDTA (pH 7.6) : 2butoxyethanol. Pellets were washed with 70% ethanol, 0.1Kacetate/acetic acid (pH 6.0), then with absolute ethanol. After dr y, pellets were resuspended in 1m l water, and 10g DNA digested with restriction enzymes. An a liquot of one of the treatments (T28) was EcoRI-digested before and after treatment with 150g/ml Proteinase K and with phenol:chloroform. A second attempt to isolate digestible DNA using the strawberry protocol was made, addi ng antioxidants 4% PVP and 5mM ascorbic acid to the extraction buffer (T32-T35). 10. Several attempts were made to determine which isolated variable in the strawberry protocol plays the major role in DNA yield. The possi bilities raised were: i, the SDS, rather than CTAB, nature of the protocol. Treatment nu mbers T16, T18, and T24 used SDS, therefore testing this variable; ii, the boric acid, instead of HCl, used to adjust the pH of Tris; iii, the reextraction of interphase formed after chlorofo rm treatment; iv, the dilution that raised Na concentration to 80mM prior to DNA precipitati on; v, precipitation by 2butoxyethanol in place of isopropanol or ethanol. The isolated roles of boric acid and 2-butoxye thanol in DNA isolation were addressed by using a buffer similar to the one proposed by Murray and Thompson, but adjusting the pH of Tris to 7.6 with boric acid rather than HCl (buffer: 200mM Tris/borate, 200mM EDTA, 2.2M NaCl, 2% CTAB, 2% 2-mercap toethanol, 2% PVP), and precipitating one treatment with isopropanol (T30) and the other with 2-butoxyethanol (T31). 11. The strawberry protocol suggests two differe nt dilutions (2.5 volumes or 4 volumes) to elevate the Na+ concentration to 80mM. The chosen d ilution here was the 2.5vol. An experiment was set up to test the merits of the combinations of two factors: i, re-extr action of the interphase by extraction buffer and chloroform; and ii, DNA precipitation by 2-butoxyethanol. The former factor was tested by keeping each, the first a nd the second extraction rounds, as separate treatments, therefore determining the gain in DNA yield given by the second extraction. The latter factor contrasted the use of isopropanol versus 2-butoxyethanol, where T32=first extraction round/isopropanol; T33=first extraction round/2-butoxyethan ol; T34=second extraction round/isopropanol; T35=second ex traction round/2-butoxyethanol. 12. Finally, 2-butoxyethanol was used in the gua nidine thiocyanate protocol (number 3). The treatments were essentially the same as desc ribed for T8 in protocol number 3, except that 2% 2-mercaptoethanol was added to the extrac tion buffer and the Tris was adjusted by boric acid, not HCl. 100mg of tissue processed by 6ml bu ffer. Nucleic acids precipitations were done by isopropanol (control, T36), and 2-butoxyethanol (T37). 13. According to an article th at proposes a method to isol ate DNA from cashew (Rout et al., 2002), boric acid can be used in replacement of Tris, instead of assuming the role of simply adjusting the pH of a Tris solution. The buffer composition used in treatment T38 was 1M boric acid pH 8.0, 2mM EDTA, 1.4M NaCl, 4% CTAB, 0.2% 2-mercaptoethanol. 14. Epicentre kit. Used 10mg (T39), 30mg (T 40), 100mg (T41) of Strawberry Festival leaf tissue with 300l buffer. 15. PowerPlant DNA Isolation kit from MO BIO (T42). A leaflet (350mg) of fresh FRA520 ( F. nubicola ) was ground with liquid nitrogen in mi crofuge tube. The remaining steps were carried out according to manufacturers directions. 16. Qiagen DNeasy Plant Mini kit (T43). Followe d companys directions for fresh tissue. 17. Silica-based DNA extraction. Nucl eic acids tend to adsorb to silica in the presence of chaotropic salts, such as sodium iodide (N aI) (Vogelstein and Gillespie, 1979), guanidine thiocyanate, and guanidine hydrochloride. The binding capacity depends on the solutions ionic strength and pH, being higher at concentrated solutions and pH<7.5 (GeneClean Manual). Silica

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101 columns have been used elsewhere to eliminate polysaccharide contaminants, which is verified by increase of the ratio A260/230 (Abdulova et al ., 2002). The protocol used here was based on Rogstads article (Rogstad, 2003), which uses a CTAB extraction buffer and describes the preparation of the silica binder. CTAB extrac tion buffer: 2% CTAB, 1.4M NaCl, 100mM TrisHCl pH 8.0, 20mM EDTA pH 8.0, 1% 2-mercaptoet hanol. Strawberry Fe stival leaves were ground (10mgT44 and 100mgT45) and 5 ml of extraction buffer were added. Incubation was carried out at room temper ature for 30 minutes. Equal volume of chloroform:octanol was added, samples were centrifuged, the upper phase was transferred to a new tube, and 2.5ml of silica binder were added. The mixture was agit ated thoroughly for 5 min, then centrifuged. The supernatant was discarded, and 4ml of silica wash (25% isopropanol, 25% ethanol, 100mM NaCl, 10mM Tris-HCl pH 7.4, 2mM EDTA pH 8.0) were added, vortexed to resuspend the silica. Samples were centrifuged, supernatant discarded, and a sec ond wash took place. The silica pellet was dried for 2 hours at 37C, and the DNA was eluted by 1ml of ultra pure water, vortexed, and incubated at 65C for 5 min. After centrifugation, the upper phase was transferred to a new tube, RNAse-treated, then DNA was precipitated by isopropanol. The following protocols (18-22) attempted to extract DNA from nuclei isolated from leaf tissue. Protocols 23-33 consist of variations of the protocol by Murray and Thompson and utilized leaves (rath er than isolated nuclei) for DNA extraction. DNA Extraction from Isolated Nuclei Nuclei were purified accordi ng to the procedure described by Folta and Kaufman [Folta, 2000] and nuclei were recovered from the 35/80 inte rphase of percoll gradients. Nuclei were incubated with each extraction buffer at 65C fo r at least 10 minutes. The following buffers were mixed to 50-150 l of purified nuclei in storage buffe r as an attempt to extract DNA: 18. Qiagen DNeasy Plant Mini kit. Differe nt volumes (50lT46 and 150lT47) of isolated nuclei were processed acco rding to manufacturers directions. 19. Fultons nuclei lysis buffer [Fulton, 1995], supplemented with 0.5% sodium bisulfite: 200mM Tris pH 7.5, 50mM EDTA pH 8.0, 2M NaCl 2% CTAB. Two tubes, one 50l nuclei (T48) and the other containing 75l nuclei (T49), were incubate d with 200 and 75l of nuclei lysis buffer at 65C for 45min. Phenol:chlor oform followed by chloroform extractions took place, the upper phase transferred to a ne w tube, and DNA precipitated by isopropanol. 20. Petersons procedure [Peterson, 1997]: 20% SDS was added to a final concentration of 2% and mixed with 50l nuclei (T50) or 150l (T51) by gentle inversion to lyse the nuclei. The mixture was incubated in water bath at 65C for 10 minutes, cooled to room temperature, then 5M sodium perchlorate was added to reach fina l concentration of 1M. Sodium perchlorate is used to dissociate nucleic acid-protein co mplexes [Wilcockson, 1973]. Following centrifugation, the upper phase was transferre d to a new tube using a la rge-bore tip. After a phenol deproteinization step, the aqueous phase was dialyzed twice, th e first overnight and the second for an entire day, both into TE pH 7.0 at 4C. Samples were consecutively treated with 50 g/ml RNAse for 1 hour and with 150 g/ml proteinase K. Af ter extractions with phenol:chloroform/isoamyl alcohol and chloro form/isoamyl alcohol, DNA was precipitated and resuspended. 21. Guanidine thiocyanate buffer (4M guanidi ne thiocyanate, 100mM Tris-HCl, 10mM EDTA, 0.5M NaCl, 1% sarkosyl, 1% sodium bisulfite) was used (750l) to extract DNA from 50l nuclei (T52). The buffer/nuclei were incubate d at room temperature for 10min and were

PAGE 102

102 followed by phenol:chloroform and chlorofo rm extractions. DNA was precipitated by isopropanol. 22. Use of triisopropylnaphthalenesulfonic acid (TIPS) as a hydrotr ope in a surfactant system (Bies and Folta, 2004). Hydrotropes stabi lize surfactants (e.g. SDS) to allow them to remain soluble. Nuclei (150l) were inc ubated with 1200l of extraction buffer 1 (10mM EDTA, 10mM Tris, 1%SDS) at 65C for 20min (T53) The sample was treated with Proteinase K for 1h at 37C. After a phenol:chloroform extract ion and centrifugation, the interphase was reextracted with 5 volumes of extraction buffer 2 (50mM Tris-HCl pH 8.0, 5% SDS, 1%TIPS, 2% 2-mercaptoethanol, 4% PASp-ami nosalicylic acid). The supernatan ts of both extractions were combined and nucleic acids precipitated by isopropanol. Modifications of Murray and Thom pson DNA Isolation Protocol A series of modifications of the protocol proposed by Murray and Thompson were tested. Though the original protocol included cesium chlori de gradient, this step was suppressed for all variations tested. 23. Extraction buffer: 200mM Tris, 2M NaCl 50mM EDTA, 2% CTAB, 2% PVP, 2% 2mercaptoethanol. After initial 45min incubation at 65C, solid CTAB wa s added to extraction buffer, raising CTAB concentration to 6%. Furt her incubation was necessary dissolve the CTAB. Both fresh (T54, T55) and lyophilized (T56, T57) were used, in 100mg (T54, T56) and 500mg (T55, T57) amounts. A chloroform:octanol deprotei nation step takes place, then the upper phase receives 0.1 volume of 10% CTAB. After a second chloroform:octanol extraction and transfer of the upper phase to a new tube, 3 volumes of 50mM Tris-HCl pH 8.0, 10mM EDTA, 1% CTAB were added to the aqueous phase. The concentrati on of CTAB here is maintained, but, since not salt was added, the ionic strength of the soluti on decreases from 2M NaCl to 0.5M. In low ionic strength, CTAB precipitates nucle ic acids during a 30-minute inc ubation. The pellet formed after the incubation and successive centrifugation, the s upernatant is discarded and the pellet dissolved in 0.5 volume of 1M NaCl. Prep was treate d with RNAse and downstream stages of DNA precipitation by alcohol followed as th e standard procedure cited above. 24. Increase in CTAB concentra tion to 6% as above, with th e difference that here CTAB was not added as powder, instead as e qual volume of 10% CTAB, 2 M NaCl. For DNA precipitation, 3 volumes of 6% CTAB, 100mM Tris-Hcl, 25mM EDTA were used, decreasing concentration of NaCl to 0.5M. Both fresh (T 58) and lyophilized (T59) leaves were used. 25. Pea: extraction buffer: 0.7M NaCl, 1% CTAB, 50mM Tris-HCl pH 8.0, 10mM EDTA pH 8.0, 1% 2-mercaptoethanol, 0.01% sodium bi sulfite. Departs from Murray and Thompson protocol in that DNA precipitati on is achieved only by addition of ethanol, and not by decreasing salt concentration. All protocols bellow counted with precipitation methods that differ from the first proposed by Murray and Thompson. Different tissue-to-buffer rations were tested by extracting DNA from 10mg (T60), 50mg (T61) and 100mg (T62) of tissue, keeping the extraction buffer volume constant at 7ml. 26. Sugarcane [Aljanabi, 1999]: 200mM Tris-H Cl, 50mM EDTA, 2.2M NaCl, 2% CTAB, 0.06% sodium sulfite, pH 8.0; afte r homogenization of the tissue and buffer, 0.5 volume of each 5% sarkosyl, 10% PVP, and 20% CTAB were a dded, elevating the CTAB concentration from 2% to 5% and decreasing NaCl concentration to 0.8M. Plant tissue: Straw berry Festival, fresh, mature, leaves of Strawberry Fe stival (T63) or Sw eet Charlie (T64), 3.5g, 4ml buffer/g tissue. 27. Cacti (de la Cruz et al., 1997). Combina tion of CTAB and SD S extraction buffers. CTAB buffer: 100mM Tris-HCl pH 8.0, 20mM ED TA pH 8.0, 4% CTAB, 1.7M NaCl, 4% PVP,

PAGE 103

103 5mM ascorbic acid, 10mM 2-mer captoethanol. STE buffer: 100mM Tris-HCl pH 8.0, 50mM EDTA pH 8.0, 100mM NaCl, 10mM 2-mercaptoet hanol. Fresh 100mg of Strawberry Festival leaf tissue were ground in liquid nitrogen and subsequently incuba ted for 10 min at 65C with 1ml CTAB buffer. STE buffer (4ml) and SDS (to final concentration of 2%) were added and shaken vigorously for 7 minutes. A second 10-min incubation at 65C wa s carried out; 1.25ml of cold 5M KOAc was added and incubated in i ce for 40 min, centrifuged at 3,500rpm for 10 min T65). The upper phase was transferred to a ne w tube and the nucleic acids precipitated by isopropanol. An alternative method (T66) subst ituted the addition of KOAc and ice incubation by addition of equal volume of 24:1 choloform:oc tanol, keeping steps af ter centrifugation the same. 28. Extraction buffer/plant materi al Incubation temperatures a nd durations were tested: 4C (T67-T70), 20C (T71-T74), 42C (T75-T78), 65C (T79-T82), and 0min (T67, T71, T795 T79), 5min (T68, T72, T76, T80), 30min (T69, T73, T77, T81), 60min (T70, T74, T78, T82). CTAB buffer (2% CTAB, 1.4M NaCl, 100mM Tris -HCl pH 8.0, 20mM EDTA pH 8.0, 1% 2mercaptoethanol) was incubated in water baths with the various temperature treatments. When the buffer reached temperature equilibrium with the water baths, each tube received 1.6g of liquid nitrogen-ground strawberry leaves and the mixture was incubated at the various duration treatments. When incubation duration was reached, an aliquot of 10ml of the temperature treatment was mixed with chloroform. For inc ubation time zero, an aliquot was taken right after buffer and ground tissue were mixed and ch loroform was added. Samples were centrifuged at 4,000rpm for 10min. The upper phase was transfe rred to a new tube and nucleic acids were precipitated by isopropanol. After centrifugation and discard of the supernatant, the dry pellet was resuspended in water and tr eated with RNAse. The precipitation steps were repeated to obtain virtually RNA-free DNA. DNA was qua ntified with aid of a NanoDrop ND-1000 spectrophotometer. This experiment was repeated 3 times. 29. Tissue was ground in liquid nitrogen, and an aliquot of the extraction buffer (2% CTAB, 1.4M NaCl, 100mM Tris-H Cl pH 8.0, 20mM EDTA pH 8.0, 1% 2-mercaptoethanol) was combined to the ground tissue to undergo further grinding and formation of slurry. The tissues tested were unexpanded (T83) and expanded (T84) leaves from the F. nubicola FRA520. Following formation of slurry, equal volume of 24:1 chloroform:octanol was added, vortexed, samples were centrifuged, and upper phase transf erred to a new tube. Nucleic acids were precipitated by addition of 70% isopropanol, the alcohol was decanted, and the dry pellet resuspended in water. 30. An experiment was designed to contrast the traditional method of grinding tissue in liquid nitrogen, then adding the powder to buffer (T85) versus grinding tissue in liquid nitrogen, then adding the buffer (described immediately a bove) to the tissue and further grind until slurry is formed (T86). The 100mg per treatment of FR A520 plant material was mixed before nucleic acid isolation to eliminate the l eaf age factor. A NanoDrop was us ed to quantify the nucleic acid content. 31. A factorial experiment tested interacti ons between formation (T87, T89) or not (T88, T90) of slurry and incubation temperatures of 4C (T87, T88) and 60C (T89, T90). After grinding the tissue (50mg per grin ding method) in one of the two fashions tested, the material was split to be incubated for 1 hour in the tw o different temperatures. The downstream steps were followed as described above, including quan tification of nucleic ac ids and absorbance at 230nm and 280nm by a NanoDrop ND-1000 spectrophotometer.

PAGE 104

104 32. CTAB buffer concentrations of 2% (T91), 6% (T92), and 20% (T93) were tested. The slurry was formed by breaking down 400mg of tissue in liquid nitrogen first, then adding 2 ml of buffer for further grinding. Once homogenizatio n was achieved, another 8ml of buffer were added and the mixture was incubated at 65C fo r 30min. The 10ml of buffer were split into 2 tubes (treatment replications) a nd 5 ml of chloroform:octanol we re added to each tube. Nucleic acids from centrifugation upper phase were pr ecipitated by isopropanol and the dry pellet resuspended in 1ml TE pH 8.0. Samples were quantified by NanoDrop. 33. Because homogenizing tissue in buffer seemed to have a positive effect on DNA recovery, an experimented was set up to te st Polytron homogenizer speeds (half maximum speedT95-T98; full speedT99-T103) and duration of homogenizing treatment (no polytronT94; 5 secondsT95, T99; 15 s econdsT96, T100; 30 secondsT97, T101; 60 secondsT98, T102; 120 secondsT103). Enough La boratory Festival #9 tissue for all treatments (2g) was ground in liquid nitrogen an d, by adding an aliquot of the buffer, ground to a paste consistency. The paste was divided into 10 tubes and enough buffer to reach 5ml was added to each tube just prior to treatment w ith Polytron. Samples were incubated at 65C for 30min. Downstream steps from inc ubation were as described above. The final strawberry DNA extractio n protocol is listed bellow. Strawberry DNA Extraction Protocol CTAB extraction buffer 100ml 2% CTAB 2g 1.4M NaCl 28ml of 5M NaCl 100mM Tris-HCl, pH 8 10ml of 1M Tris 20mM EDTA pH8 4ml of 0.5M EDTA 1% BME 1ml diWater to 100ml Tissue-to-buffer ratio = 40 mg/ml. For 12-ml tubes, maximum tissue processed is 200 mg. Grind 200 mg of liquid-nitrogen frozen leav es (young or unexpanded) in mortar-and-pestle Add 2 ml extraction buffer to ground sample, ma cerate in mortar until consistency of paste is achieved. Transfer the paste to a 12-ml tube, and add 3 ml buffer Homogenize utilizing a Polytr on at full speed for 2 min Incubate for 1h at 65C, with intermittent agitation Add equal volume (5ml) of 24:1 chloroform:octanol Mix by shaking vigorously Centrifuge at 4,000 rpm, 5 min Transfer the upper, aqueous phase to a new 12-ml tube Precipitate DNA with equal volume of 70% isopropanol Mix by inverting the tube several times Centrifuge at 4,000 rpm, 5 min Discard the supernatant Air-dry nucleic acids pellet

PAGE 105

105 Resuspend pellet in 500ul to 1 ml (depending on the amount required to dissolve the pellet) of deionized water or TE pH 8.0.

PAGE 106

106 APPENDIX B In silico ANNOTATION AND DISTRIBUTION OF Fragaria vesca GENES Under each fosmid name is a list of number ed potential genes predicted by FGENESH. The nucleotide intervals that had protein hits by BLASTP were used for a similarity search against the non-redundant Viridi plantae, protein database us ing BLASTX. The best matches identified by the algorith m are listed under Protein Hit. Threshold value was 10-15. Letter X under Protein Hit denotes no similarity wa s detected in the protein database. Under Orientation, + signs signify th at the query sequence is translated in the same direction it was input, where negative orientation signifies that th e complement strand is translated. EST Hits are sequences of DNA for which an EST was det ected within Rosaceae, with a minimum length of 100 nucleotides and 95% identity. Gene distributions were calcula ted by dividing each fosmid insert size by the number of genes either predicted by FGENESH or identified by sim ilarity to the non-redundant Viridiplantae protein database. Simple Sequence Repeats (SSRs) with at leas t 5 repeats of a motif are represented by color-coded triangles: in FGENESH-defined genic sequence; in FGENESH-defined intergenic sequence Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 01L02 13 7 40,302 3.1 5.8 1 unknown 2 X 3 pectin lyase + 4 unknown 5 beta-glucan binding 6 enolase 7 X 8 X 9 unknown + DV440 436.1 10 X 11 X 12 X 13 unknown DY670 952.1 05N03 8 5 34,611 4.3 6.9 1 ATP binding/adenylate cyclase + 2 X 3 Senescenceassociated + CX6614 21.1 4 hypothetical DW248 990.1 Orientation

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107 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 5 X CX6616 57.1 6 X CO8169 31.1 7 peroxidase 8 unknown 11D02 9 3 37,961 4.2 12.7 1 ATP synthase, mitochondrial DY668 653.1 2 X Not predicted X DW342 667.1 3 X DW344 738.1 4 Release Factor 2, chloroplast + DW346 600.1 5 X 6 X 7 X 8 X BQ1055 41.1 9 heat shock binding 13I03 8 8 37,707 4.7 4.7 1 hydrolase + 2 leucyl-tRNA synthetase 3 leucyl-tRNA synthetase 4 leucyl-tRNA synthetase 5 leucyl-tRNA synthetase 6 zinc finger family + 7 2OG-Fe(II) oxygenase DY670 360.1 8 integrase + DY671 649.1 15B13 7 2 23,212 3.3 11.6 1 senescenceassociated CX6613 47.1 Not predicted 26S ribosomal RNA (not a protein) CA8540 88.1 2 X 3 senescenceassociated CX6613 47.1 4 X 5 X CX6614 21.1 Orientation

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108 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 6 X 7 X CX6616 57.1 17O22 9 6 34,090 3.8 5.7 1 homeodomain + 2 X 3 X 4 oligopeptidase + BQ1046 55.1 5 hypothetical DY675 330.1 6 X 7 unknown 8 lectin protein kinase + 9 hypothetical 18A19 7 6 40,908 5.8 6.8 1 cytochrome P450 2 X 3 integrase 4 integrase 5 integrase + 6 integrase 7 transferase + 22H18 8 4 37,851 4.7 9.5 1 X 2 X 3 polyprotein 4 X 5 hypothetical + 6 X 7 unknown + 8 pre-mRNA processing factor 38 + 22L05 8 3 35,112 4.4 11.7 1 X 2 X Not predicted X DY674519.1 EST starts upstream of predicted gene 3, and spans oxidoreductase 3 oxidoreductase + DY671 565.1 4 oxidoreductase + 5 sulfate transporter 6 X CO3800 67.1 7 X Orientation

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109 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 8 X 27F10 11 8 37,110 3.4 4.6 1 kinase DY675 883.1 2 hypothetical CX6613 86.1 3 unknown DV438 706.1 4 integrase 5 integrase 6 integrase 7 unknown + 8 X Not predicted X CO3787 00.1 9 unknown 10 X 11 X 29G10 10 4 31,681 3.2 7.9 1 transposase 2 X 3 flavin-binding monooxygenase-like + DY673 408.1 4 X 5 X 6 X 7 X 8 phenylacetaldehyde synthase 9 unknown + 10 X 30I24 7 5 37,599 5.4 7.5 1 X 2 wall-associated kinase + 3 X ( E value=1e-10) arabidopsis response regulator 12 4 chitinase + CX6615 29.1 5 arabidopsis response regulator 12 + DY671 913.1 6 transferase 7 PICKLE chromating remodeling factor Orientation

PAGE 110

110 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 32A10 15 4 33,577 2.2 8.4 1 catalytic/ hydrolase DY667 800.1 2 X 3 X 4 X 5 X 6 X 7 copper ion binding + 8 X 9 MADS-box 10 X 11 X 12 X 13 pathogenesis-related 14 X 15 X 32L07 6 4 32,951 5.5 8.2 Not predicted X DY668 002.1 1 hypothetical 2 SMC2 3 disease resistance DY666 677.1 4 X 5 exostosin-like CX6620 49.1 6 X 34D20 8 6 30,034 3.8 5.0 1 RNA recognition motif + 2 cysteine-type peptidase + 3 X 4 transposase + 5 anthocyanin 5aromatic 6 X ( E value = 8e-14) anthocyanin malonyltransferase + FGENESH missed EST 7 NAC domain NAM Not predicted X DV438 498.1 8 X Orientation

PAGE 111

111 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 38H02 7 6 31,669 4.5 5.3 1 X 2 transposon protein + 3 cytochrome P450 + 4 cytochrome P450 + 5 integrase + 6 serine/threonine kinase 7 exportin 38H05 11 1 32,050 2.9 32.1 1 X 2 X Not predicted X dbj|AB2 08565.1 3 retrotransposon polyprotein Not predicted X dbj|AB2 08565.1 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 40B22 9 8 36,230 4.0 4.5 1 unknown + 2 cyclin-like F-box 3 X 4 cyclin-like F-box + 5 cyclin-like F-box 6 cyclin-like F-box 7 Arf GTPase activating 8 heavy metal transport/detoxificati on + 9 MuDR family transposase 40M11 9 5 31,718 3.5 6.3 1 X 2 cyclin 1-like F box + 3 X 4 X Orientation

PAGE 112

112 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 5 X 6 Secretory Protein SEC14 DY675 900.1 Not predicted X DY672 841.1 7 ATPase 8 unknown + 9 glycosyl hydrolase Not predicted X CX6621 88.1 43P07 10 4 43,641 4.4 10.9 1 X 2 retrotransposon polyprotein + 3 X 4 X 5 DNA cytosine-5methyltransferase DY668 476.1 6 unknown + DY668 476.1 7 methyltransferase small domain + 8 X 9 X 10 X 44J07 11 2 29,636 2.7 14.8 1 X 2 X 3 X DY672 792.1 4 X 5 X 6 X 7 disease resistance 8 unknown DY671 343.1 9 X 10 X DY650 877.1 11 X 47H15 9 4 34,817 3.9 8.7 1 X DY669 025.1 2 X 3 polyprotein 4 integrase 5 retrotransposon protein Orientation

PAGE 113

113 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 6 X 7 X 8 X 9 heat shock + 52E09 9 8 36,230 4.0 4.5 1 unknown + 2 cyclin-like F-box 3 X 4 cyclin-like F-box + 5 cyclin-like F-box 6 cyclin-like F-box 7 Arf GTPase activating 8 heavy metal transport/detoxificati + 9 transposase 63F17 6 3 28,318 4.7 9.4 1 phospholipase D + DY672 511.1 2 unknown 3 binding + 4 X 5 X 6 X 72E18 12 11 36,293 3.0 3.3 1 hydrolase + 2 hydrolase + 3 reverse transcriptase 4 hydrolase + DV438 212.1 5 X 6 hydrolase + DY669 358.1 7 unknown + DY675 437.1 8 transferase + 9 spliceosomeassociated + 10 unknown + 11 actin 7, actin 11 12 glycoprotein-like DY670 963.1 84N10 8 2 40,183 5.0 20.1 1 ribosomal L24/L26 + 2 X 3 X Orientation

PAGE 114

114 Predicted Number of Genes Putative Gene Distr (kb between genes) Fosmid ab initio Similari ty Protein Hit EST Hits (gb no.) Fosmid Insert Size (bp) ab initio Similaritybased 4 ATP binding 5 X 6 X 7 X 8 X Totals 235 129 905,491 Means 9 5 34,827 4.0 9.1 Sample Standard Deviatio n 2.1 2.4 4,426 0.9 6.0 Orientation

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115 APPENDIX C PCR PRIMERS USED TO AMPLIFY A ND SEQUENCE GENE-PAIR HAPLOTYPES GPH name Primer name Primer sequence GPH4 GPH4a ACGAGGGCTTGGAAGAAAGG GPH4b GCCCAACAACAGAAAGACC GPH5 GPH5#2a CAATGCCATGGTCTCCGGTC GPH5#2b TGCCGTTGCACACACCTTCC GPH5A2 GCTCTTTGGTGTTCAAAGTTGGAT GPH5B#2 ATCCAGCCAAACTGAAGGTG GPH5A3 CAGCCATGAAGTCAAGGTCA GPH10 GPH10a GGCTTCTTCTTGTCCGGCAGC GPH10b GAACTCCAGGTCAGATCTTCG GPH10c CTCGCTGCAAATCAGCTACC 10ABCol7Rev2 GAGTTTGTCGAGCTGATC 10ABCol32Rev2 ATAGAGGCGATGTTGTAG 10AB#3 GGCCCTGATCACTCGACA 10AB#4 GGTTTGGTTGGTTAAGGTG 10AB#5 GACAGTACCTGAAAATTTGG 10AB#6 AAGTATCATTAACAGGC 10AB#7 ATCATATATGCGGGTGTG 10AB#8 TAACGAGCAGTGGCGG 10AB#9 ATCACCTCTACTCCCACGC 10AB#10 CACCGTAACAGCTGAGCAAG 10AB#11 ACACAAATGCCTCATCCACA 10AB#12 ACTAAAGCCCAGCAACCCTC 10AB#13 TTCTCTGTCAACCCTGCCTT 10AB#14 GGGGCAAAGTTTACATAGCA 10AB#15 AACTCGCCGGAAGACACTTA 10AB#16 GCCGGAAGACACATATCGAT 10AB#17 GCATCCCCTTTACATCCAAA 10AB#18 GTTAGAGACGACGACGGGAG 10AB#19 TGCCTGGCAAAGTAAACCTC 10AB#20 GGCGTGTCAATTTGTGAATG 10AB#21 TCATCTTCCTCTGTATGCGACT 10AB#22 GGTTTTGTTTTTGGTGGGAA 10AB#23 GTCGAGTGATCAGGCCGTA 10PPR1F AACGGAGAAGAAGACTGTCG PPR1R1 GATCGAACGGCTGATATTAAA 10PPR1R2 TGTAGCTCATACTTTTGTTCTC GPH23 GPH23F CTTGAGGGCCATCAGCAC GPH23R TACACCCACGCCTTCATCTC GPH23F2 GAACTGCGAAGATCTATCTGA 11D02 11D02F GAGCTGCTGTGTGAACCAAA 11D02R GTTCAACTCCAGATGAAGTGAGG

PAGE 116

116 GPH name Primer name Primer sequence 17O22 17O22F AAAATGGGTTGCACGAGTTC 17O22Rb GGGTTTCCTCACAAACTTCG 27F10 27F10F CCTGCAGGGTTTTTCATCAT 27F10R TGGAAATGTATTCTGGTTCTCC 29G10 29G10F TGGCCTTGTTTCCTAAACTCTT 29G10R AGAAGAAGGCAGCACCCAAT 32L07 32L07F GAGTTGAAAAACGGGTCGAA 32L07Rb AGGAAAATGCGGGAGAAAGT 34D20 34D20Fb GCAGAAAGAAACTGATGTGCTT 34D20Rc CGCAGTCGTAAAAATTCGTCT 34D20Fb2 TGGGTGTGGATGAACTATACG 40M11 40M11Fd CAACATTTTGGTGGCCTTCT 40M11Rd CGGCCTATGAAACCACAGTT 63F17 63F17F GCAGAAAGAAACTGATGTGCTT 63F17R CGCAGTCGTAAAAATTCGTCT 72E18 72E18Fb GCAGAAAGAAACTGATGTGCTT 72E18Rb CGCAGTCGTAAAAATTCGTCT 72E18Fb2 GCAGCAATCAAATCATTCCA

PAGE 117

117 APPENDIX D SEQUENCES GENERATED DURING CHAR ACTERIZATION OF GENENPAIR HAPLOTYPES >GPH4_ananassa_13 ACGAGGGCTTGGAAGAAAGGAGGTCAATTTGGTTAAGGTGTGTTGGAGTCGCCAAGTTGAGGGTGATGCATTCTTGG GAGTTAGAGTCGGATATGAGGGCTAAGTACCCCAAGTTGTTTCTTTTTGAGTTAGTATCTTAAAATTTCGGGGACGA AATTTCTTTAAAGAGGGTAGAGTGTAATACCCCAGAAATTTGATATTAGTTTCTAATTTTATTTAGGAATTTTTGAG TTAGAAGTTAGCGTGTTTTGAAGTTTGAAGGAAGAACGGAAGGGTTCGGATGCATAAATTGCTGAACCGGTTTTATG GTTCTGAAAGGTCAAGAGTTGACTTTCTAATCCGTTGGGTTTCTCGAGAAACTTCCTTCACGGAAGTTGTAGAGCAC GACGATACGAGTTCGTAGACACGTGGCACGCGTAAAACGGACTTCGTATGAGAAAGTTATGGTCAGCAGAAGTTGTG GCTTTTCGGGAATATTTAGGTTAAATAGGAAATTTTCGTTTTGGGTTCTATTATTTTTCAGAAATTCCTTTCTTCCC CTTCTTCTCTCTCCCCGACCCCGAGAGAACCCAAGCTTCCCAGCCGACCCGACCCGGACCCGGTTGACCCCACCCGG ATTTTCCGGCCATCTCCGGCCGACCCAGGCAACGGCACTGGTCGGGTTCTCTTCCTCTCCTCCGTCAGAGCTGACCT GTGGCGGTGATGTGCGCCGTTTCGGTCCCGAGGTGGTGACCCGAAGCTCGGAAGTTCGGGTTAGAGACCCGGTCGGA TTCCTTCATCCGGCGGCGGCGACAAGGTAGGAGACCGATCGGGATAGAAACCCCTTGGCGTCTTGGTCCGATTGCTG GTGGCCTTGAAGTGCGACACACGGCGGAGAGTGGCAGTGGTGGATCCTAATCTTTCCGGCGAGTTTCCGGCAAATTC CCGGCCGATTTGGTTTCGACCTCAGGTATGGAAGTTGCTCTCCTTGCTCTGAGCTATATTTTTGGTGTTGGAAGTTT GTCCGTTTTCGAAGGTTAGTGGGGTGGCGCGTGGGACCCACGTGCAGTCGCTAAGGGCAGCGCGTAGCGGCGCGTCC GTAGGTGGTTGTGGTCTTTCTGTTGTTGGGC > GPH4_ananassa_15 ACGAGGGCTTGGAAGAAAGGAGCCTTTGTCTGTGAATATGTTGGGGAGATAATGACCTACAAGTACTTGTATAATCG GGGAAGACACACATACTCAATCACACTGGATGCCGGTTGGGGCATTGCTTAGCCTTGTGTTTTTGGTTAAGCAGATC ATATTTCCCAGAATTAGATCTGCATAATAATCCATTTTCACGCCAGTCATTTGGTGCCTGTGGTTTAGAACTTAGAT TTCAGAAGTTCTATCAAGTTTGTCACTTCCTCACCTCTTGTGATGAGAGAAATTTTCAATTCGTTGATGTTGACAAA GATCATCTGACTATAAATTTGCCCATGTAATCGTATTCTGTTTCTCCTAAAAATATTGTTCTTGTAAATTTGGGGAA ATCCGGAAAAGGCTATACTGTCATTTGCTTCCTAACTTGTCTTGAGCAATGACCTAATGATTTTCCTATAGCTTTTG TTGGTTTTTTTCTCGTTCTTTCTTTCTCTGACGTTATGTTTAATTCCCTCAACAACTCCAGATGCTATGATGAAAAC TTGGTTGATATCCCAGTTCAAGTGGATACTCTTGCTCGCTATTATTACCATGTATGAATTTGGCTGCTTCCTTTCTA AATGGTCTTTCTGTTGTTGGGC >GPH5_vesca_clone21 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAGAAGCTT CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT TGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATATGAAATAGAAGGTAATATGCATG ATATAAATATCTAGTTAATTGTACAATGATATTTGTAACCAGTGAAAATAATGACAATCTTTATAACAAAATTTCAG TTATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAG ACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGACAA AGAATCCTTTTGTCATATGGATTGAATCTGAATTAGTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCATGT TACATGTCTCATGATGTCTTCATTTGGTGACAAAAGCTAAATCTTAACCTGACCTAAGTATCAAGACATATTGGACA ATTGGGCTTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTATAGAAAACACTCTGTGATCT TCACCATTGAGGAGTCAAGTTACTCAGCCCTGAAGTAAAAGTCCAGTCAGTAGTGCAGTTGAGTTCAACTTGTTCTG GGTTCTTCAAAGTTTGAAACTTTAAGCTTCGATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATG TGGTTGGCATATATAAAAGCCTGAAAAGATTGCCCAAAACCCAAGCTGGGTTCATCCGAAAAAGTTTTTGAATCTTT TTAAAGCCCTTTTTTAATAATTTGGAATNCCACCTTCCnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnGTATTAGATGAAAACTAGTTTT TCTAAGAACTTGATGAGTTGATGGAGGATTACATATGAGGTTTGGTTATGTTTTTAGGTATGCAATCCTTCTGTAAG

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118 TGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGAGTACCTTGTCTCGCATGGGCTTAAGAATGCTTCATATA CTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGAT TTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTGCATATGATATTA AATCTTTTGTTTGGTTCCTCATTGTATAATTTGGTTCTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTA ATTCAGGGTTCAAAATTGCACCTGATCAGCTTACACGAAAGAGAATGCCGGATGTGATAAATTCAGGTGTCAATGAC CCTCCACAAAAGAGATCACTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTATGATAGAAGATCTTCTAT TTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATTTTTCAGCAGCCAGCGTCTTCTATGGTTAATTCAGGCTT CAAAATTGCACCTCATCAGCTTACGCGAATGAGATTGCCGGATGTGGTAAATTCAGGTGTCAATGACCCTCCACAGA GGACATTGCCGGATGTAAGTATCTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACC TTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGATGTTTTTTGGCTT CCAGTTTTTCTTCACATAAGCATTTTAAAGCTGATCATTGTAATCGAACTCGAATTATTCTACTACTGGTGTAAGTT GCCTTGTGTCACCACCACTAAGATCACAATTTCGTATTTTATGATCAACACCGAAGACCTATGTCTAGTGTCGTGAT TATGGTCATGTGAAGTGGATTTCTTAATATATGCCTCGTCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTG ATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTTCGTGCTGAAGATGGGAGTGTTGTATGT GGAGTTCATGAAAACATTGTGAATTTTTTTGATATATATTTTTGTTTTTGTAAAAATGGATCTCTTCATAACATTGG GGTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAACGGCA >GPH5_viridis_clone5 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTG GTGTTCAAAGTTGGATTCATTTACATGTGACACAGTTACCATTTTCCCATATGAAATAGAAGGTAATATGCATGATA TAAATATCAAGTTAATTGTACAGTGATATTTGTAACCAGTGAAAATAATGACAATCTTTATAACAAAATTTCAGTTA TCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGAACAGCAACACCAAACAAACAGAACCAGACC AAATCACACCAATATAAAACATAATTGGATTTTCATGAAAGGCAGCAAGGCATGATCAATGAAGGAGAAGACAAAGA ATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCATGTTAC ATGTCTGCATTTGGTGACAAAAGCTAAATCTTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTGAG CCCAAATCTGTACTAGCCCATAATATGCTTTTTATAGAAAACACTCTCTGTGATCTTCGCCATTGAGGAGTCAAGTT ACTCAGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAAC TTTAAGCTTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGC TTGAAGGATTGCCAAAACCAAGCTGGTTCATTGGTAAAAGTTTTGATCTTTTAAGCCTTTTATAATTTGATCACTCT CATTGTTTTATCAATTTnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnGAACTTGATGTTATTTACTTGATGAGTTGACGGAGGATTACATATAAGGCTTGGTTATG TTTTTAGGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGAGTACCTTGTCT CGCATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTTGGAAGCCTTGTTGCT TGTCCTTACCAGGTTTGAATTGATGATTTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTG GCGTGGAGCTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAAGTCGGTTTTTTATTATAATCT TTCAGCAGCCAGCATCTTCCATGGTTAATTCAGGGTTCAAAATTGCACCTAATCAGCTTACACGAAAGAGAATGCCG GATGTGGTCAATTCAGGCGTCAATGACCCTCCACAAAAGAGATCGCTGGATGTAAGTATCATATGCTACATGGAACT TTTGTAGTTTGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTTTCGGCAGTC AGCATCTTCTATGGTTAATTCAGGCTTCAAAATTGCACCTTACCAGCTTGCACGAATGAGATTGCCGGATGTGATAA ATTCAGGTGTCAATGACCCTCCACAGAGGACATTGCCAGATGTAAGTATCTTATGCTACATGGAAATTTTGTTCTGG CCAGATTGGCATGAAAATCCAGATACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATT TATTCTGCAAATGATGTTTTCAGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAACTC GAGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGGTCACAATTTCGTATTTTATGATCAACAC TGAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAAGTGGATTTCTTAATATATGCCTCATCTATGTCTTCAT CCAGCAATCCTGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAGTCCTGGATTATCTGGTGCAGGAGCTGTACTCC GTGTTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGGATATATATATTTGTTTTTG TAAAAATGGATCTCTTTATAACATTGGGGTTACTGTAGTTGCACCGGCTGCGGGAAGGTGTGTGCAACGGCA >GPH5_iinumae_clone5

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119 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATC C T CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT CAAACAAAGC A TCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT TGGTGTTCAAAGTTGGATTCAATTACATGTAGACATAGTTACCATTTTCCCATTTGAAATAGAAGGTAATATATCAA GTTAATTGTACAATAATATTTGTAATCAGTGAAAATAATGACAATCTTTATAACAAAATTTCAGTTATCTTTTCACT GCTGTATGAACTGTCACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACC AATATAAAACAGAATTGGATTTCCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGACAAAGAAACCTTTTGT CATAGGGATTGAACCTGAATTATCTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCATGTTACATGTCTGCAT TTGGTGACAAAAGCTAAATCTTAAGAATTTAGACGTATTAGACCATTGGGCTTAATCATCGTCCGAGCCCAAATCTG CACTAGCCCGTAATATGCTTTTTATAGAAAACAGACTCTCTGTGATCTTCGCCATTGAGGAGTCAGGTTACTCAGCT CTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAACTTTAAGCT TCAATGGAGGAAGATAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGCTTGAAGGA TTGCCAAAACCAAGCTGGTTCCTCGGTAAAGTTTTGATCTTTTAAGCCCTTTATAATTTGATTACTCTCATTGTTTT ATCAATTTTTGATTTCCCATTTGATnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnACTA GTTTTTCTAAGAACTTGATGTCATTTACTTGATGAGTTGATGGAGGATTACACATGTGGTTTGGTTTTGTTTTTAGG TATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGAGTACCTTGTCTCACATGGG CTTAAGAATGCTTCATATACTATCAGCGCCAGTGATGTGAAAGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTA CCAGGTTTGAATTGATTTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAGCTA GTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAATCTTTCAGCAGCCA GCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTAATCAGCTTACACGAAAGAGAATGCCGGATGTGGTAAA GTCAGGCGTCAATGACCCTCCACAAAAGAGATCATTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTTTG ATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTTTCAGCAGCCAGCGTCTTCTA TGGTTAATTCAGGCTTCAAAATTGCACCTTATCAGCTTACACGAATGAGATTGCCGGATGTGGTAAATTCAGGTGTC AATGACCCTCCACAGAGGACATTGCCGGATGTAAGTATCTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCA TGAAAATCCGGATACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAA TGATGTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAACTCGAGTAATTCT GCTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATTTCGTATTTTATGATCAACACTGAATACCTA TGTCTAGTGTTGTGATTATGGTCATGTGAAGTGGATTTCTTAATATATGCCTCATCTATGTCTTCATCCAGCAATCC TGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTCCGTGCTGAAGA TGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGGATATATATTTTTGTTTTTGTAAAAGTGGA TCTCTTTATAACATTGGGTTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAACGGCA >GPH5_nubicola_clone7 CAATGCCATGGTCTCCGGTCTATTTCAAC C GGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT CAGAATCAGATTTGTTTGC T CTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAACTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAATGAACTGTAAGAGACATATTTCAAGC TCTTTGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAATAGAAGGTAATACG CATGATATAAATATCAAGTTAATTGTACAATGATATTTATAATCAGTGAAAATAATGACAATCTTTATAACAAAATT TCAGTGATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGACCAACAACACCAAACAAACAGAA CCAGACCAAATCACACCAATATAAACAGAACTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGA CAAAGAATCCTTTCGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCGTATGCAGGCA TGTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGC TTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATTCTTTTTATAGAAAACAGAGATTCTCTGTGATCTT CACCATTGAGGAGTCAAGTTACTCGGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTG GGTTCTTCAAAGTTTGAAGCTTTAAGCGTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATG TGGTTGGCATATATAAAAGCTTGAAGGATTGCCAAAACCAAGCTGGTTCATCGGTAAAGTTTCGATCTTTTAAGCCT TTTATAATTTGATCACTCTCATTGTTTTAnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn

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120 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnTAAGAACTTGATGTTATTTGCTTGATGAGTTGAT GGAGGATTACATATGAGGTTTGGTTATGTTTTTAGGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTG CCTAAGGAGGCCGAGGAGTACCTTGTCTCGCATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAA AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCATGTGTTCTAGTTTCTGTT TGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCAT TGTATAATTCGGTTTTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACC TGATCAGCTTACACGAAAGAGAATGCCGGATGTGATAAATTCAGGTGTCAATGACCCTCCACAAAAGAGATCATTGG ATGTAAGTATCATATGCTACATGGAACTTTTGTAGTATGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTC GGGTTTTTATTATAATTTTTCAGCAGCCAATGTCTTCTTTGGTTAATTCAGGCTTCAAAATTGCACCTCATCAGCTT ACGCAAATGAGATTGCCGGATGTGGTAAATTCAGGTGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTAT CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAGTTTGGCTGGATTATGGA GTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGATTTTTGGCTTCCAGTTTTTCTTCACATAAGCATTT TAAAGCTGGTCATTGTAATTGAACTCGAATAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATC ACAATTTCGTATTTTATGATCAACACCGAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAAGTGGATTTCTT AATATATGCCTTGTCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGG ATTATCTGGTGCAGGAGCTGTACTCCGTGCTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAGCATTGTGAATT TTTTTTTATATATATTTTTGTTTTTGTAAAAATGGTTTTATAACATTGGGGTTACTATAGTTGCACCGGCTGCGGGA AGGTGTGTGCAACGGCA >GPH5_nilgerrensis_clone19 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGAC T GGAAGATCAT CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAACTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT TGGTGTTCAAAGTCGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAACAGAAGGTAATATGCATG ATATAAATACCAAGTTAATTGTACAATGATATTTGTAATCAGTGAAAATAATGAAAATCTTTATAACAAAATTTCAG TTATCCTTCCATTGCTGTGTGAACTGTTACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAG ACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAGAAGAC AAAGAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCAT GTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAACACATATTGGACCATTGGGCT TAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTATAGAAACAGAGATTCTCTGTGATCTTCA CCATTGAGGAGTCAAGTTACACAGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGG TTCTTCAAAGTTTGAAACTTTAAGCTTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTG GTTGGCATATATAAAAGCTTGAAGGATTGCCAAACCAAGCTGGTTCCTCGGTAAAGCTTTGATCTTTTAAGCCTTTT ATAATTTGATTACCCTTATTGTTTTATCAAnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnGTTTTTCTAAGAACTTGATGTTATTTACTTGATGAGTTGATGGAGGATTAC ATATGTGGTTTGGTTTTGTTTTTAGGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGG CCGAGGAGTACCTTGTCTCACATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAAAGATGGTCTG TTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATTTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTA TTTTCATGGCATGTGGCGTGGAGCTAGTTGCATATGGTATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGT TTTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTGATTCAGGGTTCAAAATTGCACCTAATCAGCTTACA CGAAAGAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAGATCGCTGGATGTAAGTATCAT ATGCTACATGGAACTTTTGTAGTTTGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCAGGTTTTTATTAT AATCTCTCAGCAGCCAGCGTCTTCTATGGTTAATTCAGGCTTCAAAATTGCACCTTATCAGCTTACACGAATGAGAT TGCCGGATGTGGTAAATTCAGNGTGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGCATCTTATGCTACAT GGAAATTTTATTCTGGCCAGATTGGTATGAAAATCCAGATACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATC ACTTGTTTATTGTATTTATTCCGCAAATGATGTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGA TAATTGTAATCGAACTCGAGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATTTCG TATTTTATGATCAACACTGAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAAGTGGATTTCTTAATATATGA CTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGG TGCAGGAGCTGTACTCCGTGCTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGGAT

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121 ATATATTTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTATAGTTGCACCGGCTGAGGGAAGGTG TGTGCAACGGCA >GPH5_mandshurica_clone1 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGGAGCAGGTTAGCTAAAAGCTTC AAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTTT GGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAATAGAAGGTAATACGCATGA TATAAATATCAAGTTAATTGTACAATGATATTTGTAATCAGTGAAAATAATGACAATCTTTATAACAAAATTTCAGT GATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGA CCAAATCACACCAATATAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGACAAAG AATCCTTTCGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCATGTTA CATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAAT CATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATTCTTTTTATAGAAAACAGAGGTTCTCTGTGATCTTCACCA TTGAGGAGTCAAGTTACTCGGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTC TTCAAAGTTTGAAACTTTAAGCGTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGCTCCAACGGGAGATGTGGTT GGCATATATACAAGCTTGAAGGATTGCCAAAACCAAGCTGGTTCATCGGTAAAGTTTTGATCTTTTAAGCCTTTTGT AATTTGATCACTCCCATTGTTTTATCAATTTTAGATTTCCCATTTGATTACATTACTGGCTCTTGTTTATTTTGTTG AACTAACTATGCCCTTTCGTTCTAACATGCAACTGAAAATAACTGCTAGATTGTATAGCTGAGCCTTTATGGTGTTC ATTATGTAAAAGAGAATGAATTCTGGTGGTGGGTATAAAGCACCTCCCTGAGATTATATGAGATACTATGCTTCTGG AAAATGTTATAAGATGAAAACAACTTTTTCTAACAACTTGATGTTATTTACTTGATGAGTTGATGGAAGATTACGTA TGTGGTTTGGTTTTGTTTTTAGGTATTCAATCCTTCTGAAATTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCG AGGAGTACCTTGTCTCGCATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTT GGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTA TTTTCATGGCATGTCGCGTGGAACTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGT TTTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTGATCAGCTTACA CGAAAGAGAATGCCGGATGTGATAAATCCAGGTGTCAATGACCCTCCACAAAAGAGATCACTGGATGTAAGTATCAT ATGCTACATGGAACTTTTGTAGTATGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTAT AATTTTTCAGCAGCCAGGGTCTTCTATGGTTAATTCAGGCTTCAAAATTGCACCTCATCAGCTTACGCGAATGAGAT TGCCGGATGTGGTAAATCCAGGTGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTATCTTATGCTACATG GAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCA CTTGTTTATTGTATTTATTCTGCAAATGATGTTTTTTGGCTTCCAGTTTTTCTTCACATAAGCATTTTAAAGCTGAT CATTGTAATCAAACTCGAATAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATTTTGT ATTTTATGATCAACACCGAATACCTATGTCTAGTGTCGTGATTGTGGTCATGTGAAGTGGATTTCTTAATATATGCC TCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGT GCAGGAGCTGTACTCCGTGCTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATTTTTTTGATAT ATATTTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTGCTATAGTTGCACCGGCTGCGGGAAGGTGTG TGCAACGGCA >GPH5_ananassa_clone2 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT CGGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGAGAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATCCTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGCTGAACTGTAAGAGACATATTTCAAGCTCTT TGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTTCCATTTGAAACAGAAGGTAATATGCATG ATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAATAAGTGAGAATAATGACAATCTTCATAACAAAATTT CAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGAACAACTACACCAAACAAACAGAAC CAGACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCGGCAAGGCACAATCAATGAAGGAGAAGA CAAAGAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCA TGTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGC TTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTATAGAAAATACTGTGATCTTCACCATTG

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122 AGGAGTCAAGTTACTCAGCCATGAAGTCAAGGTCAAGCCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTCTTC AAAGTTCGAAACTTTAAGCTTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGC ATGTATAAAAGCTTGAAGGATTGCCAAAACCAAGCTGGTTCATCGGTAAAGTTTTGATCTTTTAAGCCTTTTGTAAT TTGATCACTCCCATTGTTTTATCAATTTTTGATTTCCCATTTGATTACATTACTGGGTCTTGTTTATTTTGTTGAAA TAACTATGCCCTTTCGTTCTAGCATGCAACTGAAATTTACTGCTAGATTGTATTGTTGTGCCGTTATGGTGTTCATT ATGTAAAAGAGAATGAATTCTGGTGGTGGGTATAGAGTACCTCCCTGATTTTTTATGAGATACTATGCTTCTGGAAA ATGTTATAAAGATGAAAACTAGTTTTTCTAAGAACTTGATGTTATTTACTTGATGAGTTGATGGAGGATTACGTATG TGGTTTGGTTTTGTTTTTAGGTATTCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAG GAGTACCTTGTCTCACATGGGCTTAAGAATGCTTCATGTACTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTTGG AAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATTTCATGTGTTCTAGTTTCTGTTTGGGCATCTGTTATTTTC ATGGCATGTGGCGTGGAGCTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCAGTTTTTT ACTATAATCTTTCAGCAGCCAGCATCATCTATGGTTAATTCAGGGTTCAAAATTGCGCCTAATCAGCTTACACCAAA GAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAGATCGCTGGATGTAAGTATCATATGCT ACATGGAACTTTTGTAGTTTGATAGAAGACCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCT TTCAGCAGCCAGCGTCTTCTATGGTTAATTCAGGCTTCAAAGTTGCACCTGATCAGTTTACACGAATGCGATTGCCG GATGTGGTAAATTCAGGTGTCAATTACCCTCCACAGAGGACATTGCCGGATGTAAGTATCTTATGCTACATGGAAAT TTTGTTCTGGCCAGATTGGCATGAAGATCCAGACACCTTCAGTCTGGCTGGATTATGGAGTTGCGTTGATCACTTGT TTATTGTATTTATTCTGCAAATGATGTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTG TAATCGAACTCAAGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATTTCGTATTTT ATGATCAACACTGAATACCTATGTCTAGTGTCATGATTATAGTCATGTGAAGTGGATTTCTTAATATATGCCTCATC TATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGTGCTTCAAAAGGAAATCCTGGACCATCTGGTGCAGG AGCTGTACTCCGTGCTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGGATATATAT TTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGCTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCA ACGGCA >GPH5_ananassa_clone6 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGGAGAGAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAAAAGAAAAATGGGATCTACAG AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn

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123 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnACTGGTGTAAGTTGCCTTGTGTCACCTCCACTAAGATCACAATTTCGTATTTTATGATCAACACT GAATACCTATGTCTAGTGTCATGATTATAGTCATGTGAAGTGGATTTCTTAATATATGCCTCATCTATGTCTTCATT CAGCAATCCTGCATACTTGAGTTTGATGGTGCTTCAAAAGGAAATCCTGGACCATCTGGTGCAGGAGCTGTACTCCG TGCTGAAGATGGGAGTGTTGTATGTGGATTTCATGAAAACATTGTGAATCTTTTAGGATATATATTTTTGTTTTTGT AAAAATGGATCTCTTTATAACGTTGGGGTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAACGGCA >GPH5_ananassa_clone7 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT CGGAATCAGATTTGTT C GCGCTTGCAGAAAGAGAATCCTCGAGTGAAGAGAAGATCCTAAGGAGGAACTCCGAGTCT GGTTTAGAATCCTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG AAAAGAGCTTGCTCTTGCTTTCC A CCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT TGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTTCCATTTGAAACAGAAGGTAATATGCATG ATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAATAAGTGAAAATAATGACAATCTTTATAACAAAATTT CAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCACACACAAGAGCAACAACACCAAACAAACAGAAC CAGACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGA CAAAGAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCA TGTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGC TTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTGTAGAAAATACTGTGATCTTCACCATTG AGGAGTCAAGTTACTCAGCCATGAAGTCAAGGTCAAGCCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTCTTC AAAGTTCGAAGCTTTAAGCTTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGC ATATATAAAAGCTTGAAGGATTGCCAAAACCAAGCTGGTTCATCGGTAAAGTTTTGATCTTTTAAGCCTTTTGTAAT TTGATCACTCCCATTGTTTTATCAATTTTTGATTTCCCATTTGATTACATTACTGGGTCTTGTTTATTTTGTTGAAA TAACTATGCCCTTTCGTTCTAGCATGCAACTGAAATTTACTGCTAGATTGTATTGTTGTGCCGTTATGGTGTTCATT ATGTAAAAGAGAATGAATTCTGGTGGTGGGTATAGAGTACCTCCCTGATTTTTTATGAGATACTATGCTTCTGGAAA ATGTTATAAGATGAAAACTAGTTTTTCTAAGAACTTGATGTTATTTACTTGATGAGTTGATGGAGGATTACGTATGT GGTTTGGTTTTGTTTTTAGGTATTCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGG AGTACCTTGTCTCACATGGGCTTAAGAATGCTTCATGTACTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTTGGA AGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATTTCATGTGTTCTAGTTTCTGTTTGGGCATCTGTTATTTTCA TGGCATGTGGCGTGAAGCTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCAGTTTTTTA TTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCGCCTAATCAGCTTACACCAAAG AGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAGATCGCTGGATGTAAGTATCATATGCTA CATGGAACTTTTGTAGTTTGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTT TCAGCAGCCAGCGTCTTCTATGGTTAATTCAGGCTTCAAAATTGCACCTGATCAGTTTACACGAATGCGATTGCCGG ATGTGGTAAATTCAGGTGTCAATTACCCTCCACAGAGGACATTGCCGGATGTAAGTATCTTATGCTACATGGAAATT TTGTTCTGGCCAGATTGGCATGAAAATCCAGACACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTT TATTGTATTTATTCTGCAAATGATGTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGT AATCGAACTCAAGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACACTAAGATCACAATTTCGTATTTTAT GATCAACACTGAATACCTATGTCTAGTGTCATGATTATAGTCATGTGAAGTGGATTTCTTAATATATGCCTCATCTA TGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGTGCTTCAAAAGGAAATCCTGGACCATCTGGTGCAGGAG CTGTACTCCGTGCTGAAGATGGAAGTGTTGTATGCGGAGTTCATGAAAACATTGTGAATCTTTTAGGATATATATTT TTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAAC GGCA >GPH10_ananassa_clone2 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT AGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTGGAGAA AGCTTTCATCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTTATCTAAAAGGCAGGTTTAATATCAGCCGTTAG ATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACA TATATTTTATTTTTTTAAAGTAACTAAATGACTATGTACATCGTTTAACAAAAGAAACAATTGAAGTTAAATTAAGA GCACCATAACAGCTGAGAAAGAGTACGAGAACAAAAGTATGAGCTAAAACAAATAGAGAAAATATAGAGGCGATGTT

PAGE 124

124 GTAGAAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAA AGCGGCTTCATATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATT CCCAATTTTCATTTCCCACCAAAAACAAAACCCACACGACGCCGTTTTGCTCCAATCCCCCTTTCTTCTTCAACCCC ATAGTCGCCTCAGCTCAGTTCCATTTGTCTCAGATGCGATGGCCTCCGGCGACCCAATCTCCGACTACACCCAAACA CATCGCATTGTCCTTCTAATCGACCTCAACCCACTCCTCCATCTCCAAGATCCAACCCAATTCCTCACCTCTGTCCT CTCCTCAATCAAAACCCTAACCTCCTTCCCTTCTCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCTCCTCT CCTCTCCGCCTCCAAGCTCCCGTCTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACATATCGATCCCTAT CTCAAACCCTGGCGTCTCTCTCGTTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAACGCTTGTTGCGGCT GCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAGCAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGATACGTT TTCGAATTGCTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAATTTGTGAATGAGTTCTTGA ATTGTGAGGGTTTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAGGCATAT GTGTATAGAGATATTCAATTGAGTCGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGTTGGATT GAAATGTGGTGTTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGCTTGGTT CGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTGGGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATAT AAGAAGGTTAGAGCGCATTTGAGTCTTGAGATATCGGATGTAAAGGGGATGCCTTTGGAGTGCAAGTTTTGTGATCT TGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATCGCTTGTTTTCGGTGGAAGGCATGAGCTCGC AGACAAGAGGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAATGGAGTGTCGAAGATTCAGGTTAAGGCT TTGCAGAAGGATAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCTGATTCTGGTCTATGAAGTTTCAGGAAA AGATGGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCTATCAGTGGAATTGGGTGAGTTTG TACCGAGGAAATTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTTTCT ATTTCAAATGATAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATTTTTGTTAT GGATGAAGGAATTCACCCTCATAAAAAAGGGCATGGCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAAAGATGA AGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCATCTAATAAG CATTCTGCTGAGATTGATGGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCACCTGGAGTTC TTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAA AAAAGTTGAAATTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAATCTAAGGTG CACCAGGAAAAACAAAAGGAGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCGTCATC TGGTTCAGCTGGAGAAATTTCTTTCCCTGTGGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAGATTACAAA CCTCAGAAGATTTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCA TTCGCACATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCACAACAACCCCTTCAGAAGATCA AACTCCTGTAAAATATGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGG ATATGGTTGCCAGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCCTGGATGTGAAGGCTTTACTTCAGAA ATAATAGTTCGAGAGTATCCTTTCATTTCTCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCTAC TTTAATGCTATGTAAACTTTGCCCCTTGTTAGTGTTACACTTTTCCTTCACTAGCACAAAGATATGAATTACAGATA CTTTTCCGGATGGAGATTTTACAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTGAAACA TATTTGCACGCTTTTGGAGACCATTCGTGCTCGGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAA ATTATGCTGGAAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTATTTTCT AATGAAATTGTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAATATATGTTTATGGT TTGCCTGTTAATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATTTGTATG CCCTCCCAAAAGGCCTTTGGGGGTAGTATGAAGAAGGGAGACATTGACAGTCAAAAATATTATCTCCTTATTTTACG TACAAAATTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTCCTTTGC ATAATTTCATATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATGCAT ACAACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACTGCCCAAA TTTATGTGAAAACACTGCATTTATGTTTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTTT CAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATGATGAGG AAGAACTCCCTAATAATTTATTCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATGAGGTG GGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAACATTACAATGGAAG GCCAAGTGCTCAAGTACTTAAACAAGAAGAGCATGCTCGCAAGTTGATGAAAGCTCAAGAGAGTAGAGAGAGGGCTT GGAGAATTGCTTCTTTCACAAGTCGGGTAGCTGATTTGCAGCGAG >GPH10_ananassa_clone7_(same_r estriction_pattern_as_clone20) GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT AGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTGGAGAA AGCTTTCGGCTTTGAAGTGGAGTGTAGGGTAATAACAAACTCGTGATTAAAAGACAGGATTAATGTCAGTGAGGTTT GGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTTCAAACCTCACGACATATGTAGGGTGTATGAATTAT

PAGE 125

125 TAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACGGCCTGATCACTCGACATATGTTGATATACGCCCA ACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTTTTTTAAAATAATTAAATAACTATTTACGTTGTTT AACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAGCTGAGCAAGAGTACGAGAACAAAAGTATGAGCTA CATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAA AAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCATATCATCCGCTTGATCATATATGCGGGT GTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAATAAAACCCACACG ACGCCGTTTTGCTCCAATCCCCCTTTCTTCTTCAACCCCATAGTCGCCTCAGCTCAGTTCCATTTGTCTCAGATGCG ATGGCCTCCGGCGACCCAATCTCCGACTACACCCAAACACATCGCATTGTCCTTCTAATCGACCTCAACCCACTCCT CCATCTCCAAGATCCAACCCAATTCCTCACCTCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTTCTCTCT CTTCCTCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCTCCTCTCCTCTCCTCTCCGCCTCCAAGCTCCCGT CTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACATATCGATCCCTATCTCAAACCCTGGCGTCTCTCTCG TTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAACGCTTGTTGCGGCTGCGATGCGGCAGCTGGTACATGA TTACGCTTGGGAGCAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGGTACGTTTTCGAATTGCTGTGGTTTGAGGT CTAATTTGGCTGTTGTGTTTTTACCGGCGTGTCAATTTGTGAATGAGTTCCTGAATTGTGAGTTGAATTGTGAGGGT TTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAGGCATATGTGTATAGAGA TATTCAATTGAGTTGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTG TTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGCTTGGTTCGGCTCTTGTT CCATTTGGTTTGATTTATCCAGAGATTGGGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAG AGCGCATTTGAGTCTTGAGATATCGGATGTAAAGGGGATGCCTTTGGAGTGCAAGTTTTGTGATCTTGAGTTGGCTG ATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATCGCTTGTTTTCGGTGGAAGGCATGAACTCGCAGACAAGAGGT CATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAATGGAGTGTCGAAGATTCAGGTTAAGGCTTTGCAGAAGGA TAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCTGATTCTGGTCTATGAAGTTTCAGGAAAAGATGGAAAAG AAGTTTCTGGTGGTTTGTTTGTAGATAGGGTTCTTGAAATGCTATCAGTGGAATTGGGTGAGTTTGTACCGAGGAAA TTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTTTCTATTTCAAATGA TAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATTTTTGTTATGGATGAAGGAA TTCACCCTCATAAAAAAGGGCATGGCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAAAGATGAAGAATGAGATG TGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGA GATTGATGGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCACCTGGAGTTCTTTCTGTAAGG CAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAAA TTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAGTCTAAGGTGCACCAGGAAAA ACAAAAGGAGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCGTCATCTGGTTCAGCTG GAGAAATTTCTTTCCCTGTCGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAGATTACAAACCTCAGAAGAT TTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCACACATCG GCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCACAACAACCCCTTCAGAAGATCAAACTCCTGTAA AATCTGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGATATGGTTGCC AGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCCTGGATGTGAAGGCTTTACTTCAGAAATAATAGTTCG AGAGTATCCTTTCATTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCTACTTTAATGCTAT GTAAACTTTGCCCCTTGTTACTGTTACACTTTTCCTTCACTAGCACAAAGATATGAATTACAGATACTTTTCCGGAT GGAGATTTTACAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTGAAACATATTTGCACGC TTTTGGAGACCATTCGTGCTCGGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGA AAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTATTTTCTAATGAAATTTG TATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAATATATGTTTATGGTTTGCCTGTTA ATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATTTGTATGCCCTCCCAAA AGGCCTTTGGGGGTAGTATGAAGAAGGGAGACATTGACCGTCAAAAATATTATCTCCTTATTTTACGTACAAAATTG ATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTCCTTTGCATAATTTCAT ATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTCTCTGACACTATGTATGCATACAACTTTTG CAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACTGCCCAAATTTATGTGAA CACACTGCATTTATGTTTGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTTTCAGGTACTGTCAG ACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATGATGAGGAAGAACTCCCTAA TAATTTATTCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATGAGGTGGGTGAAAATAGTA GAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAACATTACAATGGAAGGCCAAGTGCTCAA GTAGTTAAACAAGAAGAGCATGCTCGCAAGTTGATGAAAGCTCAAGAGAGTAGAGAGAGGGCTAGGAGAATTGCTTC TTTCACAAGTCGGGTAGCTGATTTGCAGCGAG >GPH10_ananassa_clone18

PAGE 126

126 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT AGCGAACTGAGTGGTTTGGATTTGAGAAGAGGATGAAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTAGAGAA AGCTTTCAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTTATCTAAAAGACAGGTTTAATATCGGCCGTTAG ATCACATTACGGCCCTGATCACTCGACATATGTTGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACA TTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTTTTTTTAAAGTAACTAAATGAC TATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAAGAGCACCATGACAGAGTACGAGAACAAAAGTATG AGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATGTAGAGGCGATGTTGTAGAAATAATTGAACATTAG AAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACAAACTCGTAACCTAAAAGCGGCTTCATATCATCCACT GGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCA AAAACAAAACCCACACGACGCCGTTTTGCTCCAATCCCCCCCCCCTTTCTTCTTCAACCCCATAGTCGCCTCCTCAG CTCAGTTCCATTTGTCTCATGCGATGGCTTCCGACTCGAATTCCGGCGACCCAATCTCCTCCTACACCCAAACCCAT CGCATCGTCCTTCTAATCGACCTCAACCCACTCCTCAATCTCCAAGATCCAACCCAATTCCTCACCCCTGTCCTCTC CTCAATCAAAACCCTAACCTCCTTCCCTTCTCTCTCTTCATCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCT CTCCTCTCCTCTCCGCCTCCAAGCTCCCGTCTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACTTATCGA TCCCTATCTCAAACCCTGGCGTCTCTCTCTTTTGACCGCAAGTTGGCCGGGTCCGATTCGCCGCGGGGAACGCNTGT TGCGGCGGCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAGCCGGTGATCTGCGACGCCGCGGCGGCGGAGACCG GTACGTTATCGAATTGCTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAATTTGTGAATGAG TTCTTGAATTGTGAGGGTTTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGA GGCATTTGTGTGTAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAG TTGGATTGAAATGTGGTGTTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTG CTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTGGGGTGTCATCTAGGATTTTCGGGTGTAATGA TCGATATAAGAAGTTTAGAGCGCATTTGAGTCTTGAGATATCGGATGGAAAGGGGATGCCTTTGGAGTGCAAGTTTT GTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATGGCTTGTTTTCGGTGGAAGGCATG AACTCGCAGACAAGAGGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAACGGAGTGTTGAAGATTCAGGT TAAGGCTTTGCAGAAGGATAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCCGATTCTGGTCTATGAAGTTT CAGGAAAAGATGGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCTATCAAGTGGAATTGGG TGAGTTTGTACCAAGGAAATTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCAT TAGTGTCTATTTCAAATGATAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATT TTTGTTATGGATGAAGGAATTCACCCTCATAAAAAAGGGCATGTCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCC AAAGATGAAGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCAT CTAATAAGCATTCTGCTGAGATTGATGGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCACC TGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGCTTACTTTGCCAGGCAACGTAG CAGCTCAAAAAAGTTGAAATTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAGT CTAAGGTGCACCAGGAAAAACAAAAGGAGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATG TCATCATCTGGTTCAGCTGGAGAAATTTCTTTCTCTGCGGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAG ATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACT TGGGGGCATTCGCACATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAGCATAGCTCAACAACCCCTTCA GAAGATCAAACTCCTGTAAAATCTGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTACTCAGAGAT CCCAAGGATATGGTTGCCAGGCACAAAAGCTATGATCCATCTTCTCAAGCATCTGATCCTGGATGTGATGGCTTTAC TTCAGAAATAATAGTTCGAGAGTATCCTTTCATTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCA ATTCTACTTTAATGCTATGTAAACTTTGCCCCCTGTTACTGTTACACTTCCTTCACTAGCACAAAGATATGAATTAC AGATACTTTTCCGGATGGAGATTTTACAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTG AAACATATTTGCACGCTTTTGGAGACCATTCGTGCTCAGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCT AGAAAATTATGCTGGAAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTAT TTTCTAATGAAATTTGTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAGTATATGTT TATGTTTTGCCTGTTAATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATT TGTATGCCCTCCCAAATGGCCTTTGGGGGTAGTAAGAAGAAGGGAGACATTGACAGTCAAAAATATTATCTCCTTAT TTTACGTACAAAATTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTC CTTTGCATAATTTCATATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGT ATTCATACAACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACGG CCCAAATTTATGTGAAAACACTGCATTTATGTTTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCA AATTTTCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATG ATGAGGAAGAACTCCCTAATAATGTATTCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGAT GAGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAACATTACAA TGGAAGGCCAAGTGCTCAAGTAGTTAAACAAGAAGAGCATGCTCGCAAGTTGATGGAAGCTCAAGAGAGTAGAGAGA GAGCTAGGAGAATTGCTTCTTTTACAAGTCGGGTAGCTGATTTGCAGCGAG

PAGE 127

127 >GPH10_ananassa_clone19 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT AGCGAACTGAGTGGTTTGGATTTGAGAAGAGGATGAAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTAGAGAA AGCTTTTAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTTATCTAAAAGACAGGTTTAATATCAGCCGTTAG ATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACA TTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTTTTTTTAAAGTAACTAAATGAC TATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAAGAGCACCATGACAGAGTACGAGAACAAAAGTATG AGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATATAGAGGCGATGTTGTAGAAATAATAGAACATTAG AAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACAAACTCGTAACCTAAAAGCGGCTTCATATCATCCACT GGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCA AAAACAAAACCCACACGACGCCGTTTTGCTCCAATCCCCCCCCCTTTCTTCTTCAACCCCATAGTCGCCTCCTCAGC TCAGTTCCATTTGTCTCATGCGATGGCTTCCGACTCAAATTCCGGCGACCCAATCTCCTCCTACACCCAAACCCATC GCATCGTCCTTCTAATCGACCTCAACCCACTCCTCAATCTCCAAGATCCAACCCAATTCCTCACCCCTGTCCTCTCC TCAATCAAAACCCTAACCTCCTTCCCTTCTCTCTCTTCATCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTC TCCTCTCCTCTCCGCCTCCAAGCTCCCGTCTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACTTATCGAT CCCTATCTCAAACCCTGGCGTCTCTCTCTTTTGACCGCAAGTTGGCCGGGTCCGATTCGCCGCGGGGAACGCTTGTT GCGGCGGCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAGCCGGTGATCTGCGACGCCGCGGCGGCGGAGACCGG TACGTTATCGAATTGCTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAATTTGTGAATGAGT TCTTGAATTGTGAGGGTTTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAG GCATTTGTGTGTAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGT TGGATTGAAATGTGGTGTTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGC TTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTGGGGTGTCATCTAGGATTTTCGGGTGTAATGAT CGATATAAGAAGTTTAGAGCGCATTTGAGTCTTGAGATATCGGATGGAAAGGGGATGCCTTTGGAGTGCAAGTTTTG TGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATGGCTTGTTTTCGGTGGAAGGCATGA ACTCGCAGACAAGAGGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAACGGAGTGTTGAAGATTCAGGTT AAGGCTTTGCAGAAGGATAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCCGATTCTGGTCTATGAAGTTTC AGGAAAAGATGGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCTATCAGTGGAATTGGGTG AGTTTGTACCAAGGAAATTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTA GTGTCTATTTCAAATGATAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATTTT TGTTATGGATGAAGGAATTCACCCTCATAAAAAAGGGCATGTCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAA AGATGAAGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCATCT AATAAGCATTCTGCTGAGATTGATGGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCACCTG GAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGTTTACTTTGCCAGGCAACGTAGCA GCTCAAAAAAGTTGAAATTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAGTCT AAGGTGCACCAGGAAAAACAAAAGGGGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATGTC ATCATCTGGTTCAGCTGGAGAAATTTCTTTCTCTGCGGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAGAT TACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTG GGGGCATTCGCACATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAGCATAGCTCAACAGCCCCTTCAGA AGATCAAACTCCTGTAAAATCTGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATC CCAAGGATATGGTTGCCAGGCACAAAAGCTATGATCCATCTTCTCAAGCATCTGATCCTGGATGTGATGGCTTTACT TCAGAAATAATAGTTCGAGAGTATCCTTTCATTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAA TTCTACTTTAATGCTATGTAAACTTTGCCCCCTGTTACTGTTACACTTCCTTCACTAGCACAAAGATATGAATTACA GATACTTTTCCGGATGGAGATTTTACAATCAGAAGTTGGGGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTGA AACATATTTGCACGCTTTTGGAGACCATTCGGTGCTCAGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCT AGAAAATTATGCTGGAAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTAT TTTCTAATGAAATTTGTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAATATATGTT TATGTTTTGCCTGTTAATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATT TGTATGCCCTCCCAAATGGCCTTTGGGGGTAGTAAGAAGAAGGGAGACATTGACAGTCAAAAATATTATCTCCTTAT TTTACGTACAAAATTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTC CTTTGCATAATTTCATATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGT ATTCATACAACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACGG CCCAAATTTATGTGAAAACACTGCATTTATGTTTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCA AATTTTCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATG ATGAGGAAGAACTCCCTAATAATGTATTCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGAT

PAGE 128

128 GAGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAGCATTACAA TGGAAGGCCAAGTGCTCAAGTAGTTAAACAAGAAGAGCATGCTCGCAAGTTGATGGAAGCTCAAGAGAGTAGAGAGA GAGCTAGGAGAATTGCTTCTTTTACAAGTCGGGTAGCTGATTTGCAGCGAG >GPH10_ananassa_clone20 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT AGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTGGAGAA AGCTTTCGGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTGATTAAAAGACAGGATTAATGTCAGTGAGGTTT GGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTTCAAACCTCACGACATATGTAGGGTGTATGAATTAT TAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACGGCCTGATCACTCGACATATGTTGATATACGCCCA ACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTTTTTTAAAATAATTAAATAACTATTTACGTTGTTT AACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAGCTGAGCAAGAGTACGAGAACAAAAGTATGAGCTA CATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAA AAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCATATCATCCGCTTGATCATATATGCGGGT GTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAATAAAACCCACACG ACGCCGTTTTGCTCCAATCCCCCTTTCTTCTTCAACCCCATAGTCGCCTCAGCTCAGTTCCATTTGTCTCAGATGCG ATGGCCTCCGGCGACCCAATCTCCGACTACACCCAAACACATCGCATTGTCCTTCTAATCGACCTCAACCCACTCCT CCATCTCCAAGATCCAACCCAATTCCTCACCTCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTTCTCTCT CTTCCTCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCTCCTCTCCTCTCCTCTCCGCCTCCAAGCTCCCGT CTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACATATCGATCCCTATCTCAAACCCTGGCGTCTCTCTCG TTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAACGCTTGTTGCGGCTGCGATGCGGCAGCTGGTACATGA TTACGCTTGGGAGCAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGGTACGTTTTCGAATTGCTGTGGTTTGAGGT CTAATTTGGCTGTTGTGTTTTTACCGGCGTGTCAATTTGTGAATGAGTTCTTGAATTGTGAGTTGAATTGTGAGGGT TTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAGGCATATGTGTATAGAGA TATTCAATTGAGTTGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTG TTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGCTTGGTTCGGCTCTTGTT CCATTTGGTTTGATTTATCCAGAGATTGGGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAG AGCGCATTTGAGTCTTGAGATATCAGATGTAAAGGGGATGCCTTTGGAGTGCAAGTTTTGTGATCTTGAGTTGGCTG ATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATCGCTTGTTTTCGGTGGAAGGCATGAACTCGCAGACAAGAGGT CATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAATGGAGTGTCGAAGATTCAGGTTAAGGCTTTGCAGAAGGA TAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCTGATTCTGGTCTATGAAGTTTCAGGAAAAGATGGAAAAG AAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAGTGCTATCAAGTGAAATTGGGTGAGTTTGTACCGAGGAA ATTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTTTCTATTTCAAATG ATAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATTTTTGTTATGGATGAAGGA ATTCACCCTCATAAAAAAGGGCATGGCATTGGTGCAGAGAATAAGGGTCAGTCTCGTCCAAAGATGAAGAATGAGAT GTGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTG AGATTGATGGAAAGAAAAAAAGTAGCGAAAGAAGTTCACATTCACTCAAAGATCTCACCCGGAGTTCTTTCTGTAAG GCAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAA ATTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAGTCTAAGGTGCACCAGGAAA AACAAAAGGAGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCGTCATCTGGTTCAGCT GGAGAAATTTCTTTCCCTGTCGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAGATTACAAACCTCAGAAGA TTTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCACACATC GGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCACAACAACCCCTTCAGAAGATCAAACTCCTGTA AAATCTGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGATATGGTTGC CAGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCCTGGATGTGAAGGCTTTACTTCAGAAATAATAGTTC GAGAGTATCCTTTCATTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCTACTTTAATGCTA TGTAAACTTTGCCCCTTGTTACTGTTACACTTTTCCTTCACTAGCACAAAGATATGAATTACAGATACTTTTCCGGA TGGAGATTTTACAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTGAAACATATTTGCACG CTTTTGGAGACCATTCGTGCTCGGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGG AAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTATTTTCTAATGAAATTT GTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAATATATGTTTATGGTTTGCCTGTT AATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATTTGTATGCCCTCCCAA AAGGCCTTTGGGGGTAGTATGAAGAAGGGAGACATTGACCGTCAAAACTATTATCTCCTTATTTTACGTACAAAATT GATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTCCTTTGCATAATTTCA TATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATGCATACAACTTTT

PAGE 129

129 GCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACTGCCCAAATTTATGTGA ACACACTGCATTTATGTTTGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTTTCAGGTACTGTCA GACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATGATGAGGAAGAACTCCCTA ATAATTTATCCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATGAGGTGGGTGAAAATAGT AGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAACATTACAATGGAAGGCCAAGTGCTCA AGTAGTTAAACAAGAAGAGCATGCTCGCAAGTTGATGAAAGCTCAAGAGAGTAGAGAGAGGGCTAGGAGAATTGCTT CTTTCACAAGTCGGGTAGCTGATTTGCAGCGAG Polymorphic segment of GPH10: fragme nt between primers 10PPR1 and 10AB22 >10PPR1AB22_vesca AACGGAGAAGAAGACTGTCGACATTTTAAGAGAAAGCTTTCAGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT TATCTGAAAGACAAGTTTAATATCAGCCGTTGGATCATATTACGGCCCTGATCGCTCGACATAATTCGATATATATA TATATATATTTTTTTTTTTTCTAAAAAAAAAAATCGATATACAGTATATTTTTTTTGAATTAATTAAATGAGTATTT AGATCGCTTAAAAAGATAAACAATCGAAATTGGTTTAAGAACACCATAGGAGCAAGAGTATGAGAACAAAAGTATGA GCTACACTGTTTGCTCATCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATAAACAATTAAACATTACA AAATCAAATTACCTAACAATGAACCATTTTCAGACATGTAAAATCATAAAATTAAAAGGTTCGAGTCGCATATGAGT TTGTCGAGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGTAACGAGTCAAACGAGCTAAAAACGAGT CGAATAAAAATCGGGCACCATCAATATCGAGACTATGTAAGAGCCGAGGAGTAAAATAATAACAAACTCGTTATCTA AAAGACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCCT CATTCCCAATTTTCATTCCCACCAAAAACAAAACC >10PPR1AB22_nubicola AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT TATCTGAAAGACAGGTTTAATATCAGCCGTTGGATCATATTACGGCCCTGATCGCTCGACATAATTCGATATATATA TATATTATTTTTTTCTAAAAAAAAAAATCGATATACAGTATATTTTTTTTGAATTAATTAAATAAGTATTTAGATCG CTTAAAAAGATAAACAATTGAAGTTGGTTTAGAAGCATCATAGGAGCAAGAGTACGAGAACAAAAGTATGAGCTACA CTGTTTGCTCGTCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATAAACAATTAAACATTACAAAATCA AATTACTTAACAATGAACCATCTTCAGACATGTAAAATCAGAAAGTTAAAAGGTTCGAGTCGCATATGAGTTTGTCG AGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGTAACGAGTCAAACGAGCTAAAAACGAGTCGAATA AAAATCGGGCACCATCTATATCGAGACTATGTAAGAGCCGAGGAGTAAAATAATAACAAACTCGTTATCTAAAAGAC AGGTTTAATATCAGCCCTTGGACCATATGTACGGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCCTTCC AATTTTCATTTCCCACCAAAAACAAAACC >10PPR1AB22_mandshurica AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT TATCTGAAAGACAGGTTTAATATCAGCCGTTGGATCATATTACGGCCCTGATCGCTCGACATAATTCGATATATATA TATTATTTTTTTCTAAAAAAAAAAAAATCGATATACAGTATATTTTTTTTTGAATTAATTAAATGAGTATTTAGATC GCTTAAAAAAAATAAACAATCGAAGTTGAATTAGGAGCACCATAGGAGCAAGAGTATGAGAACAAAAGTATGAGCTA CATTGTTTGCTCGTCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATAAACAATTAAACATTACAAAAT CAAATTACTTAACAATAAACCATCTTCAGACATGTAAAATCAAAAAGTTAAAAGGTTCGAGTCGCATATGAGTTTGT CGAGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGTAACGAGTCAAACGAGCTAAAAACGAGTCGAA TAAAAATCGGGCACCATCAATATCGAGACTATGTAAAAGCCGAGGAGTAAAATAATAACAAACTCGTTATCTAAAAG ACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCATT CCCAATTTTCATTCCCACCAAAAACAAAACC >10PPR1AB22_nilgerrensis AACGGAGAAGAAGACTGTCGACATATCTAGAGAAAGCTTTCAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG TTATCTGAAAGACAGGTTTAATATCAGCCGTTGGATTATATTCCGGCCCTGATCTCTCGACATATGTTGATATACGC CTGACTCAAATTCTATATACATTTTCGAAAGAATTTTTGTTGTTGAAGTAACTAAATGACTATACGATTGAAGATAG ATTAAGAGAAACATAGCAACTGAGTAAAAAGTATGAGAACAAAAGTATGAGCTACATTGTTTGCTCCTCGGTCTGTT TATATGGAGAAAATATAAATGTGATGTTGTAAAAATAATTGAACATTAAAAAAAATTAAATTATCTAATAACGAATC ATCTTTAGACGTACGTACAAAATCAGAGAGTTAAGAGATTCGAGTTGCTCAAATACCATATTTTACTTGACTTAACA

PAGE 130

130 ATACGAGTCAAACGAGCTAAAAACGAGTCGATTAAATCTAAGAGCCGAGGAGTAAAGTAATAACAAACTCGTTATCT AAAATACAGGTTTAATATCAGCCGTTGGATCATATATACAGGTGTGATTCGAAAACCGAAGTTAACCCGCCAAACCC TCATTCCCAATTTTCATTCCCACCAAAAACAAAACC >10PPR1AB22_viridis AACGGAGAAGAAGACTGTCGACATTTCTAGAGAAAGCTTTCAGCTTTGAAGTGTAGGATAATAACAAAGAAACTCGT TATCTGAAAGACAAGTTTAATACCAGCCGTTGGATCATATTACTGCCCTGATCGCTCGACATAATTCGATATATATA TATATATTATTTTTTTCTAAAAAAAATAAATCGATATACAGTATATTTTTTTTTGAAGTAATTAAATGATTATTTAA ATCGCTTAAAAAGATAAACAAGAAGTTGGTTTAGGAGCACCATAGGAGCAAGAGTATGAGAACAAAAGTATGAGCCA CACTGTTTGCTCTTCGGTTTGTTTATACAGAGAAAATATAAAAGTGATGTTGTAGAAACAATTGAACACTAAAAAAT CAAATTACCTAACAACGAACCATCTTCAGACATACAAGATCAGAAAGTTAAGAGGTTCGAGTCGCACATGAGTTTCT TGAGGCGATCAAATACCACAGTTTACTTGACTCAACAACTTTACGCATACGAGTCAAACGAGCTAAAAACGAGTCGA ATAAAAATCGGGCACCATCAATATCGAGACTATGTAAGAGCCGAGGAGTAAAAAATAATAACAAACTCGTTATCTAA AAGACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTGTGATTCGAAAACCGAGTTAACCCGCCAAACCCTCC TTCCCAATTTTATTTCCCACCAAAAACAAAACC >10PPR1AB22_iinumae AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAACTTTGAAGTAGTGTAGGATAATAACAA ACTCGTTATCTAAAAGACAGGTTTAATATCAGCCGTTAGATCCTATTAAGAGCCGAGGAGTAAAATAATAACAAAGT CGTAACCTAAAAGCGGCTTCATATCATCTACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCC AAACCCAATTTTCATTTCCCACCAAAAACAAAACC >10PPR1AB22_ananassa_clone2 AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCATCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG TTATCTAAAAGGCAGGTTTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGC CCAACTCAAATTCGATATATATTTTCGATATACATATATTTTATTTTTTTAAAGTAACTAAATGACTATGTACATCG TTTAACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCATAACAGCTGAGAAAGAGTACGAGAACAAAAGTATGAG CTAAAACAAATAGAGAAAATATAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCC GATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCATATCATCCGCTTGATCATATATGCGGGTGTGAT TCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACC >10PPR1AB22_ananassa_clone7_(sam e_restriction_patte rn_as_clone20) AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAAGTGGAGTGTAGGGTAATAACAAACTCG TGATTAAAAGACAGGATTAATGTCAGTGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTTC AAACCTCACGACATATGTAGGGTGTATGAATTATTAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACG GCCTGATCACTCGACATATGTTGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTT TTTTAAAATAATTAAATAACTATTTACGTTGTTTAACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAG CTGAGCAAGAGTACGAGAACAAAAGTATGAGCTACATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAG AAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCG GCTTCATATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCA ATTTTCATTTCCCACCAAAAATAAAACC >10PPR1AB22_ananassa_clone18 AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG TTATCTAAAAGACAGGTTTAATATCGGCCGTTAGATCACATTACGGCCCTGATCACTCGACATATGTTGATATACGC CTAACTCAAATTCGATATATATTTTCGATATACATTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTC GATATACATTTTTTTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAA GAGCACCATGACAGAGTACGAGAACAAAAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATG TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACAAA CTCGTAACCTAAAAGCGGCTTCATATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCG CCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACC >10PPR1AB22_ananassa_clone19 AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTTAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG TTATCTAAAAGACAGGTTTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGC

PAGE 131

131 CTAACTCAAATTCGATATATATTTTCGATATACATTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTC GATATACATTTTTTTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAA GAGCACCATGACAGAGTACGAGAACAAAAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATA TAGAGGCGATGTTGTAGAAATAATAGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACAAA CTCGTAACCTAAAAGCGGCTTCATATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCG CCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACC >10PPR1AB22_ananassa_clone20 AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCG TGATTAAAAGACAGGATTAATGTCAGTGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTTC AAACCTCACGACATATGTAGGGTGTATGAATTATTAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACG GCCTGATCACTCGACATATGTTGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTT TTTTAAAATAATTAAATAACTATTTACGTTGTTTAACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAG CTGAGCAAGAGTACGAGAACAAAAGTATGAGCTACATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAG AAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCG GCTTCATATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCA ATTTTCATTTCCCACCAAAAATAAAAC >GPH23_ananassa_clone3 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACGACCTTTACAGTGAGAGTGTGACCAGAG GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGATAAAA GACCTGCCAGAATCCACGCCACCAAACATCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCA TCAACATGCAATAATGTGGGTCCATAAGAACCATGTGCATGACATAAGATTCCTCAAGCTTCGATTTCCTAATTTGC TTCAAAAGAAGTAAGTTCAGAGTCACTCAAACCCTAATATAGATCTCAAATTTAATGAAACATATTCCTAAGAGCCT ACACAAATATAAAATCGTAACTGAACTTAATCTGAAACTGTCGTATAAATTGTAAATCGATCAAAACCAAACTTCAT GTTCAGATTCACAGACCGTATCAGAGATAGCATACAAGTGACCTCTGAAACAAAACATAATTCCAACAAGATCGCAA ACATTCGAGATTAAATACGATGAGCTATGAGACAACTATTCCATGCAAATCTAACAAAAAAGAATAAAGGGATCTGG AGAATTATGGGTTAGAGGTGACCTTCAGAGTTTGGGTGAAACACAACTGGGGACGACCTACACCGAGGAGGAACTGC CAAAATCTATCTGAAACCTAACAAATAAAAAGGGTCTATCTGTCAATAAACGAGCTCCCTATCGTCCATCTCCAATC TTGTAAGGGGTGATCCTTACGCTTCCCTTTGTCCTCTCCCCCATCTACTAAGTAGACGCTAGCTGCGGATTGTTATT ATGTTTTGGATAGAATACCTTTGCAAAATTGGAAGCTCCAGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAAT ATCAGACCGTTCCGACGCCGGAGCTTCCTCAGAAAGCCTACCATCCGCAACATCGTTGCCAGGCCTTGCGAGGTTTG CCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTATAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGATGAA TGAATCATCAAATTAATTAATCAAGGTGACAAAGAAAGATTATATCCTTCCATTCCTAAGTCAAAAACCATTACAAT GTACCGCCGGCAAAATGCTGCTAATAGAATTGACATTGTAAGTGGGGGATAGTGTCACGAGCTGCAATAGGAGGTAG TGTCATCGTGACACTCTAGAGGTGATGCTTAAGAGGGTCACAAGGTCAATGGCAAAGCATGAGGGTTAAAGAGGATG TTTACTGACATGTTGAAAGACAATGTCGTAATTAGTTAAAGTGAAGTACTGTGAAGTTAGTATTTCGAAAAACTGTA ACATCGGAAGGGGTTCAATACATTTGACGACATATTTTTATGAAGTTATTAGGAATTAGTTACGAGAGATGGTTTTT TCTTAGAATATTTTGATTTTGATGTTTCCTTGACACACATTATATTTCTCCATGTTCTTCTATGTATAAGTAATTTT CTGTATCACTTAGAAACATTTCTTACTCTTTCCAGAAGCATCTCCAAACATCCCCCTAAACCAATAGCCCTAACATG TCAATGTCACATGTCAATAGATGAAAGATCAACCTAAATGGTACCATATGTCCATACATAAAAGGACCCAAAAAAAT AAATAAAGAAATAAATATGCACCTTCATTTTTAAGCGCCAGAAAAAGTAGAGAAGAATATAAGGTTTGAAGTGATCA AGGGGATAAGCAGTTTAAGGTCGACTTGTTTGGAAACAATGCTAACCACCACCACTGCCACTCACAGTCTCAGCTCC TCCTCCTCTGCTTCCCAACTCCCATCGCTCTTCCACTCTCTATCACAAAACCCAATCTCCCTCAGATTCTCCTCCAC ATTAAAGCTAACCAAAACCAGAACCAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTGCTCTGA GAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCCTCCACAGTGCCGTCGGAGATGAAGGCGTGGGTG TA >GPH23_ananassa_clone4 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACGACCTTTACAGTGAGAGTGTGACCAGAG GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGATAAAA GACCTGCCAGAATCCACACCACCAAACTCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCAT CAACATGCAATGATGTGGGTCCATAAGAACCATGAGTATGACATAGAGTCTTCAAGCTTCGATTTCCTTATTTGCTT CGAAAGAAGCAAGTTCAGAGTCACACAAACCAGAATATAGATCTCAAATTTAATGAAACATATTCCTAAGAGCCTAA AGAAATATAAAATCGTAACTGAACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCAAAAACAAACTTCAAGT

PAGE 132

132 TCAGATTCAGAGAGCAGATCAGAGATAGCGTACAAGTGACCTAAGAAACAAAACAAAATTCCAACAAGATCGCAAAC ATTCGAGATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAAGAGAATAAAGGGATCTGGAG AATTAGGGGTTAGAGGTGACCTTAAGAGTTTGGGTGAAACACAACTGGGGAAGACAGAGACAGAGGAGGAACTGCGA AGATCTATCTGAAACCAAACAAAGTAAAAAGGGTTTAGCTGTCAGTAACGAGCTCCTAACCGTCCATCTCCAATCTT GTCAGGGGTGATCCTACGCGTCCACTTGTCCTTTCCCAGTTCTAACTATGTAGACGCTAGCTGCGGATTGTTATTAT GTTTTGGATAGAATACCTTTGCAAAATAGGAAGCTCCTCCTTGTTTTTCTGCAAGAGAAAGGCCAAAATATCTGACC ATTCCGACGCCGGAGCTTCCTCAGAAAGCCAGTACCATCCGCAACATCGATGCCAGGCCTTGCGAGGTTTGCCTCCG CTTCTTTGGATTGTGTTTTTCGTGGTTTAGGAGATTGTTGAACAAAAAAGAAAAACATATATGATAAATGAATTATC AAATTAATTAATCAAGGTGACGATACAAGATGAGAACACCAAAGGTTCAATAGTGTGTACTCTCAAGCCTAATACTA ACACAACAAAGAAAGATTCTATCCTTCCATTCCCAGATCAAAAACCACTCTAATGTACCGCCGGCAAAGTGCTGCTA ATAGAATTGACATTGTAAGTAGGGGATAGTGTCACGAGCAGCTCTAGGAGGTAGTGTCATCGTGACACTTTATTGGG GTGGATGCTAAGGGGGTTCAGGTTATGAGCAAGCATGGGGGGTAAGGGGGATATCTACTGGCATTTTGAATGACAAT GTTGTAAATGAAAACTTATATTTCAAGGTATTTTGATTTAATATTTAGAAAAACTGTAACATCAAAAGGGGTTCAAT ACATTTGCCGACATATTTTTATGAGGTTTTTATGAATTAGTTATGAGAGATGGTTTTCCTTGGACTATTTAGATTTT GATGTTTCCTTAACACACATTATATTTCTCCATTTTCTTGTAAGTAATTTTCTGTATCACTTAAAAACATTTCTTAC TCTTCCCAGAAACATCTCCAAACATCCCTAAACCGATAGCTCTAACATGTCAATGTCAATAGATGAAAGATCAACCT AAATGGTACCATATGTCCATACATAAAAAGACCCAAAAAGAAAATAAATAAGCACCTTCATTTTTAAGCGCCATAAA AAGTAGAGAAGAATACAAGGTTTGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTCGGAAACAATGCTA ACCACCACCACTGCCACTCTCAGCTGCTCCTCCTCCTCTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACC AAACCCAATCTCCCTCAGATTCTCCTCCACATTACAGCTAACCAAAACCAGAACCAGACCAACCCTTAAAACTCTCA CTCGCCAAAAATGCCAGCTCCCTGCTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCCTCC ACGGTGCCGTCGGAGATGAAGGCGTGGGTGTA >GPH23_iinumae_clone2 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACGACCTTTACAGTGAGAGTGTGACCAGAG GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGATAAAA GACCTGCCAGAATCCACGCCACCAAACTCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCAT CAACATGCAATAATGTGGGTCCATAAGAACCATGAGTATGACATAGAGTCTTCAAGCTTCGATTTCCTTATTTGCTT CGAAAGAAGCAAGTTCAGAGTCACACAAACCAGAATATAGATCTCAAATTTAATGAAACATATTCCTAAGAACCTAA AGCAATATAAAATCGTAACTGGACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCAAAAACAAACTTCAAGT TCAGATTCACAGAGCAGATCAGAGATAGCATACAAGTGACCTAAGAAACAAAACAACATTCTAACAAGATCGCAAAC ATTGGAGATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAAGAAACTAAAGGGATCTGGAG AATTAGGGGTTAGAGGTCACCTTAAGAGTTTCGGTGAAACACAACACAACTGGGGAGACAGAGACAGAGGAGGAACT GCGAATATCTATCTGAAACCAAACAAAGTAAAAAGGGTTTAGCTGTCAGTAACGAGCTCCTAACCGTCCATCTCCAA TCTTGTCAGGGGTGATCCTACGCGTCCACTTGTCCTCTCCCAGTTCTAACTATGTACACGCTAGCTGCGGATTGTTA TTATGTTTTGGATAGAATAGAATACCTTTGCAAAATAGGAAGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAA TATCTGACCATTCCGACGCCGGAGCTTCCTCAGAAAGCCGGTTCCGTCCGCAACATCGATGCCAGGCCTTGCGAGGT TTGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTTTAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGAT GAATGAATTATCAAATTAATTAACCAAGGTGACAATACAAGATGAGAACACCAAGGGTTCAATAGTGTGTACTCTCA AGCCTAATACTAACACAACAAAGAAAGATTTCTATCCTTCCATTCCCAAATCAAAAACCACTACAATGTACCGTCTA ATTGAATTGACATTGTAAGTGAGAGATAGTGTCACGAGCTGCATTGGGAGATAGTGTCATCGTGACACTCTATGAAG GGGATGCTTAAGAGGGTCGCATCAATGACAAACATGAGGGCAAATAGAAGGTCTACTGGCATGTCGAATGACAATGT CGTAATTAGTTAAGTGAAACTTATATTTCAAGGTACTTTGACTTAGTATTTAGAAAAACTGTAACATCGAAAGGAGT TCAATACATTTGACGACATATTTTTATGAGGTTTCTATAAATTAGTTATGAGAGATGGTTTTCCTTGGACTATTTTG ATTTTGATGTTTCCTTAACACACATTATATTTAAAGTAATTTTCCGTATCACTTAAAAACATTTCTTACTCTTTCCA GAAGCATCTCCAAACATCTCCCTAAATGTCAATGTCAATAGATGAAAGATCAACCTAAATGGTACCATATGTCCATA CATAAAAAGACCCAAAAAGAAATAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAGTAGAGAAGAATATAAGGTT TGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTCGGAAACAATGCTAACCACCACCACTGCCAGTCTCA GCTGCTCCTCCTCCTCTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACCAAACCCAATCTCCCTCAGATTC TCCTCCACATTACAGCTAACCAAAACCAGAACCAGAACCAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCA GCTCCCTGCTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCCTCCACGGTGCCGTTGGAGA TGAAGGCGTGGGTGTA >GPH23_iinumae_clone5 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACGACCTTTACAGTGAGAGTGTGACCAGAG GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGATAAAA

PAGE 133

133 GACCTGCCAGAATCCACGCCACCAAACTCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCAT CAACATGCAATAATGTGGGTCCATAAGAACCATGAGTATGACATAGAGTCTTCAAGCTTCGATTTCCTTATTTGCTT CGAAAGAAGCAAGTTCAGAGTCACACAAACCAGAATATAGATCTCAAATTTAATGAAACATATTCCTAAGAACCTAA AGCAATATAAAATCGTAACTGGACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCAAAAACAAACTTCAAGT TCAGATTCACAGAGCAGATCAGAGATAGCATACAAGTGACCTAAGAAACAAAACAACATTCTAACAAGATCGCAAAC ATTGGAGATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAAGAGACTAAAGGGATCTGGAG AATTAGGGGTTAGAGGTCACCTTAAGAGTTTCGGTGAAACACAACACAACTGGGGAGACAGAGACAGAGGAGGAACT GCGAATATCTATCTGAAACCAAAACAAGTAAAAAGGGTTTAGCTGTCAGTAACGAGCTCCTAACCGTCCATCTCCAA TCTTGTCAGGGGGGATCCTACGCGTCCACTTGTCCTCTCCAGTTCTAACTATGTACACGCTAGCTGCGGATTGTTAT TATGTTTTGGATAGAATAGAATACCTTTGCAAAATAGGAAGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAAT ATCTGACCATTCCGACGCCGGAGCTTCCTCAGAAAGCCGGTTCCGTCCGCAACATCGATGCCAGGCCTTGCGAGGTT TGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTTTAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGATG AATGAATTATCAAATTAATTAACCAAGGTGACAATACAAGATGAGAACACCAAGGGTTCAATAGTGTGTACTCTCAA GCCTAATACTAACACAACAAAGAAAGATTTCTATCCTTCCATTCCCAAATCAAAAACCACTACAATGTACCGTCTAA TTGAATTGACATTGTAAGTGAGAGATAGTGTCACGAGCTGCATTGGGAGATAGTGTCATCGTGACACTCTATGAAGG TGATGCTTAAGAGGGTCGCATCAATGACAAACATGAGGGCAAATAGAATGTCTACTGGCATGTCGAATGACAATGTC GTAATTAGTTAAGTGAAACTTATATTTCAAGGTACTTTGACTTAGTATTTAGAAAAACTGTAACATCGAAAGGAGTT CAATACATTTGACGACATATTTTTATGAGGTTTCTATGAATTAGTTATGAGAGATGGTTTTCCTTGGACTATTTTGA TTTTGATGTTTCCTTAACACACATTATATTTAAAGTAATTTTCTGTATCACTTAAAAACATTTCTTACTCTTTTCCA AACCATCTCCAAACATCTCCCCAAATGTCCATGGCCATAATAGATGAAAGATCAACCTAAATGGTACCATATGTCCA TACATAAAAAGACCCAAAAAGAAATAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAGTAGAGAAGAATATAAGG TTTGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTCGGAAACAATGCTAACCACCACCACTGCCAGTCT CAGCTGCTCCTCCTCCTCTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACCAAACCCAATCTCCCTCAGAT TCTCCTCCACATTACAGCTAACCAAAACCAGAACCAGAACCAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGC CAGCTCCCTGCTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCCTCCACGGTGCCGTTGGA GATGAAGGCGTGGGTGTA >GPH23_mandshurica_clone3 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACGACCTTTACAGTGAGAGTGTGACCAGAG GTGCCTGGGCGGAGCTGCCCAACCTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGTGTCTGCCATTTGATAAAA GACCTGCCAGAATCCACACCACCAAACTCTTTAGCACCAATCCAATCCATAACAACTTCATAAAACACACATAGCAT CAACATGCAATAATGTGGGTCCACAAGAACCATGAGTATGACCATAGTCTTCAAGCTTCGATTTCCTTATTCGCTTC GAAAGAAGCAAGTTCAGAGTCACACAAACCAGAACATAGATCTCAAAAATTTAATGAAACATATTCCTAAGAACCTA AAGAAATATAAAATCGTAACTGAACTTATTCTGAAATTGTCGTATAAATTGTAAACCGATCAACACAAACTTCAAGT TCAGATTCACAGAGCAGATCAGAGATAGCATACCAGTGACCTAAGAAACAAAACAACCTTCTAACAAGATCGCAAAC ATTGGAGATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAAGAGAATAAAGGGATCTGGAG AATTAGGGGTTAGAGGTCACCTTAAGAGTTTGGGTGAAACACAACCTGGGGAGACAGAGACAGAGGAGGAACTGCCA AGATCTATCTGACCACCAAACCAAGTaAAAAGGgTtaaGCTATCAgTAACgAgCtCCtAACCGTCCaTCTCCAATCt TGTCagGgGtGAtCCTACGCGTCCACTTGTCCTCTCCCAGTTCTAACTGTGTAGACGCTAGCTGCGGATTGTTATTA TTTTTTGGATAGAATACCTTTGCAAAATAGGAAGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAATATCTGAC CATTCCGACGCCGGAGCTTCCTCAGAAAGCCAGTACTATCCGCCACATCGATGCCAGGCCTTGCGAGGTTTGCCTCC GCTTCTTTGGATTGTGTTTTTCGTGGTTTAGGAGATTGCTGAACAAAAAAGAAAAACATAACATATGATGAATGAAT TATCAAATTAATTAATCAAGGTGACAATACAAGATGAGAACACCAAAGATTCAATAGTGTGTACTCTCAAGCCTAAT ACTAACACAACAAAGAAAGATTCTATCGTTCCATTCCCAAATCAAAAACCATTACAATAGACCGCCGGCAAAGTGCT GCTAGGAGGTAGTGTCATCGTGACACTCTATGAAGGTGATGCTTAAGAGGGACACGTCAATAGCAAGTATGAGGGAA AAAAAGGATGTTTACTGGCATGTCGAATGATAATTTCGTAATTAGTTAAGTGAAACTTATATTTCAAGGTACTTTGA CCTAGTATTTGGAAAAACCGTAACATCTAAATACACTTGACGACATATTTTTATGAGATTTCTATGAATTAGTTATG AGAGATGGTTTTCTTTAGACTATTTTGATTTTGGATGTTTCCTTAACACACATTATATTTCTCCATTTTCTTTTTAT GTATAAGTAATTTTCTGTATCACTTAAAAACATTTCTTACTCTTTCCAGAAGCATCTCTATCCCCCTAAACCAAATA CACTAACATGTCAATGTCCACCCAAAAGAAATAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAATAGAGAAGAA TATAAGGTTTGAAGTGAACAAGGGGATAAGCAGTTTAAGGTCGACTTGTTCGGAAACAATGCTGACCACCACCACTG CCACTCTCAGTCTCAGCTCCTCCTCCTCTACTTCCCAACTCCCACCACTCTTCCACTCTCTAACACCAAACCCAATC TCCCTCAGATTCTCCTCCACATTACAGCTAACTAAAACCAGAACCAGACCAACCCTTAAAACTCTCACTCGCCAAAA ATGCCAGCTCCCTGCTCTGAGAGTGTCTGCTAAATACGAAGCTTCCCCTGCCACAGCTGAGGCCTCCACGGTGCCGT CGGAGATGAAGGCGTGGGTGTA

PAGE 134

134 From fosmid 10B08 >FvescaParent_10B08Fb GTATGTCCTATTGATTAGATCGTAAATGATTAATTAGATAGGTAATTACTTTCTTGAAAGCGGGCAAGCCGTGATAG TCTTGAAAGAGAGCGAGCTTTCTGAAGATGGATTTCCCGTTTGTTTTGAGCCCCGCCGCGTCTGGGTTGCTTGCAAC CCACGACTCAAGTAGGTCAAGCGAAAGCTGCAAACAAGTGTGTTCATGACCATCAGTACATAGTCTGGAAGTTCAAT GCTTACCTAATCAACACTACTCAAGTCAAGTGTCCAGTACTGATCGACCTACTTAGACCACGTACGTCATATATTTT TCTTCTATATATTTTCAAGGCTAGAATGAGAACTAAGAACCTAGCTAGCTAGCTAACCTGATTCTCTGCAAGTCCCA TCTGGATGATTCCGGTGGGATTTTTAACTTGATCATACGGGTTCTTCTCGTATTCTTCCCACCCTAGGAAGTATGAC GAGTTCTGGCCATGAGAGTCGCAGCTAGCTTTCTTCGACAACATTTTCGAGCAGTTAAACAGATCAAGAGCTTTTCA ATTCTGAGAGAGAGAGGGACAGAGGAGCAAAGAGAGCTAGACGTAGATAGAGAAGGTTTTGTAAGCAGCTCAGTTTG GTTTGTGGAAAATATTTAGGTAAGGCTGCATGGATATAAATAGGTGCTGTTGATTCGTTTTACTTATTAGCTTAAAC AAACACATATGAGTTGGACCTCATCCGAATCTTTTATCTTAATTCTACTCGTACTTTTTTTTTTTTTT >FnubicolaParent_10B08Fb AGTTAGTTAATAATATGTCCTATTGATTAGATCGTAAATGATTAATTAGATAGGTAATTACTTTCTTGAAAGCGGGC AAGCCGTGATAGTCTTGAAAGAGAGCGAGCTTTCTGAAGATGGATTTCCCGTTTGTTTTGAGCCCCGCCGCGTCTGG GTTGCTTGCAACCCACGACTCAAGTAGGTCAAGCGAAAGCTGCAAACAAGTGTGTTCATGACCATCAGTACATAGTC TGGAAGTTCAATGCTNACCTAATCAACACTACTCAAGTCAAGTGTCCAGTACTGATCGACCTACTTAGACCACGTAC GTCATATATTTTTCTTCTATATATTTTCAAGGCTAGAATGAGAACTAAGAACCTAGCTAGCTAGCTAACCTGATTCT CTGCAAGTCCCATCTGGATGATTCCGGTGGGATTTTTAACTTGATCATACGGGTTCTTCTCGTATTCTTCCCACCCT AGGAAGTATGACGAGTTCTGGCCATGAGAGTCGCAGCTAGCTTTCTTCGACAACATTTTCGAGCAGTTAAACAGATC AAGAGCTTTTCGATTCTGAGAGAGAGGGACAGAGGAGCAAAGAGAGCTAGACGTAGATAGAGAAGGTTTTGTAAGCA GCTCAGTTTGGTTTGTGGAAAATATTTAGGTAAGGCTGCATGGATATAAATAGGTGCTGTTGATTCGTTTTACTTAT TAGCTTAAACAAACACATATGAGTTGGACCTCATCCGAATCTTTTATCTTAATTCTACTCGTACTTTTTTTCTTTTT TCTTTTTCATGTGCATAAGCAAATGCATTTCCGATTAAACATAAAAATGTACTGTCGAAACATCATTTCCAGCCAAA TCCAAACATTCGCTCTAAAAAAGCTACATTTGCTATTAGATTCAATAACACAAAACCAAGCAAACATTAATCTTCAT AAATACCAAAATTGGCCTCAATACCCAACTTAAAAACGACCTCAGTCCAGAGGAACCTCAACGTCTCCGTCGGTGAT CTCCTGCTGCAAGTCCTTGGGGTCCTTCCCATCCAC >11D02_vesca GAGCTGCTGTGTGAACCAAATGGTACAGAGAAGCCGTTTGCCAAACCTACCCATGATCCAATCAAATGCATAAACTT TAAGAACTCAAATACCAAACGATCAAACATAATGACTGAAATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAA AACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCAGCCATT CCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAATTTCATACAGA AGTCTTCCCACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATAGTATAAGAAGGA GATGAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTG GTTTCCTTTCAGTCTCTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCAAT TTTCTTTTCTAGGTGATTTTTTTTCTTTTATAATTAATTTGGTTTTATTTTTCCAAATAATACCTGAAAGACTTTTT TTTTCGATAGGATTGCAGTAATTTTTTTTGGACAGTATTACGGGACACTGTGACAGCTTTAGAGTTTGAATCTTAGG TTGGATGATTTAAGTATCTTAGTTGAATGGATGTTATGACATATTGGTGATTAGTATTAGAGTAATGAGAAAGAGAA AATAAAATGAAAATACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAATGCAATAGATTGACAAAGAAA TGAAGACCTAAAAAAACCATTGCATTAATGCAATAGTGTTGATATTCCAATCTCTCCTGAATAGTATTACAACTCTC CTGGACAAGTCATAACTGTGGGGGGTAATGGTGTAAACAAACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTT GCTGGGCAGACATAGCACCCCATATATCATATCAGATGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTA AACCTAATTTTGTCTAAAGAATGCTAAAATCGAACTCCCAAGCAACCGAATCTTCTGTTCCCCTGCTTTAGTATGTT GTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGGAGTTACAGCATGAAGCAGCTCATCTCTTGTT GCAGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATGAAGCTGCTCGTCTGG AGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAGGTCCCAACACTTACCAGGTAGGTTAGTCTCTT CTGCGTCGAGTAACCATGCGGGCACCTGGTGAGAAAAGCGTAACATCTCTCTTCTCGGAATCCATAGAATGGCGCTT CTGTCCGTATCAGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGGTGAGACACAAACACCAATCTCTTC AGAGTAATCATCAAGAACCTCCACCATTTGATATTGGTGCCTCACTTCATCTGGAGTTGAAC >11D02_viridis

PAGE 135

135 GAGCTGCTGTGTGACCAAATGGGTACAGAAGAAGCCNGTTTGCCAAACCTACCCATGATCCAATCAATGCATAAACT TTAAGAACTCAAATACCAAACGATCAAACATAATGACTGAAATGAACAAAAATCAAATGAGCAAAGACTAAATGAGA AAACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCTGCCAT TCCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAAACACAATTTGATACAGAAGT CTTCACACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATAGTATAAGGAGAAGAT GAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCCTAACTTGGTT TCCTTTCAGTGTCTTCGACCTTCTTCTAAAACGACGGAGTCGGTGAAACTGTGCAAGTCTTCTTGTGAATTTTCTTT TCTAGGTGATTTTTTTTTTCTTTTATAATTAATTTGGTTTTATTTTTCCAAATAATACCTGAAAGACTTTTTTTTTT TTTTTTTGATAGAAATACCTAAAAGACTTCATAAAAGCTGTTAAGGCTTCATTTAGGATTGCAGTAATTTTTTTTGG ACAGTATTACGGGACACTGTGACAGCTTGAGTTTGAATCTTAGGTGGGATGATTTAAGTATCTTAGTTGAATGGATG TTATGACATATTGGTGATTAGTATTAGAGTTATGAGAAAATAAAATGAAAATACAGTACTGGCAATAAACACAATAC GGTGGAGCAATCAACAAAGCAATAGATTGACAAGAAATGAAGACCTAAAAAAAACCATTGCATTAATGCAATAGTGT TGATTTTCCAATCTCTCCTGAATAGTATTACAACTCTCCTGGACAAGTCATAACTGTGGGGGGTAATGGTGTAAGCA AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACATAGCACCCCATAAATCATATCAGATGG GGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAAACCTAATTTTGTCTAAAGAATGCTAAAATCGAACTCCC AAGCAACCAAATCTTCTGTTCCCCTGCTTTAGTATGTTGTGATTATGCCTCTGCTTCCCCAGCAGCATGAATCCGCT CGTCTGGAGTTACAGCATGAAGCAGTTCGTCTCTTGTTGCAGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGT AGCTCGTCTGGCATTGCAGCATGAAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGG ATCTAGGTCCCAACACTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNN >11D02_iinumae GAGCTGCTGTGTGAACCAAATGGTACAGAGAAGCCGTTTGCCAAACCTACCCATGATCCAATCAAATGCATAAACTT TAAGAACTCAAATACCAAACGATCAAACATAATGACTGCAATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAA AACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCTGCCATT CCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAATTTGATACAGA AGTCTTCACACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATACTATAAGAAGGA GATGAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCTTAACTTG GTTTCCGACGGAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCAATTTTCTTTTCTAGGTGTTTTTTTTTCTTTT ATAATTAATTTGGTTTTATTTTTCCAAATAATACCTGAAAGACTTTTTTTTTTTCGATAGAAATACCTGTTAAGACT TAAGACTTCATTTAGTATTGCAGTAATTTTTTTGGACAGTATTACGGGACACTGACAGCTTTAGAGTTTGAATCTTA GGTTGGATGATTTAAGTATCTTAGTTGAACGGATGTTATGACATATTGGTACTTAGTATTAGAGTTATGAGAAAAGA AAAAATGAAAATACAGTACTGGCAATAAACACAATTCGGAGGAGCAATCAACAATGCAATAGATTGGCAAGAAATGA AGACCTAAAAAAACCATTGCATTAATGCAATAGTGTCGATTTTCCAATCTCTCCTGAATAGTATTACAACTCTCCTG GACAAGTCATACCTGTGGGGGGTAATGGTGTAAACAAACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCT GGGCAGACGTAGCACCCCCTAAATCATATCAGATGGGGTTAATACTACCCAGGTGTGACATATTTGTACAGTTAAAC CTAATTTTGTCTAAAGAATGCTAAAATCGAACTCCCAAGCAACCGAATCTTCTGTTCCTCTGCTTTAGTATGTTGTG GTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGGAGTTACAGCATGAAGCAGCTCGTCTCTAGTTGCA GCATGAGGTAGCTCGTCTCTTGTTGCAGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCGTCTGGCAT TGCAAGATGAAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAGGTCCCAAC ACTTACCAGGTAGGTTAGTCTCTTCTGCGTCGAGTAACCATGCGGGTACCTGGTGGGAAAAGCGTAACATCTCTCTT CTCGGAATCCATAGAATGGCGCTTCTGTCCGTATCAGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGG TGAGACACAACACCAATCTCTTCAGAGTAATCATCAAGAACCTCCACCATTTGATATGGTGCCTCACTTCATCTGGA GTNNNNN >11D02_nubicola GAGCTGCTGTGTGACCAAATGGTACAGAGAAGCCNGTTTGCCAAACCTACCCATGATCCAATCAAATGCATAAACTT TAAGAACTCAAATACCAAACGATCAAACATAATGACTGAAATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAA AACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCAGCCATT CCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAATTTCATACAGA AGTCTTCCCACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATAGTATAAGAAGGA GATGAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTG GTTTCCTTTCAGCCTCTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCAAT TTTCTTTTCTAGGTGATTTTTTTTCTTTTATAATTAATTTGGTTTTATTTTTCCAAATAATACCTGAAAGACTTTTT

PAGE 136

136 TTTCGATAGGATTGCAGTAATTTTTTTTGGACAGTATTACGGGACACTGTGACAGCTTTAGAGTTTGAATCTTAGGT TGGATGATTTAAGTATCTTAGTTGAATGGATGTTATGACATATTGGTCATTAGTATTAGAGTTATGAGAAAGAGAAA ATAAAATGAAAATACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAATGCAATAGATTGACAAAGAAAT GAAGACCTAAAAAAACCATTGCATTAATGCAATAGTGTTGATATTCCAATCTCTCCTGAATAGTATTACAACTCTCC TGGACAAGTCGTAACTGTGGGGGGTAATGGTGTAAACAAACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTG CTGGGCAGACATAGCACCCCATAAATCATATCAGGTGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAA ACCTAATTTTGTCTAAAGAATGCTAAAATCGAACACTCCCAAGCAACCGAATCTTCTGTTCCCCTGCTTTAGTATGT TGTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGGAGTTACAGCATGAAGCAGCTCGTCTCTTGT TGCAGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATGAAGCNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN Clones from unspecific amplification The size fragments amplified with primers for the vector were not the expected, i.e., they had lower molecular weight than the that had been amplicon cloned. >11D02_nilgerrensis_unspecific ATAAAACCTTAAAACCAGTTTTGGAATATCTAATAACAATCACAATAAACTTCTGATAATGAGATATTAATCCTCAC ATATCTTATTCAACAAGNCCTTAAACAACATTTCAAACCTCACTTAAAAATATTCCTCCACTTTTCATAGGTGTGAC AATAGGTGCAGTTTTTCCCGTTTGCGCAAAAACTTTTCCCCTCATTGAAATATTTACATTTTTTCTTCTTATAGAAA GAGTCACGAGGCACCGGAGCTTCCACCGCTGGCTCAGGATGCTGCTGTGCGAACGCCGCCTAATGATGGGGAGGACA AACCGCCTGGAGATGCTGAGGATAACTCTGATAACCCGCTTGAGGATAGAGAGAATAACTCACCTGAGGATGGGGAT GATAACTCTGATAACCCGCCTGGGGATGGAGAGGATAATCAATATCCATAAATCAACACAAACCATAAATTGTTCAA ATAAGTTATTGGTGTGAACCAAATCATAAAACCTTAAACCAGTCTAGAAATGTCTAATAACAAGCACAATAAACTTC TGGTAATGAGGCATTAATCCTCACATCTTATTCAACAGGCCTTAAACAACATCTCAAACCTCACTTCATCTGGAGTT GAAC >11D02_F_ananassa_3_unspecific AAAGACGTTTTGCAGATGTTCACTCCAGATGAAGTGAGGGAGTGTGATGCCATTAAATGGCAGCAACTATAAGACTT GGAGAACTGAGTTAGACCTGAACCTAGCTCAGCAGAATGCAGATTGGTGTCTAAGTGTTCCAATGCCTACTGAACTT GGCGCAGCTAGAGATAATTGGCTGAGAGCTAATAAGATCTGTAAGCTTACCATTATACGGACTATGACAGATGTTGT GAAAGGTGGTATCCCTGAGAAAGAGGTAGCTAGTGAGTTTTTGGAGGCCATAGCTGAGAGGTTTGCTGTGAGTGACA AGGCTGAAACCAGCATGTTGCTGGATCAGCTGCATAGCATGAAGTATGACATGAAGCTGAACATTAGGGAGTATATC CTGAAGATGATAGACATAGCCTCTAAGCTTACTGCCCTTAAGATGACCATAGAGGAGGACTTGGTGGTTATGCTGGT TCTGAAATCCCTGCCTGTAGAATTTGATCAGCTGAAAACTGCCTATAACACCCAGAAGGATAAGTGGTCGTTGAATG AGTTAATTGCTGTGTGTGTCCAGGAGCATGAGAGGATTAAGAGGGTAACCATTCACCTAGTTACCACCAAACCTCAG TGGAACAAAGCTGAGAAGAAAACTGGTCCTTCTAAGAACTTGGGAGTTGGCAAGAAGGCCATGAAGGTCAGTGGTAA CAAGGGAGGCATTAAGTGTTTTTTCTGCAAGGTTAAGGGTCACATGAAACCTGACTGTGGCAAGTATAAGACCTGGA AAACTATGATAGGAAATGAACCAGTCAAAAACTTTTATGTTTAGGTTAATTTTAGCTAATGTTCCAACTGAAACTTG TAGTTTT >11D02_R_ananassa_3_unspecific ATCTAAAGGCAGCGTGTGACCAATCCAAAGGGTTGTACTTCTACTTGCTTTCTTTTTAGATTGTTTAGCAAATTTAC CCTTAACACACTCGATGCAATCCACCAAATCAGAAAAGTCTAAATCCGGCAAAATTCCGCTGTTTTACCAACAATTT TAATCTCTCCTTTGAGACATGTCCAAGTCTCCTGTGCCAAAGAAAAGCAGACTTCTCATTCATTTTTCTTTTATTTC CAGTAATAGAATCATCAACACCATTCTCAATCAACAAAACTTCAGTGTGGCAAGTATTAGGTAAAGACCAGTAATCA TCAATAAACTGAGCAGTAGCAAGATAATGAGAAGCACGACTTATTTTCATTCCATAAGAATCAATCAAAAACTGACA ATTGTCTTTAACCAAAACAGCAACTGAAATTAAGTTCCTAGACATGGAAGGAACATAATACACTTGCTCTAAAACTA GAAAAACTTCTGGCCTCAAAACTAACTTAACAAAACCAACTGCTTCTATTTCTACTCTCGTGCCTTCTCCAACATGA ATCCTGACTTCATTTTCCCTTGAACTCAACAGGCTTTGAACCCTCTTGTAAAGAATAGATAATGTGCCCCTGTGAAC CTGTATCACACTCCCAGTAACTGTATAACATTTCTTTAATTCACCACCAACTTAGAAATTCCCTTTTATACCATTAC CTTCCAATCCTTCAATCTAATATATTTTATAACTAACCCAAGATTGTTCCCATAACCTCCCTAAAAAAAATATAATA TGAACTCTCTTTTTATCTCATCATTAGCAATGACTATCTCC

PAGE 137

137 >11D02_ananassa_7_unspecific NNGGGACGTTTTGCAGATACTGCTGTGTGACCAAACCCTAAATAAACCCCACCATCCAGCCTTGCCACCTCTATTGC ATGCCTTTTGATCGTTTCCTCCTCACACTTGTTTTCTTTCTCTTCCTCCCCCAAAACAAGAACCCAAAGTCCCCCAA AACGCATCATATATAGATACAGACGCAGCGAGTGTTATTATTAACCCGAAGAAAAACCCAAAGAGCTAATCGACAGA GAAGAAGAAGAAAGAGGCTTTATATAAAGAGAGAAAACGCTTGCTGTTGATTCAGAGCAACCAGCCCTTCTCTTTTT CCCTTCTTCTCTCTGTGTGTAATATTTCAATGGCCGTTGAAGCTCGGCACCTCAATCTATTCCCCTCTCAACAACTC TTCTGCAACAACAGGTACTGATCCTCACTTCATCTGGAGTTGAAC >11D02_ananassa_9_unspecific GAGCTGCTGTGTGAACCAAAAAAGAGAAAGACAGAAAAAAGAGCAGGAGGATGGAGTTGCCAAAAAGGCTGATCTGG TGCGGCAAGGTTGATTTGTGTTTTGGTCCTTACTGATTTGTGTCCATTGGATTGCTTATAAATGACGTGTCAGCTTG TTAGTGATGTCCAAGCAGACAAGCCAAATCCTATGAAAAGAGAGTCAATTACATATAAGTCTATTTTGAGACATTTT CTCCCATATAGTTTCATCCAAACATTTTTACCCATATAAGTCCACCTAAAGCTAAATAGAGTAGTATATTAGATATT AAAGTTTAACATAATTACAGGTTGTCATCTAGCAAAAAAAAAAAAATATTTCTCTTATTTACGAATATACCACTAGA GTAAAAGGTCAACAACCACCCTCCTCTCAAAAAAAGTCTACGGTCGACTTCCAGCGATGTTTCCAATTAACTTTCGG TGAGGATTTTGGTTCACACAGCAGCTC >17O22_vesca AAAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATTTGACAACAGACA TAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCG TAGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTT CCCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATCCCCTTATC ACTGTAGGGCTTCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATAGTAGCCTAGTTTAGTTTCTTATGCT GAAGCAAAATATGTAATCACCTACGCTACAGAATAGTGTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATGA AGAACGGTACCAGTTACCCAACATAATCATAGTTATTTTGGCCTATTGATATTTTGATTAACGTGTAATTGATCGCT ACTTGAATGATGTATATTATGAATGGCACTATTTAATATTTTGGGCTGCTACCTACTCTTCAACAAACTCTAATTAA TTAACCAAACATCAGTGTCACAAGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAACCTAC CTCCCAAAAATAGTTACTTTAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGAT TTGTTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTCTGGTCATCAGTGTCAAACTAG AAGAACCGTGAATTGCGACCCCTCAGAATGTCAAAATGAGATCACTGTGATTCCTTTTAAAATTTTAACAGCGATTC TTCTACAAAAGATGGACTAAATTCCACCTTGTACTGTACAAAAAACGAGTTTGAGTAGTGGGAATCGTTCCAATATA TTTCTGCTCTGTTTACCAATTGCCAGGATGATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAGAATG AGAAGAAAGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGC AGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTTGG AACAATATCATTGATTTCAGATTTTGTCTCAGATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATG GATTCAGAAAATTTGCTACAAAAGCCCATAACTTGTAAACATCATCGAAGTTTGTGAGGAAACCC >17O22_viridis TAAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATCTGACAACGGACA TAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCG TAGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTT CCCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAAGGCAAATATGACTCTACATTCATTCGATCCCCTTATC ACTGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTCTAGTTTCTTATGCT GAAGCAAAATATGTAATCACCTAGGCTACAGAATAGTGTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATAA AGAACGGTACCAGTTACCCAACATAATCATACTTGTTTTGGCCTATTGATATTTTGATTAATATGTAATTGATCGCT ACTGGAATGATGTATATTATATTATGAATGGCACTATTTAATATTTTGGGCTGCTACCTACTCTTCAACAAACTCTA ATTAATTAACCAAACATCAGTGTCACAGGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAA CCTACCTCCCAAAAATAGTTACTTTAAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATT CTGGATTTGTTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCATCAGTGTCA AACTAGAAGAACTGTGAATTGCGACTACACCAGGATGCCTTTGGTCACTTACCAACCTCAAGAAAAGGACCCCCTCA GAATGTCAAAATGAGATCACTGTGATTCCTTTTAAAATTTTAACAGTGATTCTTCTACAAAAGACTAAATTCCACTT TGTACTGTACAAAAAACGAGTTTGAGTAGTGGGAATCGTTCCAATATATTTCTGCTCTGTTTACCAATTGCCAGGAT GATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAATGAGAAGAAAGAACTCTACAAGAATCCAAAG

PAGE 138

138 TGCGAAAACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAG AACAAAAGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCT CAGATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTGCTACAAAAGCCCAT AACTTGTAAACATCATCGAAGTTTGTGAGGAAACCC >17O22_iinumae AAAATGGGTTGACGAGTTCGTGAACAACACTTTACGACCCAAAGCGTCCAATACTTCTTAATTTGACAACGGACATA GTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCTTTTCTTATAAAAGCAATGGAATCGTA GAGACTGCATATCTTACTCAGCACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTATAACTTCC CGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATCCCCTTATCAC TGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTTTAGTTTCTTATGCTGA AGCAAAATATGTAATCACCTAGGCTACAGAAGAGTGTTACTTATTACCGGACATGTTCACAATCTTTGAAGATGAAG AACGGTACCAGTTACCCAACATAATCATAGTTATTTCGGAATATTGATATTTTGATTAATATGTAATTGATCGCTAC TTGACTGATGTATATTATGAATGTCACTATTTAATATTTTGGGCTGCTACCTACTCTTCAACAAACTTTAATTAATT AACCAAACATCAGTGTCACAAGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAACATACCT CCCAAAAATAGTTACTTTAAAGCTGGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTT GTTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTCGGTCATCAGTGTCAAACAAGAA GAACCGTGAATTGCGACTATACCAGGATGCCTTTGGCCACTTGCCAACCTCAAGAAAAGGACCCCTCAGAATGTCAA GATGAGATCACTGTGATTCCTTTTAAAATTTTGACAGTGATTCTTCTACAAAAGATGGACCAAATTCCACCTTGTAC TGTACAAAAAACGAGTTTGAGCAGTGGGAATCGTTCCAATATATTTCTGCTCTGTTTACCAATTGCCAGGATGATAC AAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAAATGAGAAGAAAGAACTCTACAAGAATCCAAAGCGCG AAAACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACA AAAGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTCGGAACAATATCATTGATTTCAGATTTTGTCTCAGA TTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTGCTACAAAAGCCCATAACT TGTAAACATCATCGAAGTTTGTGAGGAAACCC >17O22_nubicola AAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATTTGACAACGGACAT AGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCGT AGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTC CCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATCCCCTTATCA CTGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTTTAGTTTCTTATGCTG AAGCAAAATATGTAATCACCTAGGCTACAGAATAGTGTTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATGA AGAACGGTACCAGTTACCCAACATAATCATAGTTATTTTGGCCTAATTTTGATTAATATGTAATTGATCGCTACTTG AATGATGTATATTATGAATGGCACTATTTAATATTTTGGGCTGCTACCTACTCTACAACAAACTCTAATTAATTAAC CAAACATCAGTGTCACAAGTCACACCAACCTAATTAAACTTTCCATTACAAGTAGCTTTCCCAATAACCTACCTCCC AAAAATAGTTACTTTAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTGCCTATAATTCTGGATTTGTT ACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCATCAGTGTCAAACTAGAAGAA CCGTGAATTGCGACTATACCAGGATGCCTTTGGTCACTTACCAACCTCAAGAATGTCAAAATGAGATCTCTGTAATT CCTTTTAAAATTTTAACAGTGATTCTTCTACAAAAGATGGACTAAATTCCACCTTGTACTGTACAAAAAACGAGTTT GAGTAGTGGGAATCGTTCCAATATTATTTCTGCTCTGTTTACCAATTGCCAGGATGATTCAAACATCTAAACTCTAC AGGAACCCTTTTCTAGCAAAAAAATGAGAAGAAGGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTA AGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGTACAAAAGAATCAAACCAGATA AAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAGATTCTTCGTCAACAGTAGA TAGTCCGCCTTCTCTGATGAAGGAAGGATTCAGAAAATTTGCTACAAAAGCCCATAACTTGTAAACATCATCGAAGT TTGTGAGGAAACCC >17O22_mandshurica AAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATTTGACAACAGACAT AGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCGT AGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTC CCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACCACATTCATTCGATCCCCTTATCACT GTAGGGCTTCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTTTAGTTTCTTATGCTGAA GCAAAATATGTAATCACCTAGGCTACAGAATAGTGTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATGAAGA ACGGTACCAGTTACCCAACATAATCATAGTTATTTTGGCCTATTGATATTTTGATTAATGTGTAATTGATCGCTACT TGAATGATGTATATTATGAATGGCACTATTTAATATTTTGTGCTGCTACCTACTCTTCAACAAACTCTAATTAATTA

PAGE 139

139 ACCAAACATCAGTGTCACAAGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAACCTACCTC CCAAAAATAGTTACTTTAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTTG TTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCGTCAGTGTCAAACTAGAAG AACCGTGAATTGCGACTATACCAGGATGCCTTTGGTCACTTACCAACCTCAAGAAAAGGACCCCTCAGAATGTCACA ATGAGATCACTGTGATTCCTTTTAAAATTTTAACAGTGATTCTTCTACAAAAGATAAGACTAAATTCCACCTTGTAC TGTACAAAAAACGAGTTTGAGTAGTGGGAATCGTTCCAATATATTTCTGCTCTGTTTACCAAGTGCCAGGATGATAC AAACATCTAAACTCTACAGGAACCATCTTCTAGCAAAAAAATGAGAAGAAAGAACTCTACAAGAATCAAAGCGCGAA AACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGAGGAGCATCTCCAGCAAGAACAAA AGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAGATT CTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTGCTACAAAAGCCCATAACTTG TAAGGCATCATCGAAGTATTGTGAGGAAACCC >27F10_vesca CCTGCAGGGTTTTTCATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAA GCAGCTCATGATTCCTTTAAACACACACAAAAAAACCCACAGTCAAAATGAGGAAATGAACAATACCCAAGTCATGA ACACACAAAATTCAGTAAAAAAGTAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGAC AAATTTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATGAAAAAGATTAAAA CTTTAAACAAGAAAATAAAGATCAGAGAAAGAAAATATGATGGGTAGATCGGGAGAGATAAAATTACCTGAATCTGA AGTGGGGGAAGTGAGTCAGTGAAGGACTGAGTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG AAGGATGCGCGGCGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAGATGAGAATACGCTA GTGATTTTGATTATGAATTCTATAAATTCTATAAAAATTTATTTCATTTCTTAATTCTTACTCTGTTTCGGTGTTGG CCAGATTTGACTCTTCTGTGCTTCAGTTTTGACCATTTACTTTTATAACCTCAGGAAGGGTTCAAGCGCGGCCTGCC ACGTGGTGAATTCAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCCGGTAACAGTAA CTTTATCGACAAAACGCTTCTTATTTTATTTTATTTTTTTTGGCGAGCAAAACGCTTCTTATTTGTTTTGGGTCTGT ACGCTTTTGGGTCTTATTTGTCAAGTTTCAATCACTAGCAGGAAGACTTGCGTATAATTGAAATAGCCACTATCTAT ACTCTATATGCAAACACAAGAGTAGAGAAGGAGAACCAGAATACATTTCCA >27F10_viridis TGCGGGTTTTTCATCATGTAAGGACCTCCaTTGTTCGGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAAGC AGCTCATGATTCCTTTAAACACACAAAAAAAAAACACCCATAATCAAAATGAGGAAATGAACAATACCCTAGTCATG AACACACAAAATTCAGTAAAAAAGAAAAAAGGGATCCGCTTCAAGCCAATCCCATCAAACTTGCAGACCTTTGGAGA CAAATTTCGTTGCTTAATGTAATAAGCAACAAAaAATTCAGCTCAGCTGGATCAAaGCCCAGATGAAAAAGATTAAA ACTTTAAACAAGAAAATAaAGATCAGAGGAAGAAAATaTGATGGGNAGATCGGGAGAGATAAAATTACCTGAATCtG AaGTGGGGGAAGTGAGTCCGTGAAGAAGTGaGTTGGTGGANTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGC GCAAGGATGCCCGGCGCAGGATAGGAGGGAAAAGGGTGCGTAGGAtAACCCACTCCANGAACCANATGACAATGCNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTATG CAAAAaTACACTCaTATTTATGTAGAAAACGAGAATTGAACCTCTAACCTCTTACAAACAACCTATGAAATGTATAa TATATGTAAAGACGaTTAAAATATATGTATAATATATAATATATGTATTGTTTTATATTTATAACATACTATAGATA TAAATACAATTAAAATATAAAATGTTCAAATTTTAGCAAGAGGTATGTTTCGAAACCATGACTGCTCTGATGGAAAA TATGACCACTTACGATCAAAACAAAGCTATCATTGCATTATATTTGTGAAAAAAATTATATTTATCACTTCATTTTT TGGGCCACAATCTAAGTTTAGTAGAGGCCTATTACCAACCGTACCAACTAAGTCGGTATACCAACATCGATGGTTGG TTTTGATAGAGGATTTTGCCTACCAATCATAAGTTGGTTGGTACATGATATTGGTAAATAAAGTCGGTATATCTACC AATGCCAGCCCTACTTGAAACTTAGCCGGAAGACTTCATATAATTGAAATAGCTGAGATACACACTTGCTATATGCA AACACAAGAGTAGAGAAGGAGAACCAGAATACATTTCCA >27F10_iinumae CCTGCAGGTTTTTCATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAAG CAGCTCATGATTCCTTTAAACACACAAAAAAAACCCACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGAAC GCACAAAATTCAGTAAAAAAGAAAAAAGGGATCCGCTTCAATACAATCCCATCAAACTTGCAGACCTTTGGAGACAA ATTTCGTTGCTTAATGTAATAAGCAACAAAAATCCAGCTCAGCTGGATCAAAGCCCAGATGAAAAAGATTAAAACTT

PAGE 140

140 TACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGATGGGTAGATCGGGAGAGATAAAATTACCAGAATCTGAAGT GGGGGAAGTGAATCAGTGAAGGACTGAGTTGCTGGAGTCTTGGGAGATCTGAGCTCTAAAGCCGGCGAAGGATGCGC GGCGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATACGCTAGTGATTTTGA TTATGAATTCTATAAATTCTACAAAAATTTATTTCATTTCTTAATTCTTACTCTGTTTCGGTGTTGGCCAGATTTGA CTCTTCTGTGCTTCAGTCATGACTTTGACCATTTACTTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGT GGTGAATTCTGGTTCGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCGGGT AACAGTAACTTTATCGTTTTACCGCTAGCATGTCTCTGTCTGTCGACATATATAACGCTTCTTATTTGTTTTGGGTC TCTACGCTTTTGGGTCTTATTTGTCAAGTTTCAATCACTTGAAACTATTGAAATAGCTGAAATACACACTTACTATA TGCAAACACAAGGGAGAGAGGAGAACCAGATCATTCTA >27F10_nubicola CCTGCGGGTTTTTATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAAGC AGCTCATGATTCCTTTAAACACACACAAAAAAAACCCACAATCAAAATGAGAAAATGAACAATACCCAAGTCATGAA CACACAAAATTCAGTAAAAAAGAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAA ATTTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATGAAAAAGATTAAAACT TCAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGTAGATCGGGAGAGATAAAATTACCTGAATCTGAAG TGGGGGAAGTGAGTCAGTGAAGGACTGAGTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCGAA GGATGCGCGGCGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATACGCTAGT GATTTTGATTATGAATTCTATAAATTCTACAAAAAATTTATTTCATTTCTTAATTCTTACTCTGTTTCGGTGTTGGC CAGATTTGACTCTTCTGTGCTTCAGTTTTGACCATTTACATTTATAACCCCGGGAAGGGTTCAAGCGCGGCCTGCCA CGTGGTGAATTCAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCCGGTAACAGTAAC TTTATCGTTTTACCGCTAGTATGTCTCTGTCTGTCGACATGACGCTTCTTATTTCTTTTGGGTCTCTACGCTTTTGG GTCTTATTTGTCAAGTTTCAATCACTAGCAGGAAGACTTGCGTATAATTGAAATAGCCATTATCTATACTCTATATG CAAACACAAGAGAGAGAAGGAGAACCAGAATCATCCA >27F10_mandshurica CCTGCAGGGCTTTTTATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAA GCAGCTCATGATTCCTTTAAACACACACAAAAAAACCCACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGA ACACACAAAATTCAGTAAAAAAGTAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGAC AAATTTCGTTGCTTAATGTAATAAGCAACAAAAAAATCAGCTCGGCTGGATCAAAGCCCAGATGAAAAAGATTAAAA CTTTAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGTAGATCGGGAGAGATAAAATTACCTGAATCTGA AGTGGGGGAAGTGAGTCAGTGAAGGACTGAGTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG AAGGATGCGCGGCGCAGGATAGGAGGGAACAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATACGCTA GTGATTTTGATTATGAATTCTATAAATTCTATAAAAATTTATTTCATTTCTTAATTCTTACTCTGTTTCGGTGTTGG CCAGATTTGACTCTTCTGTGCTTCAGTTTTGACCATTTATTTTTATATCCTCAGGAAGGGTTCAAGCGCGGCCTGCC ACGTGGTGAATTCAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCCGGTACAGTAAC TTTATCGTTTTACCGCTAGTATGTCTCTGTCAGTACTCTGTCGACATAACGCTTCTTATTTGTTTTGGGTCTCTACG CTTTTGGGTCTTATTTGTCAAGTTTCAATCACTAGCAGGTAGACTTGCGTATAATTGAAATAGCCACTATCTATACT CTATATGCAAACACAAGAGAGAGAAGGAGAACCAGAATACATTTCCA >27F10_ananassa_2 CCTGCAGGTTTTTATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCGGAAGC AGCTCATGGACTCCTTTAAACACACAAAAAAAACCCACGATCAAAATGAGGAAATGAACAATACCTAAGTCATGAAC ACACAAAATTCAGTAAAAAAGAAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCACCCCTTTGGAGACAA ATTTCGTTGCTTAATGTAATAAGCAACAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATGAAAAAGATTAAAACTT TACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGGTGGGTAGATCGGGAGAGATAAAATTACCAGAATCTGAAGT GGGGGAAGTGAATCAGTGAAGGACTGAGTTGCTGGAGTCGTGGAAGATCTGAGCCCGGCGAAGGATGCGCGGCGCAG GATCGGAGGGAAAAGGGTGCGTAGGATAACCCAACCAATGAACCAAATGAGAACACGCTAGTGATTTTGATTATGAA TTCTATAAATTCTACAAAAATTTATTTCATTTCTTAATTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACACTTCT GTGCCTCAGTCATGACTTTGGCCATTTACTTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAAT TCTGGTTCGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCCGGCAACAGTA ACTTTATCGTTTTACCGCTAGTATGTCTCTGTCTTATTTGTTTTGGGTCCCTACGCTTTTGGGTCTTATTTGTCAAG TTTCAATCACTTGAAACATAGCAGGAAGACTTGCATATAATTGAAATAGCTGCAATACACACTTGCTATATGCAAAC ACAACAAGAGAGAGGAGGAGAACCAGAATCATCCA _____________________________________________________________________________

PAGE 141

141 >29G10_vesca TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGAGCTTTGGAGAGGTAGGAAGAGTTTATTTCTAGAGGGAGGCTACC CATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAGGAAGATGGGGAGGATAAATAAAAAGGATAGAAACTGCTC AAGTGCTTAACAATGGTTGTAGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGA TGAAGACGCCGTATCTTAAATTTTGATTTGGTTCTTCTCACACACCAGAGATTGAGTTCGGATCATCGGATCCGAAA AATCAAGTCCTTGTGTATAAAAGCACGTTACGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATTATTGTCA CGGTGGACCACGTCAGTCTGGCATATCGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCATATCAGAC CCAAGCCATTACTTGCTTCTATGAACGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATTTCTTATTC ACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGACAGTTCATTGAATTTCAGGAATCCACAATTGGGTG CTGCCTTCTTCT >29G10_nubicola TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGGGCTTTGGAGAGGTAGGAAGAGTTGATTTCTAGAGGGAGGCTACC CATTTGAAGTAGAGATTGACTAAAAACAACTTGAAAGAGGAAGATGGGGAGGATAGAAACTGCTCAAGTGCTTAACA ATGGTTGTAGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGATGAAGACACCGT ATCTTAAATTTTGATTTGGTTCTTCTCACACACCAGAGATTGAGTTCGGATCATCGGATTCGAAAAATCAAGTCCTT GTGTATAAAAGCACGTTACGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATTATTGTCACGGTGGACCATG TCAGTCTGGCATATCGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCATATCAGACCGAAGCCATTAC TTGCTTCTATGAATGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATTTCTTATTCACAACGACGATT GGCTTCTTGGTGTGTTGCGCTTTGTTAGGACAGTTCATTGAATTTCAGGAATCCACAATTGGGTGCTGCCTCT >29G10_nilgerrensis TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGGGCTTTGGAGAGTTAGGAAGAGTTGATTTCTAGTGGGAGGCTACC CATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAGGAAGATGGGGAGGATAAATAAAAAGGATAGAAACTGCTC AAGTGCTTAACAATGGTTGTGGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGA TGAAGACACCGTATCTTAAATTTTGATTTGGTTCTTCTCACACACCAGAGATTGAGTTCGGATAATCGGATTCGAAA AATCAAGTCCTTGTGTGTAAAAGCACGTTGCGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATTATTGTCA CGGTGGACCACGTCAGTCTGGCATATCTATCATCACTCTCAATCTTGTCGATCATCAATTTGGCTACATATCAGACC GAAGCCATTACTTGCTTCTATGAATGTATTTATATCATTACTAATCACCCAGAATTATGGATAATATTTCTTATTCA CAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGACCAGTTCATTGAATTTCAGGAATCCACAATTGGGTG CTGCCTTCTTCT >29G10_mandshurica TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGAGCTTTGGAGAGGTAGGAAGAGTTTATTTCTAGAGGGAGGCTACC CATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAGGAAGATGGGGAGGATAAATAAAAAGGATAGAAACTGCTC AAGTGCTTAACAATGGTTGTAGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATCATATAGAGGTGCATGAAGGA TGAAGACACCGTATCTTAAATTTTGATTTGGTTCTTCTCAGTTCTCACACACCAGAGATTGAGTTCGGATCATCGGA TCCGAAAAATCAAGTCCTTGTGTATAAAAGCACGTTACGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATT ATTGTCACGGTGGACCACGTCAGTCTGGCATATCGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCAT ATCAGACCGAAGCCATTACTTGCTTCTATGAACGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATTT CTTATTCACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGACAGTTCATTGAATTTCAGGAATCCACAA TTGGGTGCTGCCTTCTTCT >32L07_vesca GAGTTGAAAAACGGGTCGAATCCCGGCACCACCGTCCGCGTCGCGTAGGACTTGAATCCTTCCAAGGTCACCTCCTT GATGTACATAGCTGCCCTCGCCGGAGAGGTGCGGACGCTAATCGGAAGCCGATTTTGGAGAGATTTAGTGTCGGTGA TAGATCGGAACCCTAGAAATCTGAGCTTCTGGTTTTTGCTTTCGGAAGTTGAGAGTCTGAAATGACATGGTTCGAAT TTCTTTTTGTTGTTTTCCGCTTTTTTGGTGGGTTCGAATTTTTAGACCAAGGCGGGAGATATTTGGGCCAGTGATTT ATATCTTGGGCTCACTCTGGGACTCATGTCTTTGGGCCTCGTCGACCTCGAGGTGCTCATGAAGTCCGGCCGTCCTC AGGGTCGGAAACACCGCGGTACTACTGACTACTGTGTCATCGCTTTAGAATTTCATTAATTGGCTTTGCGAGCTATA AATAATTGTGATTTGGTTTGAATTTAGGTAAGTTTTAGTATTAGTATTTATCAACGGGAATTGCGGAGATGAGAAAA GTTGAGGTTGATTTGGGGGAGTGTGGTGTTGTTAGTTAGTTGAATTATTAGAAACGAAAAAATAACAGAAGAATATA

PAGE 142

142 AATGTGGATGGATTATTGGATTAAGATTTGATTCAACGGAAGAAGGAGGCGTGGTGTGTGTTTTGATAGTCTAATTT GAACTGTTTTGCTTCTGACAGCTAAAATCTATCCGGTGGTGAAAAATCAGCATCGGCTACTATGTACACTTTTAATC GGCAACGCATTAGCGATGGAGGTGACTTGTCTAATTTACTAAGTTTATTTAGGTTGTTACTGGTACATTTTATGTGT TTATTGCCGTGGATGTAGTTTGTATGGGCCAGTTGACCAGCAGTTTCAAATGGCAGGCCAATAGGGCCAACCTAGAT TGTAGTTGAATTTTGGGAAGGAAAAAAAAAAGCAAACCAAAAGACATCACCACGAGCCACTTTGGCCTATCTATATA TATTACTTCCTTGCTTAATGTGTTGCTCAATTGCTAAACAATATCATCAATGTCTAAAATAACGCGCCTCAAGGCTA AGGCAAGGGAAGGCGTGCCTTAGGACGACCTCTGAAAGACATTTGATATCAAAGGTGTGATTGAGGCGCGCGATCAA GACGACGAGGTCAAGGTGCCTAATACAACATCAGGTTATAGGTTTGAATCTCACTTTGAGAAATGTGATGGTTTGAA CGGTTAAATCTATTGTCTTTTTATATTGTATGGGCGGTAAAATTAAATGTTAAACTTCGGTAAATTGTCAAATGTTT AATAGTATAAGAATCTACATATAGTAGGTGTAAAATAGATACCGAAATGATAATATTTTGTGAATAACGTACGTCAT ATGATTTAATATTAAGACTTTGTACGATTTAACGTTACACATTAAAATTGTAGATAAAAAGTTTATATCATCATCAA CATCGATGTTCGAATAAATTTTATAACGTTCAATGCGGTACAAATCTCCCAATGACTATTATCGAGTACAACGTCCA TATCCGACACATGATATAGGCTATCAAATTATCAAAACCCTTTGATCCGATTCTGTAGCTTTGACGACTATAAGCTT AGTTAAGTTTAGTAGGACTCACCGCAATTTCGCACTAGTAGGACAAAAAGATGGTAAGATTCCTTTCATTTTTCTTC TTTACTATCCTTCTTTTCCTCAATTTTTCCCTAGAATCCTACAACAAGAAAGGACTTTGGCCCCTTGTGCTCCTTTA TCATCTTAAAAGCATCACCACCATCCCCTATATAGATGCATATTCACTATCAAGCTACCCAAGTATGCAAATTTATA GCATCTCATTATCTTGTTTCCTCTAGCTATTCTACTCAATGCATATCAACAACCTGACCCAGTTCTCCTATAATTGC TGGCAGATAGTAATACCAATTACTCCAGAATCTTCACACCCAGAACTTGAAATTACACGACCTCAATACTCCAAACA GTACAAAACAACCCAGATGATCAAAACACATAACATTCTTTATTTCATCTTATTGGGAAAATCTCTATATCTATTAT CTTCATTATTCAATTTTTCTACACTGCATGCTATACATGTTACAAAAGAGAAAGAAAAGACACTAGTCCATATCACA TAGGCCATGTCCTTCCCAATTCTAACCCAACAATTCAAGGACCACACCCATGAGTAGTGGCACTGAATCACTGAATC GTCGCCTTCACAACTACACTACCTATCCAACCCAGACTCAACACAGATGAAAATTCACAGCAGCTAAGAATATAGTA CTAGTTTTGCTCTATCTTTTTTCTTTACCAAAACAAAAAAAACCCTGTAGTAACCAATATAACCGCTAACAGCTTTT CCCATCCTGCCCATAACAGCTTTTCCCCTGCAGTATGGGAAACCCTTATCTAAAACCCCCCGATTTATAGTAACAAA AAAATAAATAAAATAATTTACTTTCCTCATTTACCATTTTACCCTCATCTTCTCCTTCATTGCCACTTGAACCCCCA CTCTCCATGCTCCTTGAACCTTCTCAACACCCTTTCTAGGGCAATGTCAAAAGCGTCTTTTACCGTCTCCAACCCCT CCTGCGGTTTCGCGTACAGAAAATTCGGTATGTAATCGATAACTTTCTCCCGCATTTTCCT >32L07_viridis GAGTTGAAAAACGGGTCGAATCCCGGCACCACCGTCCGCGTCGCGTAGGACTTGAATCCTTCCAAGGTCACCTCCTT GATGTACATAGCTGCGCTCGCCGGAGAGGTGTGGACGCTAATCGGTAGCCGATTTTGAAGAGATTTAGGGTCGGTGA TAGATCGGAACCCTAGAAAATACGTCACCACGAGCCACTTTGGCCTATCTATGTTACTTCCTTGCTTAATGTGTTGC TCAATTGCTCAACAATATCATCAATGTCTAAAATAACGCGCCTCGAGGCTAAGGCAAGGGAAGGCGTCACCTTAGGA CGACCTCTGAAAGACATTTGATATCAAAGGTGTGATTGAGGCGCGCGATCAAGACGACGAGGTCAAGGTGCCTAATA CAACATCAGGTTATAGATTTGAATCTCACTTTGAGAAATGTGATGGTTTGAACGGTTAAATCTATTGTCTCTTTATA TTGTATGGGTGGTAAAATTAAATGTTAAATTTCGGTAAATTGTCAAATGTTTAATAGTATAAGAATCTACATATAGT AGGTGTAAAATAGATACCGAAATGATAATATGTTGTGAATAATATACGTCATATGGTTTAATATTAAGACTTTGTGC GATTTAATGCTACACATTAAAATTGTAGATAAAAAATTTATATCATTATCATCATCGATGTTCGAATAAATTTTATA ACGTTCAATGCGGTACAAATCTCCCAATGACTATAATCGAGTACAACGTCCATATCGGACACATGATATAGGCTATC AAATTATCAAAACCCTTTGATCCGATTCTGTAGCTTTGACGACTATAAGCTTAGTTAAGTTTAGTAGGACTCACCGC AATTTCGCACTAGTAGGACAGAAAGATGGTAAGATTCCTTTTATTTTTCTTCTTTACTATCCTTCTTTTCCTCATTT TTTCCCTAGAATCCTACAACAAGAAAGGACTTTGGCCCCTTGTGCTCCTTTATCATCTTAAAAGCATCACCACCATC CCCTATTTAGATGCATATTCACTATCAAGCTACCCAAGTATGCAAATTAATAGCATCTCATTATCTTGTTTCCTCTA GCTATTCTACTCAATGCATATCAACAACCTGACCCAGTTCTCCTATAATTGCTGGCAGATAGTAATACCAATTACTC CAGAATCTTCACACCCAGAACTTGAAATTACACGACCTCAATACTCCAAACAGTACTGTCAGTACAAAACAACCCAG ATGATCAAAACACTTAAAATTCTTTATTTCATCTTATTGCTATCTCTATCATCTTCATTATTCAATTTTTCTACACT GCATGCTATACATGTTACAAAAGAGAAACAAAAGACACTAGCCCATATCACATAGGCCATGTCCTTCCCAATTCTAA CCCAACAATTCAAGGACCACACCCATGAGTGGCACTGAATCACTGAATCGTCACCTTCACAACCACACTACCTATCC AACCCAGACACAGATGAAAATTCACAGCAGCTAAGAATATAGTACCAATTTTGCTCTATCTTTCTTCTTTACCAAAA CAAAAAAGATCCTGTAGTAACTAATATAACAGCTAACAGCTTTTCCCATCCTGCCCATAACAGCTTTTCCCCTGCAG TATGGGAAACCCTGATCTAAATCCCCCGATTTATAGTAACAAAAAAATAAATAAAATAATTTGCTTTCCTCATTTAC CATTTTACCCTCATCTTCTCCTTCATTGCCACTTGAACCCCCACTCTCCATGCTCCTTGAACCTTCTCAACACCCTT TCTAGGGCAATGTCAAAAGCGTCTTTTACCGTCTCCAACCCCTCCTGCGGTTTCGCGTACAGAAAATTCGGTATGTA ATCGATAACTTTCTCCCGCATTTTCCT

PAGE 143

143 >34D20_vesca GCAGAAAGAAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTCAGGGGATAAAGGAAGTA TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT TATGTGAATCATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGATTATCGGC GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA ATGGATTTAGTACTTCCATTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGAACTGC AGGTGAACCGTATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGGATGAACTATACG TAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGTATGAATCTCCCTCCCGGCCACACTAGAC CACACTTTTGAACTGGCGGATTCCATCCGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCT CCTAGCCATAGTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATATGATGAATTT GGAATGAATACAAAAACTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATT CTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGT ACTTGTATAGTTGTATTGATCTGATATAACATAAATTTAATGAATCTAATAGACATTTTTCCTAGTTAACAGAGGAT AGGTCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACACAAGACTGGTCTATATA AGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCTCTTTATCTGTTTCATACTTTCATTGATCATATT GTCTAGTACTGGAAGAGCTATATTTATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGA AGGTCCTTAGTTACAATGGTGTTGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTTGTTACTTAACCGAT AGCATCATTTGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTC TGATCTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGA AAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTGGAAGCTAGGAAAATGTA TACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCACCAATAACTCCAGCTGGGACTAGTAGTTTGGCTA CCAACAACAGTTCTACAATGAGACACGAGTGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATG CTTGGGGTTTGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCATCC TTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTAATCATGGAGATGATGATGATGTTAATGAACCTTGGT ATAGAGAAGAGACTCTTATGCCTCAGCAGACGAATTTTTACGACTGCG >34D20_iinumae NNNNNNNNNAACTGATGTGCTTTCCGGAGGGACTGACAGTAGAAAAGGACAGTGCAGTTCAGGGGATAAAGGAAGTA TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT TATGTGAATCATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGATTATCGGC GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA ATGGATTTAGTACTTCCATTAACTAGTAACTGAGGAATCCAATTGCACTCTGTGTTTACATGCACAGGAGAACTGCA GGTGAACTATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGGATGAACTATAAGT AGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGCATGAGTCTCCCTCCCGGCCACACTAGACC ACCTAGATTTTGTGCCGACTATCACAATAGTGAAAGTTGGTCCTCCTAGCTATAGTTTCTAGTACTATTCTACTGAT ATCATGTTTCGTCTCAGCTTTTGACAATGGAATATGATGAATATGGAATGAACAAAACCTGCTTTGTCCATCTATTA GCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTT CATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCACTACTTGTGTAGTTGTATTGATCTAAATTTTTCCTAG TTAACAGAGGATAGGTCTCCGGCTGACGTTATCCTACAAGGAAACAGAAACGTACAATTAACGGATTCACAAGACTG GTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCTAGTACTGGAAGAGCTATATTTATCT GATAACAGAAAGTGCTTACTTGCTGGTTCCTACTCATTATGGATCCGAAGGTCCTTAGTTACAAAGGTGTTGACCTG AGCATGAGCGACTAGGATATTCTTAGAGGACCTTATTACTTAACCGATAGCATCATTCGATTCTATTTCACTTATCT TACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTCTGATCTCCTGGTAAATTCTCCGGATCCCG AGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATCTTCGCAGTGAATGATAAC AAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTGTATTTCAGAAAATCAAACGCATTCGTACA TTACGACAGCTTGGGGGGTAACAATAGTTTGGAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTC CAGCAACACAAGCACCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGACACGAG TGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTTTGGGGTTTTGTTGTCAACTA CATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAA TCACCAATAGTAATCATGGAGATGATGATGATGTTAATGAACCTTGGTATAGAGAAGAGACTCTTTGCCTCAGCAGA CGAATTTACGACTNNN

PAGE 144

144 >34D20_viridis NNNGAGAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTCAGGAGATAAAGGAAGTA TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT TATGTGAATCATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGATTATCGGC GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG ACTGCAAGTTACGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA ATGGATTTAGTACTTCCATTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTTCATGCACAGGAGAACTGC AGGTGAACCATATGTCTATATAGATATGTCGAATGTTAGATAGGATACATAGTATGAGGGTGTGGATGAACTATACG TAGAGCACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGCATGAATCTCCCTCCCGGCCACAGTAGAC CACACTTTTGAACTGGCGGATTCCATCCGGCCTAGATTTTGTGCCGACTATCACAATAGTGTAAGTTGGTCCTCCTA GCTATAGTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGATGAATATGGAA TGAACAAAAGCTGCTTTGTCCATCTTTTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTCTCTTT ATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGTATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGT ATAGTTGTATTGATCTGATATAACTCCCATTAATGAATCTAATATAAATTTTTTCCTAGTTAACAGAGGATAGGTCT CCGGCTGACCTTATCCAACAAGGAAACAGAAACATACAATTAACGCATTATACACAAGACTGGTCTATATAAGGCAT CAAATTCTCTTTATCTGTTTCATTGATCATATTGTCTAGTACTGGAAGAGCTATATTCATCTGATAACAGAAAGTGC TTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCCTTAGTTACAATGGTGTTGACCTGAGCATGAGCGACTTG GATCTTCTTAGAGGCCCTTATTACTTAACCGATAGCATCATTCGATTCTATTTCACTAATCTTAGTTCCCATTATGA TGATGATATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAG CCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATATTTGCAGTGAATGATAACGAAGATCCGAGTCGA AGCGACGGCGGAAACCATTGGAGCTTGCTGGTGTATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGG GGGTAACAATAGTTTGGAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCAC CAATAACTCCAGCTGGGACTAGTAGTTTGGTTACCAACAACAGTTCTACAATGAGACACGAGTGCCACTCTACGCAG TCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTTTGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTA CTTGCGTCTGTTTGGAAATTATCATTATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAAATCACGAATGGTAATC ATGGAGGTGATGATGATGTTAATGAACCTTGGTATAGAGAAGAGACTCTTATGCCTCAGCAGACGAATTTACGACGC G >34D20_mandshurica NNNNNGAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGAGCAGTTCAGGGGATAAAGGAAGTA TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT TATGTGAATCATCTGCAAATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGATTATCGGC GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA ATGGATTTAGTACTTCCATTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGAACTGC AGGTGAACCGTATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGGATGAACTATACG TAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGTATGAATCTCCCTCCCGGCCACACTAGAC CACACTTTTGAACTGGCGGATTCCATCCGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCT CCTAGCCATAGTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATATGATGAATTT GGAATGAATACAAAGACTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATT CTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGT ACTTGTATAGTTGTATTGATCTGATATAACATAAATTTAATGAATCTAATAGACATTTTTCCTAGTTAACAGAGGAT AGGCCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACACAAGACTGGTCTATATA AGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCTCTTTATCTGTTTCATACTTTCATTGATCATATT GTCTAGTACTGGAAGAGCTATATTTATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGA AGGTCTTTAGTTACAATGGTGTTGACTTGAGCATGAGCGACTTGGATCTTCTTAGAGTCCCTTGTTACTTAACCGAT AGCATCATTCGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTC TGATCTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGA AAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTGGAAGCTAGGAAAATGTA TACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCACCAATAACTCCAGCTGGGACTAGTAGTTTGGCTA CCAACAACAGTTCTACAATGAGACACGAGTGCCACTCTACGCAGTCGGGGCGATTTATAGACTATACCAAGACAATG CTTGGGGTTTGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCATCC

PAGE 145

145 TTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTAATCATGGAGATGATGATGATGTTAATGAACCTTGGT ATAGAGAAGAGACTCTTATGCCTCAGCAGACGAATTTTACGAGTGCG >34D20_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNATGTTGGCATCCAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGA ATTTGATTTCTACAAGGTTAGCAACAGCCTGCAAACTAGATATATTTTGTTTTTCTAACTATTACAGTGTGTATTAT GTGAATCATCTGCATATTATCTATATCTAACTCTATGGTATAATGATTCAGAACTACAAGGAAAGATTATCGGCGAG AAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATGACT GCAAGTTGCGGGCAATAGGGGCTTCAGGCCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATG GATTTAGTACTTCCATTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGTTTTCCATGCACAGGAGAACTGCAGG TGAACTTTATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGGATGAACTATACGTAG AACACCCAGAAAACCAGAGAAAGTAAAGAGGAACTGCAGGTTGGGCATGAATCTCCCTCCCGGCCACACTAGACCAC ACTTTTGAACTGGCGGATTCCATCCGGCGTAGATTTTGTGCCGACTATCACAATAGTGTAAGTTGGTCCTCCTAGCT ATAGTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGATGAATATGGAATGA ACAAAAGCTGCTTTGTCCATCTGTTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTCTCTTTATC TAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGTATA GTTATTGATCTAATATAACATAAATTTAATGAATCTAATATAAATTTTTCCTAGTTAAGTCAAAAGACCTTATCCTA CAAGGAAACAGAAACGTACAATTAACGCATTATACACAAGACCGGTCTATATAAGACATCAAAATCTCTTTATCTGT TTCATATTGATCATATTGTCTAGTACTGGAAGAGCTATATTTATCTGATAACAGAAAGTGCTTACTTGCTGGTTCAT ACTCAATATGGATCCGAAGGTCCTTAGTTACAAAGGTGTAGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCC CTTGTTACTTAACCGATAGCATCATTCGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGATATCCTTCTG GTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGA CCAACTTGGTAAAAGAAAAGTTGTGATCTTTGCAGTGAATGATAACGAAGATCCGAGTCGAAGCGACGGTGGAAACC ATTGGAGCTTGCTGGTGTATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTG GAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCACCAATAACTCCAGCTGG GACTAGTAGTTTGGTTACCGACAACAGTTCTACAATGAGACACGAGTGCCACTCTACGCAGTCGCGGCGATTTATAG ACTATACCAAGACAATGCTTGGGGTTTGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGA AATTATCATTATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAAATCACCAATAGTAATCATGGAGATGATGATGA TGTTAATGAACCTTGGTATAGAGAAGGAGACTCTTATGCCTCAGCAGACGAATTACGACGCA >34D20_nilgerrensis NGCAGAAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTCAGGGGATAAAGGAAGTA TTATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCTCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCG AATTTGATTTCTACAAGGTGAGCAACATGCCGGCAAACTAGTTATATTTTGTTTTTCTTACTATTACAGTGTGTGTT ATGTGAATCATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGATTATCGGCG AGAAGGTGTTTTGCATGCAAGCAGCAGCAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATGA CTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAA TGGATTTAGTACTTCCATTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCATAGGAGAACTGCA GGTGAACCATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGGATGAACTATACGT AGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGTATGAGTCTCCCTCCCGGCCACACTAGACC ACACTTTTGAACTGGCGGATTCCATCCGGCCTAGATTTTGTGCCGACTATCACAATAGTGTAAGTTGGTCCTCCTAG CTATAGTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGATGAATATGGAAC AAAGCTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTCTCTTTATCTAG TGCATCATGTGAGTTATCAAGTTCATGTCTATGCATTCTGCTGATTTAGGAATAAGGATTGCAGTACTTGTATAGTT GTATTGATCTGATATAACATAAATTTAATGAATCTAATATAAATTTTTCCTAGTTAAGCCAAAAGACCTTATCCTAC AAGGAAACAGAAACGTACAATTAACGCATTATACACAAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTT TCATTGATCATATTGTCTAGTACTGGAAGAGTTATATTTATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACT CAATATGGATCCGAAGGTCCTTAGTTACAATGGTGTTGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTT GTTACTTAACCGATAGCATCATTCACTTATCTTACTTCCCATTACGATGATGATATCCTTCTGGTTTCCCCTAATAT CTCTGATCTTCTGGTAAATTCTCTGGATCCTGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAA GGAAAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTG GTGTATTTCAGAAAATCAAACGCATTCGTACACTACGACAGCTTGGGGGGTAACAATAGTTTGGATGCTAGGAAAAT GTATACAGTATTCAAGAAACTTGTGGCAGCTCCAGCAACACAAGCACCAACTCCATCTGGGACTAGTAGTTTGGTTA CCAACAACAGTTCTACAATGCGACACGAGTGCTACTCTACGCGGTCGCGGCGATTTATAGACTATACCAAGACAATG CTTGGGGTTTGGGGTTTTGTTGTCAATTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCCTTC

PAGE 146

146 GAAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTAGTAATCATGGAGATGATGAAGAGCGTAATGATGATGATG TTAATGAACCTTGGTATAGAGAAGAGACCGTTATGCCTCAGCAGACGAATTTACGACTNNN >34D20_ananassa NNNNNNNNNAACTGATGCGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTCATGGGATAAAGGAAGTA TTAATGTTAGGCATCCTAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC GAATTTGATTTCTACAAGGTGATCAACATGCCTGCACACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT TATGTGAATCATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACCAGGAAAGATTATCGGC AAGAAGGTGTTTTGCATGCATGCAGCACAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA ATGGATTTAGTACTTCCATTAACTAGTAACTGAGGAATACAATTGCACTCTGTGTTTTCCATGCACAGGAGAACTGC AGGTGAACTATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGTATGAACTATAAG TAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGCATGAGTCTCCCTCCCGGCCACACTAGAC CAC ACTTTTGAACTGG CGGATTCCATCCGGCCTAGATCTTGTGCCGACTATCACAATAGTGTAAGTTGGTCCTCCTA GCTATAGTTTCTAGTACTATTCTACTGATATCATGTTTCGTCTCAGCTTTTGACAATGGAATATGATGGATATGGAA TGAACAAAACCTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTTTCTTT ATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGCATTCTGCTGATTTAGGATTTAGGATTGCACTACTTGT ATAGTTGTATTGATCTAAATTTTTCCTAGTTAACAGAGGATAGGACTCCGGCTGACCTTATCCTACAAGGAAACAGA AACGTACAATTAACGGATTCACAAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATT GTCCAGTACTGGAAGAGCTATATTTATCTGATAACAGAAAGTGCGTACTTGCTGGTTCATACTCAATATGGATCCGA AGGTCCTTAGTTCCCAAGGTGTTGACCTGAGAATGAGCGACTTGGATCTTCTTAGAGGCCCTTATTACTTAACCGAT AGCATCATTCAATTCTATTTCACTTATCTTACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTC TGATTTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGA AAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG TATTTCAGAAAATCAAACGCATTGGTACATTACGACAGCTTGGGGGGTAACAATAGTTTGGATGCTAGGAAAATGTA TACAGCATTCAAGAAACTTGTGGCAGTTCCAGCAACACAAGCACCAACTCCAGCTGGGAGTAGTAGTTTGGTTACCA ACAACAGTTCTACAATGGGACACGAGTGCTACTCTACGCAGTCGCGGCGGTTTATAGACCATACCAAGATAATGCTT CGGGTTGGGGGTTTTGTTGTCAAATACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATCATCATCCTTC GGAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTAATCATGGAGATGATGAAGAGCTTAATGATGATGATGTTA ATGAACCTTGGTATAGAGAAGAGACTCTTATGCCTCGGCAGACGAATTTTACGACTGCG ____________________________________________________________________________ >40M11_vesca CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGATGCATCAGAACAGTATG TGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTCATTAATCTGCAAACAATAAGATTTTTTAGGCAAA GCAGAACTATGAGTTCCCCAAACTAATAGCTTTCAAACAAGTAGAGGAGCACATTTACTAAAGATACCTTTGCCTGC TGCTCTTCACTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTTTTGAGGGG AAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACAGTTCCCTTCGGTCTCCCATCACCAT CAAATACAATAAATTTCAATATATTTAACAAAAAAATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATT GTTCAATATATCATTTGAAATTAACAACTTTTATTCTTCTAGTCAACCACATTTCGCAGCTACTTGTTTAACTCATA AACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTTCACAACGAATCCAACA CAAAAGGATCAAAAAAACCATCCAAAACTCATGCACAACATAATCAACCAAATATTTTAACCACAAAAACAAGCACA ATTCTCCAAAGTACAAAAAGAAATGGGCTTTAGACACCAGGAAGGCATATCAAACCGGCCCACACACGTTAAAGGGA TACAAAGATCTCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAGCTCCTCA GATGTCGGAGAGACCCATCTGAACCCAAGTCAACTGCACTGTTACAGCAACTACAAAACGCAAAGATAGAGAGAGAG AGAGAGAGAGAGAGAGAGAGAGAGAGAGAAGATGAGAATATTACACAGGGATCTGGGATCAGAGTGATGATGCTGTG TTTGTGTTTGAGTTTGAAACTGAAGCCCAACCACGACCAACACGTGCTATTTGTAAGCCGAACCCATCTGCCTTCCT TTCCTTCCATGTCTGTTCTGTGGTGTAGACTTTTCGGACAAAGCTTCCGTGGTGTCGTTTTTTTCCCAGGGTGGAAA GGTCTGAACTGCCCCGGGGGACAACGGCGTGGTGTGGTGTGGCCGCTGCCCTTTTGAGGAAATTCACGTGGATTATG GTGTGTCCTGCTTGTACTGTTGTCGGAGTTTACTAGGAACAATGAAATCATATCTATTTCTCATAAAAGGAAGCGTA TTCTTTTTTATTAACCTTTTATTAATTACCTAGATTAGCTATTTCAAGTCAAAATTCATATATCGAAATATATGCTT CTTGTCGCTAGACGCTATATTAGGTAACGAACATTTTAGGTAGTCGAACTGGTAGGCAGCGGCGGAGCTAGGATTTG TTATTAGAGAGGTTAAGATGTAAAAGGTAAGTTTATGTATATGACACTTATTTATATATTTTTCCATTTTTAATGTA AAATTCTATTATGATTTGGAAATCTGAGAAATATCTTATTTTCATTTGAGAAATGTCTTATTTTCATATGAGAAATG GTGTGTGTGGGAGCTTTTGGGGGGGGGGGGGGGAGCTAAAGGCATTTTTCTATTTATATTAGTTGTTTAGAAGAAAA AGAATGAACTATTTGGACATGACCCCTCGACCCAGCATGACCTGATTTTGAGATTAGAAGGGCACAAGTAACCAAAA

PAGE 147

147 ATGTTGTTAGTTGGGACTAAAAATCTTTCAATTGATCTTATGGCAGGTGGGCAAAAATTGTAAGCATTGCCCCCCAA TTTCGAAGAGATGCCTTGAACTAAAAACCAGGAGTAGATGCAAAACAAAGACAAGAGCAACAAATTGGTAATAAACT AGAGTGCATCTGAAACGTGCCCTATGCCAGCCAGCTTTGTCCATCGTAAGGACGAGAGCGACAATAACTATGTGACC ACATGTGATGATGTGAACGGTACTCTTGTCATTTCGACGATCTTTCTTCTGAGATTTTTTCTATTCCTGGGTCACAT ATGCGAAAGAAAACAGAGACAAAAGCATAGAAAAGAATTTGATATGATTGATTCCATAAGTGAAAGATGTATGTACA TGTTAGCCGACACTACAAATGCTGCTAGAAAGAAATATACTACCTCCTAATACAATTTCTGTACCCCCATTTCTACA CAGAATGTTGTAGCAAACTAGCAATGCCACCTTTCTGGTTTAAATTTCAAACAACAGAAACATTCTCCTTCATGTGT TTGCATGTTTCCGAAAAATGTACAGAGCATCATTCAAATGGGATCTTTCTGCAATTGAAGCACCAGTATAGAACCAA AGAGTCAGCAGTGTGTTTGTCGGAAGAGAACTATCGAGCCATGACTGTTTTCGCATCACAGGTTGCAGCTCAGATAT TACAGCGTACAAGCTTTGGCGACACCACCATTTGGATTCCTGGGTTTCATTTGGTAGCCATGCATGAGAAGAAATGA CAACAGAGGAGAAAAGAAAACTACGCGCATTCATAACTAAGTTCTATGCTTTTGTCTTTTATTTCTTGTAAGAGAAG TTTTACCTCTGACGAATGTAAGCTGGTGGATTAAGGGTATGATTTTTCCACCATCTACCCAAAAGCATCCAGCTGCT TGCAAGAAATCAAAGGAACAACATCAGTCAAGATGATTGTCTTCTTGCACAAGCCGAAAGCCCCAGCCAGCCCTCCT CCCCCTTATTGCCAAACTTCACAACTAACCAAAAGCTCATCTAGCACCAAGTTTGGCTCTTCTAACTCAAGGAGTCC GGCATCTGTAGTCACCACAGGAAACCAGCACTCTAAGCTCTGTGGCAGAAACATTACACGGTAGGAAAACAAAGAAT GCCACCGAGCTCCTATGATGCAGAAAGAGGATTCATAACACCCATCAATCACTAGTTAGATCTCTCTCTTTCCAACA ACCCCAGCAGGACTATGCTTCCTTTGTATACCATATTGCCAAGCATCTCTAGACTTATCACCCGACTGATCAAAGTC GTCCAAAGAATTCTCAAACTGTGGTTTCATAGGCCG >40M11_mandshurica CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGATGCATCAGAACAGTATG TGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTCATTAATCTGCAAACAATAAGATTTTTTAGGCAAA GCGGAACTATGAGTTCCCCAAACTAATAGCTTTCAAACAAGTAGAGGAGCACATTTACTAAAGATACCTTTGCCTGC TGCTCTTCACTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTTTTGAGGGG AAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACAGTTCCCTTCGGTCTCCCATCACCAT CAAATACAATAAATTTCAATATATTTAACAAAAAAATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATT GTTCAATATATCATTTGAAATTAACAACTTTTATTCTTCTAGTCAACCACATTTTGCAGCTACTTGTTTAACTCATA AACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTTCACAACGAATCCAACA CAAAAGGATCAAAAAAACCATCCAAAACTCATGCACAACATAATCAACCAAATATTTTAACCACAAAAACAAGCACA ATTCTCCAAAGTACAAAAAGAAATGGGCTTTAGACACCAGGAAGGCATATCAAACCGGCCCACACACGTTAAAGGGA TACAAAGATCTCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAGCTCCTCA GATGTCGGAGAGACCCATCTGAACCCAAGTCAACTGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN >63F17_vesca CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGTCCTACCAACCTCATCATACATGGGCAAGAAATCA TTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTAGATGCAGGTTTTGAATTATTAGAGTCTATAAAGGGAC ATAGTTACAACTGTTTGTATGCTTTTCCATTTTTTTTATTTTTTTATTTTTTGAGAATGTATGCTTTTTCACTTGTA TGGCCTGAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGCACACAGGAACC GTTGAGGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAGTTAGAGCAATGGGTGAGATGAACTGGAA ACAATTTGCTGCTGAGGAGGTTACAGAGATGAGGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCA AAGTCACATCCCTTCCTGGATGTGAGAGTTTCCCCGATGCAGGAGGAAATATAACCGGTTCTTTCCTTGGCATTCAA GAAAATTTGACAATTTGATCACCAGTTCAGTTTTATAGAAGAACTCAGTTAGTACAGTTTTGAAACGTTTTTTTGTT GTATTTAGCAAACCCATAGGAGGATAGGGTTTTCTTTTATTCAACAGGGATATAGGCGCTTTTAGGGTTTCTTTTCC TATTCAATTTCGTTCTTTGGTAGACCAAGTCGCTTCTTTGGCATTCAAGGAAACCTGAGCATTTGATCTGCCTGTCA TCACATCCAGAGTTGCAGATTGTTTAGAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAACTTTTGGTAT TCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGATATGCAGGTAACATCTCTGACTATGCAT CTTTGCTTTTTCTTCTTTTTTTGAGAACAAGGCATCTTGTTTATGTGTAGCCAACTTGAAGCACTGTATTTAAATAA TGCTAAAACAGTGTTAATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGAACTTGATAAGAAA ATAGATCCCAGAGATGGTCTATCTACTACCCTTGACTACACAAGTTACTCATTCTTTACATGTGAAATGGCTATCCA GAGCAGTCGTATTTCATGAGATATTAACAAGCTTTGGACGTCCAGTTGCCAGGTTCATCTCTACTCGGAGGCCAAGT CGAGCAAGGGCAGGCACATCAACAGACCCCTTAA >63F17_viridis

PAGE 148

148 CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGGTCCTACCAACCTCATCATACATGGGCAAGAAATC ATTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTATATGCCGGTTTTGAATTATTAGAGTCTATAAAGGGA CTAGTTACAACTGTTTTTTTCCACTTTTTTTTTTTTTTTTTTTTTTTTGAGAATGTATGCTTTTCACTTATATGGCC TGAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGCACACAGGAACCGTTGA GGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAGTTAGAGCAATGGGTGAGATGAACTGGAGACAAT TTGCTGCTGAGGAGGTTACAGAGATGAGGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCAAAGTC ACATCCCTTCCTGGATGTGAGAGTTTCCCCGATGCAGGAGGAAATATAACCGGTTCTTTCCTTGGCATTCAAGAAAA TTTGACAATTTGATCACCAGTTCAGTTTTATTGAAGAACTCGGTACAGTTTTGAAACGTTTTTTTGTTGTATTTAGC AAACGCATAGGAGGATAGGGTTTTCTTTTATTCAACAGGGATATAGGCGCTTTTAGGGTTTCTTTTCCTATTGAATT TCGTTCTTTGGTATACCAAGTCCCTTCTTTGGCATTCAAAGAAACCTAAGCATTTGATCTGCCTGTCATCACGTCTA GAGTTGCAGATTGTTTAGAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAACTTCTGGTGTTCAACAACG CATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGATATGCAGGTAACATCTCTGACTATGCATCTTTGCTTT TTCTTCTGTTTTTGAGAACAAGGCATCTTGTTTATTTGTGGCCAACTTGAAGCACTGTATTTAAATAATGCTAAGAC CGTGTCAATTTTGTTACAAAAGTCTAGGCAACAATGAACTTGAACTTGATAAGAAAATAGATCCTAGAGATGGTCTA TCTACTACCCTTGACTACACAAGTTGCTCATTCTTTACATGTGAAATGGCTATCCAGAGCAGTCGTAAATTTCATGA GATATTAACAAGCTTTGGACGTCCAGTTGCCAGGTTCATGTCTACTCGGAGACCAAGTGAGCAAGGGCAGGCACATC AACAGACCCCTAAA >63F17_mandshurica CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGGTCCTACCAACCTCATCATACATGGGCAAGAAATC ATTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTAGATGCAGGTTTTGAATTATTAGAGTCTATAAAGGGA CATAGTTACAACTGTTTGTATGCTTTTCCATTTTTTTATTTTTTTATTTTTTGAGAATGTATGCTTTTTCACTTATA TGGCCTGAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGCACACAGGAACC GTTGAGGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAGTTAGAGCAATGGGTGAGATGAACTGGAA ACAATTTGCTGCTGAGGAGGTTACAGAGATGAGGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCA AAGTCACATCCCTTCCTGGATGTGAGAGTTTCCCCGATGCAGGAGGAAGTATAACCGGTTCTTTCCTTGGCATTCAA GAAAATTTGACAATTTGATCACCAGTTCAATTTTATAGAAGAACTCAGTTAGTACAGTATTGAAACGTTTTTTCGTT GTATTTAGCAAACCCATAGGAGGATAGGGTTTTCTTTTATTCAACAGGGATATAGGCGCTTTTAGGGTTTCTTCTCC TATTCAATTTCATTCTTTGGTAGACCAAGTCGCTTCTTTGGCATTCAAGGAAACCTGAGCATTTGATCTGCCTGTCA TCACATCCAGAGTTGCAGATTGTTTAGAGAAGAATTCCAATTCCTTTTGTACAGTTTGGTTAACTTTTGGTGTTCAA CAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGATATGCAGGTAACATCTCTGACTATGCATCTTT GCTTTTTCTTCTATTTTTGAGAACAAGGCATCTTGTTTATGTGTGGCCAACTTGAAGCACTGTATTTAAATAATGCT AAAACAGTGTTAATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGATAAGAAAATAGATCCTA GAGATGGTCTATCTACTACTCTTGACTACACAAGTTACTCATTCTTTACATGTGAAATGGCTATCCAGAGCAGTCGT AAATTTCATGAGATATTAACAAGCTTTGGACGTCCAGTTGCCACGTTCATCTCTACTCGGAGGCCAAGTCGAGCAAG GGCAGGCACATCAACAGACCC >63F17Rrc_ananassa_2 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNTTCAATTTCATTCTGGGATAGACCAAGTCACTTTTTTGGCGTTCAAGGAAACCTGAGCATTTGATCTGCCGG TCATCACATCCAGAGTTGCAGATTGTTTATAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAACTTTTGG TGTTCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGATATGCAGGTAACATCTCTGACTATG CATCTTTGCTTTTTCTTCTTTTTTTGAGAATAAGGCATCTTGTTTATGTGTAGCCAACTTGAAGCACTGTATTTAAA TAATGCTAAAACAGTGTTAATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGAACTTGATAAG AAAATAGATCCCAGAGATGGTCAATCTACTACCCTTGACTACACAAGTTACTCATTCTTTACATGTGAAATGGCTAT CCAGAGCAGTCGTATTTCATGAGATATTAACAAGCTTTGGACGTCCAGTTGCCAGGTTCATCTCTACTCGGAGGCCA AGTCGAGCAAGGGCAGGCACATCAACAGACCCTCAA

PAGE 149

149 >72E18_vesca GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAATCCGGCCTAAGCATTAATATCAAATCAGTCCTTGAGAT TCGACATGCATAAAAAAGACAATAAAGGGTACAAAAACAACCACTCAAACAATCACAACATAATATCATTCAATACC TTGACCATTCCGGTTCCATTATCACACACAAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAACA TACACCACAATCAATGACAACAATGCCTCATTCACACAACAAAGAAATAGACATTCAAAAACAAAACACAATACACA CTACTAATGTGGCACAGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACATAGTTCTCTCACAATAGTAA AGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTAAAGAAACGATCTTTCAGATGGGAAATACCCAAATTT GATTGCTACATGCATAAAACCCTCAAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAAA ATTCAAGAAAAAAAGAGAGAGAAAATTACAGATTTAAAGCGACGAACAATGAAAAGGAATGAGAGGCAAAGAGAAGA GATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGGGAGAGAGAGAGAGAGATCGACGACGAAGCAGAGCGAAA GAGACGAGTGTGGTGTTTGTGAGTTGAGGCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCA GCCGTTTGATTTATGGACCGCGTCTATTGAGCCCTTGTGGGGCCATTACAGCTCCTTCCGCTGTTCCAGTCATTTTT TTCTCCACCTTCTTTACCTTTTTGCCCCTCAGTCCCTTCCCTTTTCTCCCAATTCTTTCTCAACTCTTCTTAAACCT AATTGCATTTCCCTAATTGCATTTTCATTTTAGTGCTTAGATCAATATGATTAAGAAGCTTCATTTTGTCAACACAA GGCAACAAGGACACAGGGGAGCATTTCGATCATCGTTCCAGTCATTTTCGTATATAATTTGGGCTTGAAATGGTTGA TCGGTCGTAAAATTTAAAATGACGTTTTGATATGATATCTATAAAGAGGACATAATTTACTTTGTATGTCAGGTTTT AATATGGAACGAAGAGTATGGGTGAAAGTGTCATCCCACACATTTTAAAAGAGCTGTAATGTAGGGTAATGAGCACA ACTGCAAGCTGCATCCTATAATGGATCAATCAGAACATTAAACAACGTAAAGAGGAAGGTATTTGCTTTACACAACC TTATAAAATGATGAGGATCTACTCAAAATCCAGACTACCATGGTTGGCAAAATTAGATCCTCACTGTAACCAGCTAG GCATTGGTAATGCATAATAGCTATAGCTAACTATAGGTGGGAGACTCATCATTGAGATCATAGAAAAACAAAGATGA AAGAAAGAAATGAAGAAACAAGCAACAGCTATTCGAAAGCAAGTACAGAAGGGATTGTTCATGAAGTGTTCACCAAG TCACAGCTTAGGGCATTCTTAGAAGTAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATATTAA CCATCCAACGAAAACATCCAAAGGTACTGTGACAGAAAGCTCAAGGGGATATCTGTGTTTAAAGCCACAACATGACT AAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCAAATGACAAAACAGTTCAAATTGACTGCATAAA TAGATTACTCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAAT ATTGAAATTGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTGAACACTTGA CACCAAAGAGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGGGGGTAGAATCACCCATCGACGTGGAT ACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGA GCTTCTACATACAAACTCAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAG AGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAAGGTGAGTTTAAATAGAC CAAGATGTTAGCTAAAAAAAACAGACAAAACATTTAAGCAAAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAA CATATTTGGGTTGGAGGACAAAGTAGTATAGAGGAGTGTACCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATT GGTCATGATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAAACCCAACCACT GATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCACGTGGTATTTGATAGCCTCCACTT CCATCAGGACTTCGCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAA CCCA >72E18_viridis nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnTTAATATCAAAATCAGTCCTTGAGA TTCAACATGCATAACAAAGACAATAAACGGTACAAAAACAACCACTCAAACAATCACAACATAATATCATTCAATAC CTTGACCATTCCGGTTCCATTATCACACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAAC ATACACCACAATCAATGACAACAATGCCTCATTCACACAACAAAGAAATAGACATTCAAAAACAAAACACAATACAC ACTACTAATGTGGCACAGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACATAGTTCTCTCACAATAGTA AAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTAAAGAAACGATCTTTCAGATGGGAAATACCCAAATT TGATTGCTACATGCATAAAACCCTCAAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAA AAAATTCAAGAAATAAAGAGAGAGAAAATTACAGATCTAAAGTGACGAACAATGAGAAGGAATGAGAGGCAGAGAGA AGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGGGAGAGAGAGAGAGATCGAAGACGAAGCAGAGCGAA AGAGACGAGTGTGGTGTTTGTGAGTTGAGGCGAAGAATTGNACCNNNATANAGGAGTGNGATTGACNAGTTATCTCN GCNGNTTGATTTATGGACCGCGTCCATTGTGCCNTTGTGGGGCCATNACNGCTCCTNCCNCTGTNCCNGCCATNTTT ATCTCCACCTTCTNCACNTTTNTGCCNCNCNTGNCCNTCCCTTTTCTCCCNNTTCTNNCTCANATCTTCTNAANGCT NATNGCNTTTNCCNNANNGCATTTGNATTTNNGNGCTTCNATCAATNCGNNNNNAANGCTNCGTNTTGTCNTNNCAN GGNTNCAAGGANCCTNNGGGANGCNNGTTGATCGTCAANTNCGGCCTGGTNATANANAATATNNGACNTNAAATGGT TGATTGNNTCGTTAAATTNGAAATAAnnnnnnnnnnnnnnnnnnnnnnAAAAGGNCNTAATTTANTNNGTANNTCAA

PAGE 150

150 GGTTTTAANTAGGGAAATGGAGGAGTGNGGGTGAAAAGTGTCCATCCCACACGTTTTAAANGAGCNTTAANGNAGGG NAANGNGCAGNAACNGCAAGCTGCATNCCTNTAATNGGATCAATCAGAACATTAAGNCAACGTAAAGAGGAAGGTAT TTGCTTTACACAACCTTATAAAATGATGNGGATNTACTCCAAAGTGAGGACTACCATGGTCGGCAAAATTAGTTCCT GACTGTAACCAGCTAGGCATNGGCAATGCATAATAGCTATAGCTGACTATAGGTGGGAGACTCATCATTGAGATCAG AGAAAAACNAAGATGAAAGAAAGAAATGATGAAACAAGAATAGTTATTCGAAAGCAAGTACAGAAGGGATTGTTCAT GAAGTGTTCACCAAGTCACAGCTTAGGGCATTCTTAGAAGCAACAAGCTTGCCAACTTCCATTTACTTGTTTCAAGT TCATGNTGATATTANCCATCCAACGAAAACATCCAAAGGTNCTGTGACTGAAAGCTCAAGGGGATATCNGTGTTTAA AGCCACTACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCAAATGACAAAACAGTTCT AATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCA GAAAACTAACATCAATATTGAAATTGGATAAATATGCGATCTGAACTTCTTCACGTTGATGAGCTATCGTAGGAAAT GGAATTGAACACTTGACACCAAAGAGAACAACGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGGAGGTAGAATC ACCCATCGACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACAAAAGATCA ATATTGCATGCAAAGAGCTTCTACATACAAACTCAAATGGAGATGTTCTGGCGCTTGTAGAATATAATTATGTATAC AAATATGCATGTACAGAGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAAG GTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAAAAAAAGACAAAACATTTAAGCAAAAGAAGAGCAGTAGAAG TTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAGTCGTATAGAGGAGTGTACCTTCTTTAAACGGCGGT GTTTTCCTAGGGCCCAATTGGTCATGATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACTGTCTGTGTTGCA ATATTAAAACCCAACCACTGATAAATCTCAGTTGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCACGTGG TATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTTGAT TTATTATCCCAAAACCAAACCCA >72E18_iinumae GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAACCCGGCCTAAACATTAACATCAAAATCAGTCCTTGAGA TTCGACATGCATAAAAAAGACAATAAAGGGTACAAAAACAACCACTCAAACAATCACAACATAATAGCATCCAATAC CTTGACCATTCCGGTTCCATTATCACACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCTAC ATACACCACAATCAATGACACCAATGCCTCATCGACACAACAAACAAATAGACATTCAAAAACAAAACACAATACAC GATGCTAACATTTCCCTAATCTCTCCTCCATCAACTAAAATCTCCATTCCAAATCACACACTACTACACAGAAACCA AAGCATGATTCAAAACAAACCAAGAACATCTACATAGTCCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAA AGGCATCGAAAGCTAGTAAAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTATATACATAAAACCCT CAAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATTAAAAAAAAATCAAGAAAAAAAGAAGAGAG AAAATTACAGATCTAAAGCGACGAACAAATGAGAAGGAATGAGAGACAGAGAGAAGAGATGAGGAAGTTGACCTTTG TGAATGAGAGTGAGAGAGAGAGAGAGATCGAAGACGAGGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATTTATGGACCGCGTCCATT GCGCCCTTGTGGGGGCCATTACAGCTCCTTCCGCTGTTCCAGTCATTTTTCTCCACCTTCTTCACCTTTCTGCCCCT CGTTCCCTTTCCCTCTTCTCCCAACTCTTCTTAGCCTAATTGCATTTTCATTTTAGTGCTTAGATCAATATGATTNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNCCGAAAGCTCAATGGGATATCTGTGTTTAAAGCCACAACATGACTAAATATA ATTGCTCCCAATTTCTAAAGTTACATTCGTTTTGTACAAATGACAAAACAGTTCAAATTGACTGCATAAATAGATTA CTCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAA CTGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTGAACACTTGACACCAAA GAGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGGGGGTAGAATCACCCATCGACGTGGATACTTGGG TCTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTA CATACAAACTCAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAGAGCTTCC ACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAAGGTGAGTTTAAATAGACCAAGATG TTAGCTAAAAAAAACAGACAAAACATTTAAGCAAAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAACATATTT GGGTTGGAGGACAAAGTAGTATAGAGGAGTGTACCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCATG ATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAAACCCAACCACTGATAAAT

PAGE 151

151 CTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCACGTGGTATTTGATAGCCTCCACTTCCATCAG GACTTCGCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA >72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNAAAAAAGAGAGAGAGATTACAGATCTANGCGACGAACAATGAGAAGGAATGAGAGGCAGAGAGAAGAGATGAGGA AGTTGACCTTTGTGAATGAGAGTGAGTGAGAGAGAGAGATCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGT TTGTGAGTTGAGGCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATTTATGG ACCGCGTCCATTGCGCCCTTGTGGGGCCATTACAGCTCCTTCCGCTGTTCCAGTCATTTTTTTCTCCACCTTCTGCC CCATTCCCTTCCCTTTTCTCCCAATTCTTTCTCAACTCTTCTTAAACCTAATTGCATTTTCATTTTANTGCTTANAT CAATATGATTAAGAAGCTCCATTTTGTCAACACAAGGCAACAAGGACNTANGGGAGCATGTCGATCATCGTTCCGGT CACTTTCGTATATAATTTGGACTTAAAATGGTTGATCGATCGTAAAATTTGAAATGACGTTTTGGTATGATATTTGT AAAGAGGACATAATTTACTATGGAACGAAGAGTATAGGTGAAAGTGTCATCCCACACATTTTAAAAGAGCTTTAATG TAGGGGTAATGAGCACAACTACAAGCTGCATCCTATAAGGGATCAATCAGAACATTAAACAACGTAAAGAGGAAGGT ATTTGCTTTACACAACCTTATAAAATGATGAGGATCTACTCAAAATCCAGACTACCATGGTTGGCAAAATTAGATCC TGCCTGTAACCAGCTAGGCATTGGCAATGCATAATAGCTAGAGCTAACCATAGGTGGGAGACTCATCATTGAGATCA TAGGAAAAAAAATGATGAAAACAAGCAACAGTTATTCGAAAGCAAGTACAGAAGGGATTGTTCATTAAGTGTTCACC AAGTCACAGCTTAGGGCATTCTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATAT TTAGTCATGTTGTTTAAAGCCACAACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCA GATGACAAAACAGTTCAAATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTT ATTACAAAGTCTAAGCAGAACACTAACATAAATATTGAAATTGGATAAATATGCGATCTGAACTTCTTCACGTTGAT GACCTATCATAGGAAATGGAATTGAACACTTGACACCAAAGAGAACAACGAAGGTAGCCTCGCCAATCACTTCTACA AGAATGGGGGTAGAATCACCCATCTACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAG GGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACTCATATGGATATGTTCTGGCGATTGCAG AATATAATTATGTATACAAATATGCATGTACAGAGCTTCTACATACAAACTCATACGAATACTTGTAAATTTAGGCA ATTTAATTCCAATAAAGGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAAGACAAAACATTTAAGCAAAAGAA GAGCAGTAGAAGTTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAATGTAGCATAGAGGAGTGTACCTTCT TTAAACGGCGGTGCTTTCCTAGGGCCCAGTTGGTCATTATAGAAGCAGCAACTGCAGCAAAAAGATAACCAGCAACC GTCTGTGTTGCAATATTAAAACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAG AAAACCACGAGGTATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAATATGGCAATACAAGT TCGCGATTTGATTTATTATCCCAAAACCAAACCCA >72E18_mandshurica GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAGACCCGGCCTAAACATTAACATCAAAATCAGTCCTTGAGA TTCAACATGCATAACAAAGACAATAAAGGGTACAAAAACAACCACTCAAACAATCACAACATAATATCATTCAATAC CTTGACCATTCCGGTTCCATTATCACACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAAC ATACACCACAATCAAATGCTACATTCACACAACAAAGAAATAGACATTCAAAGACAAAACACAAACACACTACTAAC GTGGCACGGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACATAGTTCTCTCACAATAGTAAAGAAACGA TCGTTGACAATCAAAAGGCATCGAAAGCTAGTAAAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTA TATACATAAAACCCTCAAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAAAAATTCAAG AAAAAAAGAGAGAGAAAATTACAGATCTAAAGCGACGAACAGTGAGAAGGAATGAGAGGCAGAGAGAAGAGATGAGG AAGTTGACCTTTGTGAATGAGAGTGAGTGAGGGAGAGAGAGAGAGAGATCGACGACGAAGCAGAGCGAAAGAGACGA GTGTGGTGTTTGTGAGTTGAGGCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTT GATTTATGGACCGCGTCTATTGCGCCCTTGTGGGGCCATTACAGCTCCTTCCGCTGTTCCAGTCATTTTTTTCTCCA CCTTCTTCACCTTTTTGCCCCTCAGTCCCTTCCCTTTTCTCCCAATTCTTTCTCAACTCTTCTTAAACCTAATTGCA TTTCCCTAATTGCATTTTCATTTTAGTGCTGAGATCAATATGATTAAGAAGCTTCATTTTGTCAACACAAGGCAACA AGGACACAAGGGAGCATGTCGATCATCGTTCCAGTCATTTTCGTATATAATTTGGGCTTGAAATGGTTAATCAATCG TAAAATTTAAAATGACGTTTTGATATGATATCTATAAGGAGGACATAATTTACTTTATATGTCAGGTTTTAATACGG AAGGAAGAGTATGGGTGAAAGTGTCATCCCACACATTTTAAAAGAGCTGTAATGTAGGGTAATGAGCACAACTGCAA GCTGCATCCTATAATGGATCAATCAGAACAATAAACAACGTAAAGAGGAAGGTATTTGCTTTACACAACCTTATAAA

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152 ATGATGAGGATCTACTCAAAATCCAGACTACCATGGTTGGCAAAATTAGATCCTCACTGTAACCAGCTAGGCATTGG TAATGCATAATAGCTATAGCTAACTATAGGTGGGAGACTCATCATTGAGATCATAGAAAAACAGAGATGAAAGAAAG AAATGATGAAACAAGCAACAGCTATTCGAAAGCAAGTACAGAAGGGATTGTTCATGAAGTGTTCACCAAGTCACAGC TTAGGGCATTCTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATATTAACCATCCA ACGAAAACATCCAAAGGTACTGTGACAGAAAGCTCAAGGGGATATCTGTGTTTAAAGCCACAACATGACTAAATATA ATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCAAATGACAAAACAGTTCAAATTGACTGCATAAATAGATAC TCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAAT TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTGAACACTTGACACCAAAG AGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGGGGGTAGAATCACCCATCGACGTGGATACTTGGGT CTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTAC ATACAAACTCAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAGAGCTTCCA CATACAAACTCATATGTATACTTGTAAATTTATGCAATTTAATTCCAATAAAGGTGAGTTTAAATAGACCAAGATGT TAGCTAAAAAAAAACAGACAAAACATTTAAGCAAAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAACATATTT GGGTTGGAGGACAAAGTAGTATAGAGGAGTGTTCCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCATG ATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAAACCCAACCACTGATAAAT CTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCACGTGGTATTTGATAGCCTCCACTTCCATCAA GACTTCTCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA >72E18_ananassa GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAACCCGGCCTAAACATTAACATCAAAATCAGTCCTTGAGA TTCAACATGCATAAAAAAGACAATAACGGGTACAAAAACAACCACTCAAACAATCACAACATAATATCATTCAATAC CTTGACCATTCCGGTTCCATTATCACACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAAC ATACATCACAATCAATGACAACAATGCCTCAAACAAATAGACATTCAAAAACAAAACACAATACACAATGCTAACAT TTCCCTAATATCTCCTCCATCAACTAAAATCTCCATGCCAGATCACCACAGAAACCAAAGCATGATTCAAAACAAAC CAAGAACATCTACATAGTTCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTAAA GAAACGATCTTTCAGATGGGAAATGCCCAAATTTGATTACTATATACATAAAACTCCCAAATTGATACGAAATCAAA CAATGCAGCAATCAAATCATTCCACAGAAAAAAAATTCAAGAAAAAAAAAAGAGAGAGAAAATTACAGATCTAAAGC GACGAACAATGAGAAGGAATGAGAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAGTGAGGGAGAGAGAGAG AGAGATCGAAGACGAAGCTGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAGGCGAAAGAATTGGAGCAAAATA AAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATTTATGGACCGCGTCCGTTGCGCCCTTGTGGGGCCATTGCA GCTCCTTCCGCTGTTCCAGTCATTTTTCTCCACCTTCACCTTTTTGCCCCTCATTCCCTTTCCCTCTTCTCCGATCC CAACTTTCTCAACTCTTCTTAACCCACCCAGTTGCATTTTCATTGTAGTGCTTAAATCAATATGGTTAAGAAGCTTC ATTTTGTCaacacaaggcaacaaggacataggggagcatgtggatgatcgtttttaaactacgttttggtatgatat cgtaaggaggacataatttactttgtatgtcnggtttttaatacggaatgaagagtgtgggagaaagtgtcatccca cacattcattttaaannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnCAAAATCCAGACTACCGTGGTGGGCAAAATTAGATCCTCACCTATAGTAGGGTAACTATAGGTG GGAGACTNGTCATTGTGATCATAGAAAAAGAAATGATGAAACAAGCAACAGTTACTCGAAAGCAGGTACAGAAGGGA TTGTTCATGAAGTGTTCACCAAGTCNCAGCTTAGGGCATTCTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTG TTTCAAGTTCAGGGTGACATTACCCNTCCAACGAAAACATCCAAAGGTACTGTGACTGAAAGCCCAAGGGGATATCC GTGTTTAAAGCCACAACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCAAATGACAAA ACAGTTCAAATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACAAGCAAATCTCCAAATTCTTATTACAAAG TCTAAGCAGAATACTAACATCAATATTGAAATTGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCG TAGGAAATGGAATTGAACACTCGACACCAAAGAGAACAACAAAGGTAGCCTCACCAATCACTTCTACAAGAATGGGG GTAGAATCACCCATCGACGTGGATACTTGGGTCGTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACA AAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACTCAAATGGATATGTTCTGGCGCTCGTAGAATATAATT ATGTATACAAATATGCATGTACAGAGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTC CAATAAAGGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAACAGACAAAACATTTAAGCAAAAGAAGAGCAGTAG AAGTTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAGTAGTATAGAGGAGTGTACCTTCTTTAAACGGC GGTGTTTTCCTAGGGCCCAATTGGTCATGATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTT GCAATATTAAAACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCACG TGGTATTTGATAGCCTCCGCTTCCATCAGGACTTCGCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTT GATTTATTATCCCAAAACCAAACCCA

PAGE 153

153 APPENDIX E GENE-PAIR HAPLOTYPE INDIVIDUAL LOCI ALIGNMENTS To avoid identification of untrue polymorphism s and therefore poor alignments, Ns were inserted where the chromatogram did not display distinct peaks. EcoRI sites are in green because EcoRI was used to extract inserts from TOPO vectors and detect polymorphic inserts in this vector. Simple Sequence Repeats (SSRs) are magenta-colored. Gene Pairs Detected by Microcolinearity GPH5: Single Nucleotide Polymophisms (SNPs) are in bold. SNPs that occur in more than one clone are likely to reflect real differences (as oppose to amplification or sequencing errors) and are colored red. GPH5_ananassa_clone2 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 GPH5_ananassa_clone7 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 GPH5_viridis CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 GPH5_iinumae CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 GPH5_nilgerrensis CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 GPH5_mandshurica CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 GPH5_nubicola CAATGCCATGGTCTCCGGTCTATTTCAAC C GGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 GPH5_vesca CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60 ***************************** ****************************** GPH5_ananassa_clone2 GAAGACCGGAAGATCATC G GAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAG 120 GPH5_ananassa_clone7 GAAGACCGGAAGATCATC G GAATCAGATTTGTT C GCGCTTGCAGAAAGAGAATCCTCGAG 120 GPH5_viridis GAAGACCGGAAGATCATCAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAG 120 GPH5_iinumae GAAGACCGGAAGATC C TCAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAG 120 GPH5_nilgerrensis GAAGAC T GGAAGATCATCAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAG 120 GPH5_mandshurica GAAGACCGGAAGATCATCAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAG 120 GPH5_nubicola GAAGACCGGAAGATCATCAGAATCAGATTTGTTTGC T CTTGCAGAAAGAGAATCCTCGAG 120 GPH5_vesca GAAGACCGGAAGATCATCAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAG 120 ****** ******** ** ************** ** *********************** GPH5_ananassa_clone2 TGAAGA G AAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAAT CC TTGAGCAAACTCAA 180 GPH5_ananassa_clone7 TGAAGA G AAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAAT CC TTGAGCAAACTCAA 180 GPH5_viridis TGAAGACAAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAATTGTTGAGCAAACTCAA 180 GPH5_iinumae TGAAGACAAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAATTGTTGAGCAAACTCAA 180 GPH5_nilgerrensis TGAAGACAAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAATTGTTGAGCAAACTCAA 180 GPH5_mandshurica TGAAGACAAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAATTGTTGAGCAAACTCAA 180 GPH5_nubicola TGAAGACAAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAATTGTTGAGCAAACTCAA 180 GPH5_vesca TGAAGACAAGATCCTAAGGAGGAACTCCGAGTCTGGTTTAGAATTGTTGAGCAAACTCAA 180 ****** ************************************* ************** GPH5_ananassa_clone2 GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_ananassa_clone7 GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_viridis GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_iinumae GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_nilgerrensis GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGA A CT 240 GPH5_mandshurica GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 GPH5_nubicola GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGA A CT 240 GPH5_vesca GGAACAAGAAGTAGCACCTCCC AAGAAGAAGAAGAA AAATGGGATCTACAGAAAAGAGCT 240 ********************************************************* **

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154 GPH5_ananassa_clone2 TGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 GPH5_ananassa_clone7 TGCTCTTGCTTTCC A CCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 GPH5_viridis TGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 GPH5_iinumae TGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 GPH5_nilgerrensis TGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 GPH5_mandshurica TGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 GPH5_nubicola TGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 GPH5_vesca TGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGT 300 ************** ********************************************* GPH5_ananassa_clone2 TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 GPH5_ananassa_clone7 TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 GPH5_viridis TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 GPH5_iinumae TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 GPH5_nilgerrensis TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 GPH5_mandshurica TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 GPH5_nubicola TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 GPH5_vesca TCACTTCTATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAAT 360 ************************************************************ GPH5_ananassa_clone2 AGGTGAGCAGGTTAGCTAAAAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 420 GPH5_ananassa_clone7 AGGTGAGCAGGTTAGCTAAAAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 420 GPH5_viridis AGGTGAGCAGGTTAGCTAAAAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 420 GPH5_iinumae AGGTGAGCAGGTTAGCTAAAAGCTTCAAACAAAGC A TCAATTGCCCACAGTTATTCTTTG 420 GPH5_nilgerrensis AGGTGAGCAGGTTAGCTAAAAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 420 GPH5_mandshurica AGG-GAGCAGGTTAGCTAAAAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 419 GPH5_nubicola AGGTGAGCAGGTTAGCTAAAAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 420 GPH5_vesca AGGTGAGCAGGTTAGCTA G AAGCTTCAAACAAAGCGTCAATTGCCCACAGTTATTCTTTG 420 *** ************** **************** ************************ GPH5_ananassa_clone2 ATAGATATATG C TGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 476 GPH5_ananassa_clone7 ATAGATATATGTTGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 476 GPH5_viridis ATAGATATATGTTGAA----CTGTAAGAGACATATTTCAAGCTC--TGGTGTTCAAAGTT 474 GPH5_iinumae ATAGATATATGTTGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 476 GPH5_nilgerrensis ATAGATATATGTTGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTC 476 GPH5_mandshurica ATAGATATATGTTGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 475 GPH5_nubicola ATAGATATATGTTGAATGAACTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 480 GPH5_vesca ATAGATATATGTTGAA----CTGTAAGAGACATATTTCAAGCTCTTTGGTGTTCAAAGTT 476 *********** **** ************************ ************* GPH5_ananassa_clone2 GGATTCAATTACATGTAGACACAGTTACCATTTT T CCATTTGAAA C AGAAGGTAATATGC 536 GPH5_ananassa_clone7 GGATTCAATTACATGTAGACACAGTTACCATTTT T CCATTTGAAA C AGAAGGTAATATGC 536 GPH5_viridis GGATTCA T TTACATGT-GACACAGTTACCATTTTCCCAT A TGAAATAGAAGGTAATATGC 533 GPH5_iinumae GGATTCAATTACATGTAGACA T AGTTACCATTTTCCCATTTGAAATAGAAGGTAATAT-534 GPH5_nilgerrensis GGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAA C AGAAGGTAATATGC 536 GPH5_mandshurica GGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAATAGAAGGTAATA C GC 535 GPH5_nubicola GGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAATAGAAGGTAATA C GC 540 GPH5_vesca GGATTCAATTACATGTAGACACAGTTACCATTTTCCCAT A TGAAATAGAAGGTAATATGC 536 ******* ******** **** ************ **** ***** *********** GPH5_ananassa_clone2 ATGATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAAT A AGTGA G AATAATGA 596 GPH5_ananassa_clone7 ATGATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAAT A AGTGAAAATAATGA 596 GPH5_viridis ATGATATAAATATCAAGTTAATTGTACA G TGATAT---TTGTAA C CAGTGAAAATAATGA 590 GPH5_iinumae -----------ATCAAGTTAATTGTACAAT A ATAT---TTGTAATCAGTGAAAATAATGA 580 GPH5_nilgerrensis ATGATATAAATAC CAAGTTAATTGTACAATGATAT---TTGTAATCAGTGAAAATAATGA 593 GPH5_mandshurica ATGATATAAATATCAAGTTAATTGTACAATGATAT---TTGTAATCAGTGAAAATAATGA 592 GPH5_nubicola ATGATATAAATATCAAGTTAATTGTACAATGATAT---TTATAATCAGTGAAAATAATGA 597 GPH5_vesca ATGATATAAATATCTAGTTAATTGTACAATGATAT---TTGTAA C CAGTGAAAATAATGA 593 ************* **** ** *** ***** ********

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155 GPH5_ananassa_clone2 CAATCTT C ATAACAAAATTTCAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGC 656 GPH5_ananassa_clone7 CAATCTTTATAACAAAATTTCAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGC 656 GPH5_viridis CAATCTTTATAACAAAATTTCAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGC 650 GPH5_iinumae CAATCTTTATAACAAAATTTCAGTTATCTTT T CA C TGCTGTATGAACTGT C ACCATTAGC 640 GPH5_nilgerrensis A AATCTTTATAACAAAATTTCAGTTATC C TTCCATTGCTGT G TGAACTGTTACCATTAGC 653 GPH5_mandshurica CAATCTTTATAACAAAATTTCAGT G ATCTTTCCATTGCTGTATGAACTGTTACCATTAGC 652 GPH5_nubicola CAATCTTTATAACAAAATTTCAGT G ATCTTTCCATTGCTGTATGAACTGTTACCATTAGC 657 GPH5_vesca CAATCTTTATAACAAAATTTCAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGC 653 ****** **************** *** ** ** ****** ******** ********* GPH5_ananassa_clone2 CTCTCACACAAGAACAAC T ACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 716 GPH5_ananassa_clone7 CTC A CACACAAGAGCAACAACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 716 GPH5_viridis CTCTCACACAAGAACA G CAACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 710 GPH5_iinumae CTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 700 GPH5_nilgerrensis CTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 713 GPH5_mandshurica CTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 712 GPH5_nubicola CTCTCACACAAGACCAACAACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 717 GPH5_vesca CTCTCACACAAGA A CAACAACACCAAACAAACAGAACCAGACCAAATCACACCAATATAA 713 *** ********* ** ***************************************** GPH5_ananassa_clone2 AACAGAATTGGATTTTCATGAAAGGC G GCAAGGCACAATCAATGAAGGAGA--AGACAAA 774 GPH5_ananassa_clone7 AACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGA--AGACAAA 774 GPH5_viridis AACA T AATTGGATTTTCATGAAAGGCAGCAAGGCA TG ATCAATGAAGGAGA--AGACAAA 768 GPH5_iinumae AACAGAATTGGATTT C CATGAAAGGCAGCAAGGCACAATCAATGAAGGAGA--AGACAAA 758 GPH5_nilgerrensis AACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAGAAGACAAA 773 GPH5_mandshurica A-CAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGA--AGACAAA 769 GPH5_nubicola A-CAGAA C TGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGA--AGACAAA 774 GPH5_vesca AACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGA--AGACAAA 771 ** ** ******* ********** ******** ************** ******* GPH5_ananassa_clone2 GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATC 834 GPH5_ananassa_clone7 GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATC 834 GPH5_viridis GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATC 828 GPH5_iinumae GAA A CCTTTTGTCATAGGGATTGAA C CTGAATTAT C TGGAGTGTTTCTGGCTGTCATATC 818 GPH5_nilgerrensis GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATC 833 GPH5_mandshurica GAATCCTTT C GTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATC 829 GPH5_nubicola GAATCCTTT C GTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATC 834 GPH5_vesca GAATCCTTTTGTCATATGGATTGAATCTGAATTA G TTGGAGTGTTTCTGGCTGTCATATC 831 *** ***** ****** ******** ******** ************************ GPH5_ananassa_clone2 TCATATGCAGGCATGTTACATGTCTG----------CATTTGGTGACAAAAGCTAAATCT 884 GPH5_ananassa_clone7 TCATATGCAGGCATGTTACATGTCTG----------CATTTGGTGACAAAAGCTAAATCT 884 GPH5_viridis TCATATGCAGGCATGTTACATGTCTG----------CATTTGGTGACAAAAGCTAAATCT 878 GPH5_iinumae TCATATGCAGGCATGTTACATGTCTG----------CATTTGGTGACAAAAGCTAAATCT 868 GPH5_nilgerrensis TCATATGCAGGCATGTTACATGTCTG----------CATTTGGTGACAAAAGCTAAATCT 883 GPH5_mandshurica TCATATGCAGGCATGTTACATGTCTG----------CATTTGGTGACAAAAGCTAAATCT 879 GPH5_nubicola TC G TATGCAGGCATGTTACATGTCTG----------CATTTGGTGACAAAAGCTAAATCT 884 GPH5_vesca TCATATGCAGGCATGTTACATGTCT C ATGATGTCTTCATTTGGTGACAAAAGCTAAATCT 891 ** ********************** ************************ GPH5_ananassa_clone2 TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTAAGCCCA 944 GPH5_ananassa_clone7 TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTAAGCCCA 944 GPH5_viridis TAA----------GAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCT G AGCCCA 928 GPH5_iinumae TAA----------GAATT T AGAC G TATT A GACCATTGGGCTTAATCAT C GTC C G AGCCCA 918 GPH5_nilgerrensis TAACATGACCTAAGAATTAA C ACATATTGGACCATTGGGCTTAATCATAGTCTAAGCCCA 943 GPH5_mandshurica TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTAAGCCCA 939 GPH5_nubicola TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTAAGCCCA 944 GPH5_vesca TAACCTGACCTAAG T AT C AAGACATATTGGAC A ATTGGGCTTAATCATAGTCTAAGCCCA 951 *** ** ** **** *** *************** *** ******

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156 GPH5_ananassa_clone2 AATCTGTACTAGCCCATAATATGCTTTTTATAGAAA--ATA--CT----GTGATCTTCAC 996 GPH5_ananassa_clone7 AATCTGTACTAGCCCATAATATGCTTTTTGTAGAAA--ATA--CT----GTGATCTTCAC 996 GPH5_viridis AATCTGTACTAGCCCATAATATGCTTTTTATAGAAA--ACA--CTCTCTGTGATCTTC G C 984 GPH5_iinumae AATCTG C ACTAGCCC G TAATATGCTTTTTATAGAAA--ACAGACTCTCTGTGATCTTC G C 976 GPH5_nilgerrensis AATCTGTACTAGCCCATAATATGCTTTTTATAGAAAC-AGAGATTCTCTGTGATCTTCAC 1002 GPH5_mandshurica AATCTGTACTAGCCCATAATAT T CTTTTTATAGAAAACAGAGGTTCTCTGTGATCTTCAC 999 GPH5_nubicola AATCTGTACTAGCCCATAATAT T CTTTTTATAGAAAACAGAGATTCTCTGTGATCTTCAC 1004 GPH5_vesca AATCTGTACTAGCCCATAATATGCTTTTTATAGAAA--ACA----CTCTGTGATCTTCAC 1005 ****** ******** ****** ****** ****** ********* GPH5_ananassa_clone2 CATTGAGGAGTCAAGTTACTCAGCC A TGAAGT C AA G GTC A AG C CAAGTAGTGCAGTTGAG 1056 GPH5_ananassa_clone7 CATTGAGGAGTCAAGTTACTCAGCC A TGAAGT C AA G GTC A AG C CAAGTAGTGCAGTTGAG 1056 GPH5_viridis CATTGAGGAGTCAAGTTACTCAGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAG 1044 GPH5_iinumae CATTGAGGAGTCA G GTTACTCAGCTCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAG 1036 GPH5_nilgerrensis CATTGAGGAGTCAAGTTAC A CAGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAG 1062 GPH5_mandshurica CATTGAGGAGTCAAGTTACTCGGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAG 1059 GPH5_nubicola CATTGAGGAGTCAAGTTACTCGGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAG 1064 GPH5_vesca CATTGAGGAGTCAAGTTACTCAGCCCTGAAGTAAAAGTCCAGTCA-GTAGTGCAGTTGAG 1064 ************* ***** ** ****** ** *** ** ** ************** GPH5_ananassa_clone2 TTCAACTTGTTCTGGGTTCTTCAAAGTTCGAAACTTTAAGCTTCAATGGAGGAAGAGAAG 1116 GPH5_ananassa_clone7 TTCAACTTGTTCTGGGTTCTTCAAAGTT C GAA G CTTTAAGCTTCAATGGAGGAAGAGAAG 1116 GPH5_viridis TTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAACTTTAAGCTTCAATGGAGGAAGAGAAG 1104 GPH5_iinumae TTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAACTTTAAGCTTCAATGGAGGAAGA T AAG 1096 GPH5_nilgerrensis TTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAACTTTAAGCTTCAATGGAGGAAGAGAAG 1122 GPH5_mandshurica TTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAACTTTAAGC G TCAATGGAGGAAGAGAAG 1119 GPH5_nubicola TTCAACTTGTTCTGGGTTCTTCAAAGTTTGAA G CTTTAAGC G TCAATGGAGGAAGAGAAG 1124 GPH5_vesca TTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAACTTTAAGCTTC G ATGGAGGAAGAGAAG 1124 **************************** *** ******** ** *********** *** GPH5_ananassa_clone2 GATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCAT G TATAAAAGCTTGAAG-GA 1175 GPH5_ananassa_clone7 GATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGCTTGAAG-GA 1175 GPH5_viridis GATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGCTTGAAG-GA 1163 GPH5_iinumae GATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGCTTGAAG-GA 1155 GPH5_nilgerrensis GATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGCTTGAAG-GA 1181 GPH5_mandshurica GATGCCTTTTATGTTG C TCCAA C GGGAGATGTGGTTGGCATATATACAAGCTTGAAG-GA 1178 GPH5_nubicola GATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGCTTGAAG-GA 1183 GPH5_vesca GATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGC C TGAAAAGA 1184 **************** ** ** ****************** **** **** **** ** GPH5_ananassa_clone2 TTGCC--AAAACCAAGCTGG-TTCATCGGTAAA-GTTTT--GATCTTTT--AAGCCTTTT 1227 GPH5_ananassa_clone7 TTGCC--AAAACCAAGCTGG-TTCATCGGTAAA-GTTTT--GATCTTTT--AAGCCTTTT 1227 GPH5_viridis TTGCC--AAAACCAAGCTGG-TTCAT T GGTAAAAGTTTT--GATCTTTT--AAGCCTTTT 1216 GPH5_iinumae TTGCC--AAAACCAAGCTGG-TTC C TCGGTAAA-GTTTT--GATCTTTT--AAGCC C TTT 1207 GPH5_nilgerrensis TTGCC--AAA-CCAAGCTGG-TTC C TCGGTAAA-GCTTT--GATCTTTT--AAGCCTTTT 1232 GPH5_mandshurica TTGCC--AAAACCAAGCTGG-TTCATCGGTAAA-GTTTT--GATCTTTT--AAGCCTTTT 1230 GPH5_nubicola TTGCC--AAAACCAAGCTGG-TTCATCGGTAAA-GTTTC--GATCTTTT--AAGCCTTTT 1235 GPH5_vesca TTGCCCAAAA C CCAAGCTGGGTTCATC C G A AAAAGTTTTTGAATCTTTTTAAAGCC C TTT 1244 ***** *** ********* *** *** ** ******* ***** *** GPH5_ananassa_clone2 ---G TAATTTG----ATCACTCCCATTGTTTTATCAATTTTTGATTTCCCATTTGATTA 1279 GPH5_ananassa_clone7 ---G TAATTTG----ATCACTCCCATTGTTTTATCAATTTTTGATTTCCCATTTGATTA 1279 GPH5_viridis ----ATAATTTG----ATCACTCTCATTGTTTTATCAATTTNNNNNNNNNNNNNNNNNNN 1268 GPH5_iinumae ----ATAATTTG----ATTACTCTCATTGTTTTATCAATTTTTGATTTCCCATTTGATNN 1259 GPH5_nilgerrensis ----ATAATTTG----ATTACCCTTATTGTTTTATCAANNNNNNNNNNNNNNNNNNNNNN 1284 GPH5_mandshurica ---G TAATTTG----ATCACTCCCATTGTTTTATCAATTTTAGATTTCCCATTTGATTA 1282 GPH5_nubicola ----ATAATTTG----ATCACTCTCATTGTTTTANNNNNNNNNNNNNNNNNNNNNNNNNN 1287 GPH5_vesca TTTAATAATTTGGAATN C CACCTTCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1304 ******* **

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157 GPH5_ananassa_clone2 CATTACTGGGTCTTGTTTATTTTGTTGAAATAACTATGCCCTTTCGTTCTAGCATGCAAC 1339 GPH5_ananassa_clone7 CATTACTGGGTCTTGTTTATTTTGTTGAAATAACTATGCCCTTTCGTTCTAGCATGCAAC 1339 GPH5_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1328 GPH5_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1319 GPH5_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1344 GPH5_mandshurica CATTACTGGCTCTTGTTTATTTTGTTGAACTAACTATGCCCTTTCGTTCTAACATGCAAC 1342 GPH5_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1347 GPH5_vesca NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1364 GPH5_ananassa_clone2 TGAAATTTACTGCTAGATTGTATTGTTGTGCCGTTATGGTGTTCATTATGTAAAAGAGAA 1399 GPH5_ananassa_clone7 TGAAATTTACTGCTAGATTGTATTGTTGTGCCGTTATGGTGTTCATTATGTAAAAGAGAA 1399 GPH5_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1388 GPH5_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1379 GPH5_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1404 GPH5_mandshurica TGAAAATAACTGCTAGATTGTATAGCTGAGCCTTTATGGTGTTCATTATGTAAAAGAGAA 1402 GPH5_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1407 GPH5_vesca NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1424 GPH5_ananassa_clone2 T GAATTC TGGTGGTGGGTATAGAGTACCTCCCTGATTTTTTATGAGATACTATGCTTCTG 1459 GPH5_ananassa_clone7 T GAATTC TGGTGGTGGGTATAGAGTACCTCCCTGATTTTTTATGAGATACTATGCTTCTG 1459 GPH5_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1448 GPH5_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1439 GPH5_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1464 GPH5_mandshurica T GAATTC TGGTGGTGGGTATAAAGCACCTCCCTGAGATTATATGAGATACTATGCTTCTG 1462 GPH5_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1467 GPH5_vesca NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1484 GPH5_ananassa_clone2 GAAAATGTTATAAA-------GATGAAAACTAGTTTTTCTAAGAACTTGATG-----TTA 1507 GPH5_ananassa_clone7 GAAAATGTTATAA--------GATGAAAACTAGTTTTTCTAAGAACTTGATG-----TTA 1506 GPH5_viridis NNNNNNNNNNNNN---------NNNNNNNNNNNNNNNNNNNNGAACTTGATG-----TTA 1494 GPH5_iinumae NNNNNNNNNNNNN--------NNNNNNNACTAGTTTTTCTAAGAACTTGATG-----TCA 1486 GPH5_nilgerrensis NNNNNNNNNNNNNN-------NNNNNNNNNNNGTTTTTCTAAGAACTTGATG-----TTA 1512 GPH5_mandshurica GAAAATGTTATAA--------GATGAAAACAACTTTTTCTAACAACTTGATG-----TTA 1509 GPH5_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTAAGAACTTGATG-----TTA 1522 GPH5_vesca NNNNNNNNNNNNN--------NNNNNNNNNNNNNNNNNNNNNGTA T T A GATGAAAACTAG 1536 **** GPH5_ananassa_clone2 TTT--------ACTTGATGAGTTGATGGAGGATTAC G TATGTGGTTTGGTTTTGTTTTTA 1559 GPH5_ananassa_clone7 TTT--------ACTTGATGAGTTGATGGAGGATTAC G TATGTGGTTTGGTTTTGTTTTTA 1558 GPH5_viridis TTT--------ACTTGATGAGTTGA C GGAGGATTACATAT A A GG C TTGGTT A TGTTTTTA 1546 GPH5_iinumae TTT--------ACTTGATGAGTTGATGGAGGATTACACATGTGGTTTGGTTTTGTTTTTA 1538 GPH5_nilgerrensis TTT--------ACTTGATGAGTTGATGGAGGATTACATATGTGGTTTGGTTTTGTTTTTA 1564 GPH5_mandshurica TTT--------ACTTGATGAGTTGATGGA A GATTACGTATGTGGTTTGGTTTTGTTTTTA 1561 GPH5_nubicola TTT--------GCTTGATGAGTTGATGGAGGATTACATATG A GGTTTGGTT A TGTTTTTA 1574 GPH5_vesca TTTTTCTAAGAACTTGATGAGTTGATGGAGGATTACATATG A GGTTTGGTT A TGTTTTTA 1596 *** ************* *** ****** ** ** ****** ******** GPH5_ananassa_clone2 GGTAT T CAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1619 GPH5_ananassa_clone7 GGTAT T CAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1618 GPH5_viridis GGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1606 GPH5_iinumae GGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1598 GPH5_nilgerrensis GGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1624 GPH5_mandshurica GGTAT T CAATCCTTCTG AAA T TGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1621 GPH5_nubicola GGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1634 GPH5_vesca GGTATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGA 1656 ***** *********** ** ***************************************

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158 GPH5_ananassa_clone2 GTACCTTGTCTC A CATGGG CTTAAG AATGCTTCAT G TACTATCAGTGCCAGTGATGTGAA 1679 GPH5_ananassa_clone7 GTACCTTGTCTC A CATGGG CTTAAG AATGCTTCAT G TACTATCAGTGCCAGTGATGTGAA 1678 GPH5_viridis GTACCTTGTCTCGCATGGG CTTAAG AATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1666 GPH5_iinumae GTACCTTGTCTC A CATGGG CTTAAG AATGCTTCATATACTATCAG C GCCAGTGATGTGAA 1658 GPH5_nilgerrensis GTACCTTGTCTC A CATGGG CTTAAG AATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1684 GPH5_mandshurica GTACCTTGTCTCGCATGGG CTTAAG AATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1681 GPH5_nubicola GTACCTTGTCTCGCATGGG CTTAAG AATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1694 GPH5_vesca GTACCTTGTCTCGCATGGG CTTAAG AATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1716 ************ ********************** ********* ************** GPH5_ananassa_clone2 AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGAT---TTCAT 1736 GPH5_ananassa_clone7 AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGAT---TTCAT 1735 GPH5_viridis AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1726 GPH5_iinumae AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGAT---TTCAT 1715 GPH5_nilgerrensis AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGAT---TTCAT 1741 GPH5_mandshurica AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1741 GPH5_nubicola AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1754 GPH5_vesca AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1776 **************************************************** ***** GPH5_ananassa_clone2 GTGTTCTAGTTTCTGTTTGGG C ATCTGTTATTTTCATGGCATGTGGCGTGGAGCTAGTTG 1796 GPH5_ananassa_clone7 GTGTTCTAGTTTCTGTTTGGG C ATCTGTTATTTTCATGGCATGTGGCGTG A AGCTAGTTG 1795 GPH5_viridis GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAGCTAGTTG 1786 GPH5_iinumae GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAGCTAGTTG 1775 GPH5_nilgerrensis GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGA G CTAGTTG 1801 GPH5_mandshurica GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTCGCGTGGAACTAGTTG 1801 GPH5_nubicola GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTG 1814 GPH5_vesca GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTG 1836 ********************* ********************** ***** ******* GPH5_ananassa_clone2 CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCAGTTTTTTA C TATAAT 1856 GPH5_ananassa_clone7 CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCAGTTTTTTATTATAAT 1855 GPH5_viridis CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAA G TCGGTTTTTTATTATAAT 1846 GPH5_iinumae CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1835 GPH5_nilgerrensis CATATGGTATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1861 GPH5_mandshurica CATATG A TATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1861 GPH5_nubicola CATATG A TATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1874 GPH5_vesca CATATG A TATTAAATCTTTTGTTTGGTTCCTCATTGTATAATT T GGTT C TTTATTATAAT 1896 ****** ********************************** *** **** ****** GPH5_ananassa_clone2 CTTTCAGCAGCCAGCATC A TCTATGGTTAATTCAGGGTTCAAAATTGC G CCTAATCAGCT 1916 GPH5_ananassa_clone7 CTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGC G CCTAATCAGCT 1915 GPH5_viridis CTTTCAGCAGCCAGCATCTTC C ATGGTTAATTCAGGGTTCAAAATTGCACCTAATCAGCT 1906 GPH5_iinumae CTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTAATCAGCT 1895 GPH5_nilgerrensis CTTTCAGCAGCCAGCATCTTCTATGGTT G ATTCAGGGTTCAAAATTGCACCTAATCAGCT 1921 GPH5_mandshurica CTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCT G ATCAGCT 1921 GPH5_nubicola CTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCT G ATCAGCT 1934 GPH5_vesca CTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCT G ATCAGCT 1956 ****************** ** ****** ******************* *** ******* GPH5_ananassa_clone2 TACAC C AAAGAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAG 1976 GPH5_ananassa_clone7 TACAC C AAAGAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAG 1975 GPH5_viridis TACACGAAAGAGAATGCCGGATGTGGT C AATTCAGGCGTCAATGACCCTCCACAAAAGAG 1966 GPH5_iinumae TACACGAAAGAGAATGCCGGATGTGGTAAA G TCAGGCGTCAATGACCCTCCACAAAAGAG 1955 GPH5_nilgerrensis TACACGAAAGAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAG 1981 GPH5_mandshurica TACACGAAAGAGAATGCCGGATGTG A TAAAT C CAGG T GTCAATGACCCTCCACAAAAGAG 1981 GPH5_nubicola TACACGAAAGAGAATGCCGGATGTG A TAAATTCAGG T GTCAATGACCCTCCACAAAAGAG 1994 GPH5_vesca TACACGAAAGAGAATGCCGGATGTG A TAAATTCAGG T GTCAATGACCCTCCACAAAAGAG 2016 ***** ******************* ** **** ***********************

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159 GPH5_ananassa_clone2 ATCGCTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTTTGATAGAAGA C CTTC 2036 GPH5_ananassa_clone7 ATCGCTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTTTGATAGAAGATCTTC 2035 GPH5_viridis ATCGCTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTTTGATAGAAGATCTTC 2026 GPH5_iinumae ATCATTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTTTGATAGAAGATCTTC 2015 GPH5_nilgerrensis ATCGCTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTTTGATAGAAGATCTTC 2041 GPH5_mandshurica ATCACTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGT A TGATAGAAGATCTTC 2041 GPH5_nubicola ATCA T TGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGT A TGATAGAAGATCTTC 2054 GPH5_vesca ATCACTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGT A TGATAGAAGATCTTC 2076 *** *************************************** ********** **** GPH5_ananassa_clone2 TATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTTTCAGCAGCCAGCGTCT 2096 GPH5_ananassa_clone7 TATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTTTCAGCAGCCAGCGTCT 2095 GPH5_viridis TATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTTTC G GCAG T CAGCATCT 2086 GPH5_iinumae TATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTTTCAGCAGCCAGCGTCT 2075 GPH5_nilgerrensis TATTTGGTTACTCATTGTATGATTC A GGTTTTTATTATAATCTCTCAGCAGCCAGCGTCT 2101 GPH5_mandshurica TATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAAT T TTTCAGCAGCCAG G GTCT 2101 GPH5_nubicola TATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAAT T TTTCAGCAGCCA AT GTCT 2114 GPH5_vesca TATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAAT T TTTCAGCAGCCAGCGTCT 2136 ************************* *************** ** **** ** *** GPH5_ananassa_clone2 TCTATGGTTAATTCAGGCTTCAAA G TTGCACCTGATCAG T TTACACGAATG C GATTGCCG 2156 GPH5_ananassa_clone7 TCTATGGTTAATTCAGGCTTCAAAATTGCACCTGATCAG T TTACACGAATG C GATTGCCG 2155 GPH5_viridis TCTATGGTTAATTCAGGCTTCAAAATTGCACCTTA C CAGCTTGCACGAATGAGATTGCCG 2146 GPH5_iinumae TCTATGGTTAATTCAGGCTTCAAAATTGCACCTTATCAGCTTACACGAATGAGATTGCCG 2135 GPH5_nilgerrensis TCTATGGTTAATTCAGGCTTCAAAATTGCACCTTATCAGCTTACACGAATGAGATTGCCG 2161 GPH5_mandshurica TCTATGGTTAATTCAGGCTTCAAAATTGCACCTCATCAGCTTAC G CGAATGAGATTGCCG 2161 GPH5_nubicola TCT T TGGTTAATTCAGGCTTCAAAATTGCACCTCATCAGCTTAC G CAAATGAGATTGCCG 2174 GPH5_vesca TCTATGGTTAATTCAGGCTTCAAAATTGCACCTCATCAGCTTAC G CGAATGAGATTGCCG 2196 *** ******************** ******** *** ** **** ******** GPH5_ananassa_clone2 GATGTGGTAAATTCAGG-TGTCAAT T ACCCTCCACAGAGGACATTGCCGGATGTAAGTAT 2215 GPH5_ananassa_clone7 GATGTGGTAAATTCAGG-TGTCAAT T ACCCTCCACAGAGGACATTGCCGGATGTAAGTAT 2214 GPH5_viridis GATGTG A TAAATTCAGG-TGTCAATGACCCTCCACAGAGGACATTGCC A GATGTAAGTAT 2205 GPH5_iinumae GATGTGGTAAATTCAGG-TGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTAT 2194 GPH5_nilgerrensis GATGTGGTAAATTCAGNGTGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAG C AT 2221 GPH5_mandshurica GATGTGGTAAAT C CAGG-TGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTAT 2220 GPH5_nubicola GATGTGGTAAATTCAGG-TGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTAT 2233 GPH5_vesca GATGTGGTAAATTCAGG-TGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTAT 2255 ****** ***** *** ******* ********************** ******** ** GPH5_ananassa_clone2 CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAA G ATCCAGACACCTTCAG 2275 GPH5_ananassa_clone7 CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGA C ACCTTCAG 2274 GPH5_viridis CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAG 2265 GPH5_iinumae CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCC G GATACCTTCAG 2254 GPH5_nilgerrensis CTTATGCTACATGGAAATTTT A TTCTGGCCAGATTGG T ATGAAAATCCAGATACCTTCAG 2281 GPH5_mandshurica CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAG 2280 GPH5_nubicola CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAG 2293 GPH5_vesca CTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAG 2315 ********************* *************** ***** **** ** ******** GPH5_ananassa_clone2 T C TGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGAT 2335 GPH5_ananassa_clone7 TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGAT 2334 GPH5_viridis TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGAT 2325 GPH5_iinumae TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGAT 2314 GPH5_nilgerrensis TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTC C GCAAATGAT 2341 GPH5_mandshurica TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGAT 2340 GPH5_nubicola TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGA2352 GPH5_vesca TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGAT 2375 ************************************************ ********

PAGE 160

160 GPH5_ananassa_clone2 GTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAA 2395 GPH5_ananassa_clone7 GTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAA 2394 GPH5_viridis GTTTT-C A GCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAA 2384 GPH5_iinumae GTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAA 2374 GPH5_nilgerrensis GTTTTTCGGCTTCCAGTTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAA 2401 GPH5_mandshurica GTTTTTTGGCTTCCAGTTTTTCT T CACATAAGCATTTTAAAGCTGAT C ATTGTAATCAAA 2400 GPH5_nubicola --TTTTTGGCTTCCAGTTTTTCT T CACATAAGCATTTTAAAGCTG G T C ATTGTAAT T GAA 2410 GPH5_vesca GTTTTTTGGCTTCCAGTTTTTCT T CACATAAGCATTTTAAAGCTGAT C ATTGTAATCGAA 2435 *** *************** ********************* ******** ** GPH5_ananassa_clone2 CTC A AGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATT 2455 GPH5_ananassa_clone7 CTC A AGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCAC-ACTAAGATCACAATT 2453 GPH5_viridis CTCGAGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAG G TCACAATT 2444 GPH5_iinumae CTCGAGTAATTCT G CTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATT 2434 GPH5_nilgerrensis CTCGAGTAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATT 2461 GPH5_mandshurica CTCGA A TAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATT 2460 GPH5_nubicola CTCGA A TAATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATT 2470 GPH5_vesca CTCGA A TTATTCTACTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATT 2495 *** ***** ****************************** ****** ******** GPH5_ananassa_clone2 TCGTATTTTATGATCAACACTGAATACCTATGTCTAGTGTC A TGATTAT A GTCATGTGAA 2515 GPH5_ananassa_clone7 TCGTATTTTATGATCAACACTGAATACCTATGTCTAGTGTC A TGATTAT A GTCATGTGAA 2513 GPH5_viridis TCGTATTTTATGATCAACACTGAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAA 2504 GPH5_iinumae TCGTATTTTATGATCAACACTGAATACCTATGTCTAGTGT T GTGATTATGGTCATGTGAA 2494 GPH5_nilgerrensis TCGTATTTTATGATCAACACTGAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAA 2521 GPH5_mandshurica T T GTATTTTATGATCAACAC C GAATACCTATGTCTAGTGTCGTGATTGTGGTCATGTGAA 2520 GPH5_nubicola TCGTATTTTATGATCAACAC C GAATACCTATGTCTAGTGTCGTGATTATGGTCATGTGAA 2530 GPH5_vesca TCGTATTTTATGATCAACAC C GAAGACCTATGTCTAGTGTCGTGATTATGGTCATGTGAA 2555 ****************** *** *************** ***** ********** GPH5_ananassa_clone2 GTGGATTTCTTAATATATGCCTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2575 GPH5_ananassa_clone7 GTGGATTTCTTAATATATGCCTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2573 GPH5_viridis GTGGATTTCTTAATATATGCCTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2564 GPH5_iinumae GTGGATTTCTTAATATATGCCTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2554 GPH5_nilgerrensis GTGGATTTCTTAATATATG A CTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2581 GPH5_mandshurica GTGGATTTCTTAATATATGCCTCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2580 GPH5_nubicola GTGGATTTCTTAATATATGCCT T GTCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2590 GPH5_vesca GTGGATTTCTTAATATATGCCTCGTCTATGTCTTCATCCAGCAATCCTGCATACTTGAGT 2615 ******************* ** ************************************ GPH5_ananassa_clone2 TTGATGG T GCTTCAAAAGGAAATCCTGGAC CATCTGGTGCAGGAGCTGTACTCCGTGCTG 2635 GPH5_ananassa_clone7 TTGATGG T GCTTCAAAAGGAAATCCTGGA C CATCTGGTGCAGGAGCTGTACTCCGTGCTG 2633 GPH5_viridis TTGATGGAGCTTCAAAAGGAA G TCCTGGATTATCTGGTGCAGGAGCTGTACTCCGTG T TG 2624 GPH5_iinumae TTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTCCGTGCTG 2614 GPH5_nilgerrensis TTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTCCGTGCTG 2641 GPH5_mandshurica TTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTCCGTGCTG 2640 GPH5_nubicola TTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTCCGTGCTG 2650 GPH5_vesca TTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACT T CGTGCTG 2675 ******* ************* ******* ********************* **** ** GPH5_ananassa_clone2 AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGG ATATATAT 2695 GPH5_ananassa_clone7 AAGATGG A AGTGTTGTATG C GGAGTTCATGAAAACATTGTGAATCTTTTAGG ATATATAT 2693 GPH5_viridis AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGG ATATATAT 2684 GPH5_iinumae AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGG ATATATAT 2674 GPH5_nilgerrensis AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGG ATATATAT 2701 GPH5_mandshurica AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAAT T TTTT T GATATATAT 2699 GPH5_nubicola AAGATGGGAGTGTTGTATGTGGAGTTCATGAAA G CATTGTGAAT T TTTT T TT ATATATAT 2710 GPH5_vesca AAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAAT T TTTT T GATATATAT 2734 ******* *********** ************* ********** **** ********

PAGE 161

161 GPH5_ananassa_clone2 TTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGCTACTATAGTTGCACCGGCT 2755 GPH5_ananassa_clone7 TTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTATAGTTGCACCGGCT 2753 GPH5_viridis A TTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACT G TAGTTGCACCGGCT 2744 GPH5_iinumae TTTTGTTTTTGTAAAA G TGGATCTCTTTATAACATTGGG T TTACTATAGTTGCACCGGCT 2734 GPH5_nilgerrensis TTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTATAGTTGCACCGGCT 2761 GPH5_mandshurica TTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTT G CTATAGTTGCACCGGCT 2759 GPH5_nubicola TTTTGTTTTTGTAAAAATGG----TTTTATAACATTGGGGTTACTATAGTTGCACCGGCT 2766 GPH5_vesca TTTTGTTTTTGTAAAAATGGATCTCTT C ATAACATTGGGGTTACTATAGTTGCACCGGCT 2794 *************** *** ** *********** ** ************** GPH5_ananassa_clone2 GCGGGAAGGTGTGTGCAACGGCA 2778 GPH5_ananassa_clone7 GCGGGAAGGTGTGTGCAACGGCA 2776 GPH5_viridis GCGGGAAGGTGTGTGCAACGGCA 2767 GPH5_iinumae GCGGGAAGGTGTGTGCAACGGCA 2757 GPH5_nilgerrensis G A GGGAAGGTGTGTGCAACGGCA 2784 GPH5_mandshurica GCGGGAAGGTGTGTGCAACGGCA 2782 GPH5_nubicola GCGGGAAGGTGTGTGCAACGGCA 2789 GPH5_vesca GCGGGAAGGTGTGTGCAACGGCA 2817 ********************* --------------------------------------------------------------------------------------------------------------GPH23: SNPs other than introduced by DNA polymerase are true. After preliminary sequence alignment, the putative SNPs were ve rified by observation of unambiguous peaks in the chromatograms. GPH23_iinumae_clone2 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACG 50 GPH23_iinumae_clone5 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTGCTAACG 50 GPH23_mandshurica_clone3 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACG 50 GPH23_ananassa_clone4 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACG 50 GPH23_ananassa_clone3 CTTGAGGGCCATCAGCACGTCCCTTCTGCAATACCATCTTAGTACTAACG 50 ******************************************* ****** GPH23_iinumae_clone2 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100 GPH23_iinumae_clone5 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100 GPH23_mandshurica_clone3 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100 GPH23_ananassa_clone4 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100 GPH23_ananassa_clone3 ACCTTTACAGTGAGAGTGTGACCAGAGGTGCCTGGGCGGAGCTGCCCAAC 100 ************************************************** GPH23_iinumae_clone2 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGAT 150 GPH23_iinumae_clone5 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTTGAGTCTGCCATTTGAT 150 GPH23_mandshurica_clone3 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGTGTCTGCCATTTGAT 150 GPH23_ananassa_clone4 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGAT 150 GPH23_ananassa_clone3 CTTTGTGAAGGTTGGTTTCCTCAGGGCTTGCTTCGAGTCTGCCATTTGAT 150 ********************************* ************** GPH23_iinumae_clone2 AAAAGACCTGCCAGAATCCACGCCACCAAAC-TCTTTAGCACTAATCCAA 199 GPH23_iinumae_clone5 AAAAGACCTGCCAGAATCCACGCCACCAAAC-TCTTTAGCACTAATCCAA 199 GPH23_mandshurica_clone3 AAAAGACCTGCCAGAATCCACACCACCAAAC-TCTTTAGCACCAATCCAA 199 GPH23_ananassa_clone4 AAAAGACCTGCCAGAATCCACACCACCAAAC-TCTTTAGCACTAATCCAA 199 GPH23_ananassa_clone3 AAAAGACCTGCCAGAATCCACGCCACCAAACATCTTTAGCACTAATCCAA 200 ********************* ********* ********** ******* GPH23_iinumae_clone2 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 249 GPH23_iinumae_clone5 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 249 GPH23_mandshurica_clone3 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 249 GPH23_ananassa_clone4 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATGATGTGG 249 GPH23_ananassa_clone3 TCCATAACAACTTCATAAAACACACATAGCATCAACATGCAATAATGTGG 250 ******************************************* ******

PAGE 162

162 GPH23_iinumae_clone2 GTCCATAAGAACCATGAGTATGACATA-GAGTCTTCAAGCTTCGATTTCC 298 GPH23_iinumae_clone5 GTCCATAAGAACCATGAGTATGACATA-GAGTCTTCAAGCTTCGATTTCC 298 GPH23_mandshurica_clone3 GTCCACAAGAACCATGAGTATGAC-CA-TAGTCTTCAAGCTTCGATTTCC 297 GPH23_ananassa_clone4 GTCCATAAGAACCATGAGTATGACATA-GAGTCTTCAAGCTTCGATTTCC 298 GPH23_ananassa_clone3 GTCCATAAGAACCATGTGCATGACATAAGATTCCTCAAGCTTCGATTTCC 300 ***** ********** ***** ** **************** GPH23_iinumae_clone2 TTATTTGCTTCGAAAGAAGCAAGTTCAGAGTCACACAAACCAGAATATAG 348 GPH23_iinumae_clone5 TTATTTGCTTCGAAAGAAGCAAGTTCAGAGTCACACAAACCAGAATATAG 348 GPH23_mandshurica_clone3 TTATTCGCTTCGAAAGAAGCAAGTTCAGAGTCACACAAACCAGAACATAG 347 GPH23_ananassa_clone4 TTATTTGCTTCGAAAGAAGCAAGTTCAGAGTCACACAAACCAGAATATAG 348 GPH23_ananassa_clone3 TAATTTGCTTCAAAAGAAGTAAGTTCAGAGTCACTCAAACCCTAATATAG 350 *** ***** ******* ************** ****** ** **** GPH23_iinumae_clone2 ATCTCAAA--TTTAATGAAACATATTCCTAAGAACCTAAAGCAATATAAA 396 GPH23_iinumae_clone5 ATCTCAAA--TTTAATGAAACATATTCCTAAGAACCTAAAGCAATATAAA 396 GPH23_mandshurica_clone3 ATCTCAAAAATTTAATGAAACATATTCCTAAGAACCTAAAGAAATATAAA 397 GPH23_ananassa_clone4 ATCTCAAA--TTTAATGAAACATATTCCTAAGAGCCTAAAGAAATATAAA 396 GPH23_ananassa_clone3 ATCTCAAA--TTTAATGAAACATATTCCTAAGAGCCTACACAAATATAAA 398 ******** *********************** **** ******** GPH23_iinumae_clone2 ATCGTAACTGGACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCA 446 GPH23_iinumae_clone5 ATCGTAACTGGACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCA 446 GPH23_mandshurica_clone3 ATCGTAACTGAACTTATTCTGAAATTGTCGTATAAATTGTAAACCGATCA 447 GPH23_ananassa_clone4 ATCGTAACTGAACTTAATCTGAAATTGTCGTATAAATTGTAAATCGATCA 446 GPH23_ananassa_clone3 ATCGTAACTGAACTTAATCTGAAACTGTCGTATAAATTGTAAATCGATCA 448 ********** ***** ******* ****************** ****** GPH23_iinumae_clone2 AAAACAAACTTCAAGTTCAGATTCACAGAGCAGATCAGAGATAGCATACA 496 GPH23_iinumae_clone5 AAAACAAACTTCAAGTTCAGATTCACAGAGCAGATCAGAGATAGCATACA 496 GPH23_mandshurica_clone3 ACA-CAAACTTCAAGTTCAGATTCACAGAGCAGATCAGAGATAGCATACC 496 GPH23_ananassa_clone4 AAAACAAACTTCAAGTTCAGATTCAGAGAGCAGATCAGAGATAGCGTACA 496 GPH23_ananassa_clone3 AAACCAAACTTCATGTTCAGATTCACAGACCGTATCAGAGATAGCATACA 498 ********* *********** *** ************ *** GPH23_iinumae_clone2 AGTGACCTAAGAAACAAAACAACATTCTAACAAGATCGCAAACATTGGAG 546 GPH23_iinumae_clone5 AGTGACCTAAGAAACAAAACAACATTCTAACAAGATCGCAAACATTGGAG 546 GPH23_mandshurica_clone3 AGTGACCTAAGAAACAAAACAACCTTCTAACAAGATCGCAAACATTGGAG 546 GPH23_ananassa_clone4 AGTGACCTAAGAAACAAAACAAAATTCCAACAAGATCGCAAACATTCGAG 546 GPH23_ananassa_clone3 AGTGACCTCTGAAACAAAACATAATTCCAACAAGATCGCAAACATTCGAG 548 ******** *********** *** ****************** *** GPH23_iinumae_clone2 ATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAA 596 GPH23_iinumae_clone5 ATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAA 596 GPH23_mandshurica_clone3 ATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAA 596 GPH23_ananassa_clone4 ATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAA 596 GPH23_ananassa_clone3 ATTAAATACGATGAGCTATGAGACAACTATTCCATGCAAATCTAACAAAA 598 **************************** ********************* GPH23_iinumae_clone2 GAAACTAAAGGGATCTGGAGAATTAGGGGTTAGAGGTCAC CTTAAG AGTT 646 GPH23_iinumae_clone5 GAGACTAAAGGGATCTGGAGAATTAGGGGTTAGAGGTCAC CTTAAG AGTT 646 GPH23_mandshurica_clone3 GAGAATAAAGGGATCTGGAGAATTAGGGGTTAGAGGTCAC CTTAAG AGTT 646 GPH23_ananassa_clone4 GAGAATAAAGGGATCTGGAGAATTAGGGGTTAGAGGTGAC CTTAAG AGTT 646 GPH23_ananassa_clone3 AAGAATAAAGGGATCTGGAGAATTATGGGTTAGAGGTGACCTTCAGAGTT 648 ******************** *********** ***** ****** GPH23_iinumae_clone2 TCGGTGAAACACAACACAACTGGGGAGACAGAGACAGAGGAGGAACTGCG 696 GPH23_iinumae_clone5 TCGGTGAAACACAACACAACTGGGGAGACAGAGACAGAGGAGGAACTGCG 696 GPH23_mandshurica_clone3 TGGGTGAAACACAAC----CTGGGGAGACAGAGACAGAGGAGGAACTGCC 692 GPH23_ananassa_clone4 TGGGTGAAACACAAC----TGGGGAAGACAGAGACAGAGGAGGAACTGCG 692 GPH23_ananassa_clone3 TGGGTGAAACACAAC----TGGGGACGACCTACACCGAGGAGGAACTGCC 694 ************* *** *** ** *************

PAGE 163

163 GPH23_iinumae_clone2 AATATCTATCTGAA-ACCAAACAAAGTAAAAAGGGTTTAGCTGTCAGTAA 745 GPH23_iinumae_clone5 AATATCTATCTGAA-ACCAAAACAAGTAAAAAGGGTTTAGCTGTCAGTAA 745 GPH23_mandshurica_clone3 AAGATCTATCTGACCACCAAACCAAGTAAAAAGGGTTAAGCTATCAGTAA 742 GPH23_ananassa_clone4 AAGATCTATCTGAA-ACCAAACAAAGTAAAAAGGGTTTAGCTGTCAGTAA 741 GPH23_ananassa_clone3 AAAATCTATCTGAA-ACCTAAC-AAATAAAAAGGGTCTATCTGTCAATAA 742 ** ********** *** ** ** ********** ** *** *** GPH23_iinumae_clone2 -CGAGCTCCTAACCGTCCATCTCCAATCTTGTCAGGGGTGATCCT-ACGC 793 GPH23_iinumae_clone5 -CGAGCTCCTAACCGTCCATCTCCAATCTTGTCAGGGGGGATCCT-ACGC 793 GPH23_mandshurica_clone3 -CGAGCTCCTAACCGTCCATCTCCAATCTTGTCAGGGGTGATCCT-ACGC 790 GPH23_ananassa_clone4 -CGAGCTCCTAACCGTCCATCTCCAATCTTGTCAGGGGTGATCCT-ACGC 789 GPH23_ananassa_clone3 ACGAGCTCCCTATCGTCCATCTCCAATCTTGTAAGGGGTGATCCTTACGC 792 ******** ******************* ***** ****** **** GPH23_iinumae_clone2 GTCCACTTGTCCTCTCCCAGTTCTAACTATGTACACGCTAGCTGCGGATT 843 GPH23_iinumae_clone5 GTCCACTTGTCCTCTCC-AGTTCTAACTATGTACACGCTAGCTGCGGATT 842 GPH23_mandshurica_clone3 GTCCACTTGTCCTCTCCCAGTTCTAACTGTGTAGACGCTAGCTGCGGATT 840 GPH23_ananassa_clone4 GTCCACTTGTCCTTTCCCAGTTCTAACTATGTAGACGCTAGCTGCGGATT 839 GPH23_ananassa_clone3 TTCCCTTTGTCCTCTCCCCCATCTA-CTAAGTAGACGCTAGCTGCGGATT 841 *** ******* *** **** ** *** **************** GPH23_iinumae_clone2 GTTATTATGTTTTGGATAGAATAGAATACCTTTGCAAAATAGGAAGCTCC 893 GPH23_iinumae_clone5 GTTATTATGTTTTGGATAGAATAGAATACCTTTGCAAAATAGGAAGCTCC 892 GPH23_mandshurica_clone3 GTTATTATTTTTTGGATAG-----AATACCTTTGCAAAATAGGAAGCTCC 885 GPH23_ananassa_clone4 GTTATTATGTTTTGGATAG-----AATACCTTTGCAAAATAGGAAGCTCC 884 GPH23_ananassa_clone3 GTTATTATGTTTTGGATAG-----AATACCTTTGCAAAATTGGAAGCTCC 886 ******** ********** **************** ********* GPH23_iinumae_clone2 ------TCCTTGTTTTTCGGCAAGAGAAAGGCCAAAATATCTGACCATTC 937 GPH23_iinumae_clone5 ------TCCTTGTTTTTCGGCAAGAGAAAGGCCAAAATATCTGACCATTC 936 GPH23_mandshurica_clone3 ------TCCTTGTTTTTCGGCAAGAGAAAGGCCAAAATATCTGACCATTC 929 GPH23_ananassa_clone4 ------TCCTTGTTTTTCTGCAAGAGAAAGGCCAAAATATCTGACCATTC 928 GPH23_ananassa_clone3 AGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAATATCAGACCGTTC 936 ************ ********************** **** *** GPH23_iinumae_clone2 CGACGCCGGAGCTTCCTCAGAAAGCCGGTTCCGTCCGCAACATCGATGCC 987 GPH23_iinumae_clone5 CGACGCCGGAGCTTCCTCAGAAAGCCGGTTCCGTCCGCAACATCGATGCC 986 GPH23_mandshurica_clone3 CGACGCCGGAGCTTCCTCAGAAAGCCAGTACTATCCGCCACATCGATGCC 979 GPH23_ananassa_clone4 CGACGCCGGAGCTTCCTCAGAAAGCCAGTACCATCCGCAACATCGATGCC 978 GPH23_ananassa_clone3 CGACGCCGGAGCTTCCTCAGAAAGCC--TACCATCCGCAACATCGTTGCC 984 ************************** ***** ****** **** GPH23_iinumae_clone2 AGGCCTTGCGAGGTTTGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTT 1037 GPH23_iinumae_clone5 AGGCCTTGCGAGGTTTGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTT 1036 GPH23_mandshurica_clone3 AGGCCTTGCGAGGTTTGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTT 1029 GPH23_ananassa_clone4 AGGCCTTGCGAGGTTTGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTT 1028 GPH23_ananassa_clone3 AGGCCTTGCGAGGTTTGCCTCCGCTTCTTTGGATTGTGTTTTTCGTGGTA 1034 ************************************************* GPH23_iinumae_clone2 TAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGATGAATGAATT 1087 GPH23_iinumae_clone5 TAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGATGAATGAATT 1086 GPH23_mandshurica_clone3 TAGGAGATTGCTGAACAAAAAAGAAAAACATAACATATGATGAATGAATT 1079 GPH23_ananassa_clone4 TAGGAGATTGTTGAACAAAAAAGAAAAACATA---TATGATAAATGAATT 1075 GPH23_ananassa_clone3 TAGGAGATTGTTGAACAAAAAAGAAAAACATAACATATGATGAATGAATC 1084 ********** ********************* ****** ******* GPH23_iinumae_clone2 ATCAAATTAATTAACCAAGGTGACAATACAAGATGAGAACACCAAGGGTT 1137 GPH23_iinumae_clone5 ATCAAATTAATTAACCAAGGTGACAATACAAGATGAGAACACCAAGGGTT 1136 GPH23_mandshurica_clone3 ATCAAATTAATTAATCAAGGTGACAATACAAGATGAGAACACCAAAGATT 1129 GPH23_ananassa_clone4 ATCAAATTAATTAATCAAGGTGACGATACAAGATGAGAACACCAAAGGTT 1125 GPH23_ananassa_clone3 ATCAAATTAATTAATCAAGGTGAC-------------------------1108 ************** *********

PAGE 164

164 GPH23_iinumae_clone2 CAATAGTGTGTACTCTCAAGCCTAATACTAACACAACAAAGAAAGATTTC 1187 GPH23_iinumae_clone5 CAATAGTGTGTACTCTCAAGCCTAATACTAACACAACAAAGAAAGATTTC 1186 GPH23_mandshurica_clone3 CAATAGTGTGTACTCTCAAGCCTAATACTAACACAACAAAGAAAGATT-C 1178 GPH23_ananassa_clone4 CAATAGTGTGTACTCTCAAGCCTAATACTAACACAACAAAGAAAGATT-C 1174 GPH23_ananassa_clone3 -------------------------------------AAAGAAAGATT-A 1120 *********** GPH23_iinumae_clone2 TATCCTTCCATTCCCAAATCAAAAACCACTACAATGTACCGTC------1230 GPH23_iinumae_clone5 TATCCTTCCATTCCCAAATCAAAAACCACTACAATGTACCGTC------1229 GPH23_mandshurica_clone3 TATCGTTCCATTCCCAAATCAAAAACCATTACAATAGACCGCC------1221 GPH23_ananassa_clone4 TATCCTTCCATTCCCAGATCAAAAACCACTCTAATGTACCGCCGGCAAAG 1224 GPH23_ananassa_clone3 TATCCTTCCATTCCTAAGTCAAAAACCATTACAATGTACCGCCGGCAAAA 1170 **** ********* ********** *** **** GPH23_iinumae_clone2 ------TAATTGAATTGACATTGTAAGTGAGAGATAGTGTCACGAGCTGC 1274 GPH23_iinumae_clone5 ------TAATTGAATTGACATTGTAAGTGAGAGATAGTGTCACGAGCTGC 1273 GPH23_mandshurica_clone3 ----------------GGCA----AAGTG-----------------CTGC 1234 GPH23_ananassa_clone4 TGCTGCTAATAGAATTGACATTGTAAGTAGGGGATAGTGTCACGAGCAGC 1274 GPH23_ananassa_clone3 TGCTGCTAATAGAATTGACATTGTAAGTGGGGGATAGTGTCACGAGCTGC 1220 ** **** ** GPH23_iinumae_clone2 ATTGGGAGATAGTGTCATCGTGACACTCTATGAAGGGG-ATG CTTAAG AG 1323 GPH23_iinumae_clone5 ATTGGGAGATAGTGTCATCGTGACACTCTATGAAGGTG-ATG CTTAAG AG 1322 GPH23_mandshurica_clone3 --TAGGAGGTAGTGTCATCGTGACACTCTATGAAGGTG-ATG CTTAAG AG 1281 GPH23_ananassa_clone4 TCTAGGAGGTAGTGTCATCGTGACACTTTATTGGGGTGGATGCTAAGGGG 1324 GPH23_ananassa_clone3 AATAGGAGGTAGTGTCATCGTGACACTCTA--GAGGTG-ATG CTTAAG AG 1267 **** ****************** ** ** ***** GPH23_iinumae_clone2 GGTCGC---ATCAATGACAA-ACATGAGGGCAAATA-GAAGGTCTACTGG 1368 GPH23_iinumae_clone5 GGTCGC---ATCAATGACAA-ACATGAGGGCAAATA-GAATGTCTACTGG 1367 GPH23_mandshurica_clone3 GGACAC---GTCAATAGCAA-GTATGAGGGAAAAAAAGGATGTTTACTGG 1327 GPH23_ananassa_clone4 GTTCAG---GTTATGAGCAA-GCATGGGGGGTAAGGGGGATATCTACTGG 1370 GPH23_ananassa_clone3 GGTCACAAGGTCAATGGCAAAGCATGAGGGTTAAAGAGGATGTTTACTGA 1317 * *** *** *** ** ***** GPH23_iinumae_clone2 CATGTCGAATGACAATGTCGTAATTAGTTAAGTGAAACTTATATTTCAAG 1418 GPH23_iinumae_clone5 CATGTCGAATGACAATGTCGTAATTAGTTAAGTGAAACTTATATTTCAAG 1417 GPH23_mandshurica_clone3 CATGTCGAATGATAATTTCGTAATTAGTTAAGTGAAACTTATATTTCAAG 1377 GPH23_ananassa_clone4 CATTTTGAATGACAATGTTGTAAATGA-------AAACTTATATTTCAAG 1413 GPH23_ananassa_clone3 CATGTTGAAAGACAATGTCGTAATTAGTT-----AAAGT--------GAA 1354 *** *** ** *** **** *** GPH23_iinumae_clone2 GTACTTTGAC-TTAGTATTTAGAAAAACTGTAACATCGAAAGGAGTTCAA 1467 GPH23_iinumae_clone5 GTACTTTGAC-TTAGTATTTAGAAAAACTGTAACATCGAAAGGAGTTCAA 1466 GPH23_mandshurica_clone3 GTACTTTGAC-CTAGTATTTGGAAAAACCGTAACATCTAAA--------1417 GPH23_ananassa_clone4 GTATTTTGAT-TTAATATTTAGAAAAACTGTAACATCAAAAGGGGTTCAA 1462 GPH23_ananassa_clone3 GTACTGTGAAGTTAGTATTTCGAAAAACTGTAACATCGGAAGGGGTTCAA 1404 *** *** ** ***** ******* ******** ** GPH23_iinumae_clone2 TACATTTGACGACATATTTTTATGAGGTTTCTATAAATTAGTTATGAGAG 1517 GPH23_iinumae_clone5 TACATTTGACGACATATTTTTATGAGGTTTCTATGAATTAGTTATGAGAG 1516 GPH23_mandshurica_clone3 TACACTTGACGACATATTTTTATGAGATTTCTATGAATTAGTTATGAGAG 1467 GPH23_ananassa_clone4 TACATTTGCCGACATATTTTTATGAGGTTTTTATGAATTAGTTATGAGAG 1512 GPH23_ananassa_clone3 TACATTTGACGACATATTTTTATGAAGTTATTAGGAATTAGTTACGAGAG 1454 **** *** **************** ** ** ********* ***** GPH23_iinumae_clone2 ATGGTTTTC-CTTGGACTATTTTGATTTTG-ATGTTTCCTTAACACACAT 1565 GPH23_iinumae_clone5 ATGGTTTTC-CTTGGACTATTTTGATTTTG-ATGTTTCCTTAACACACAT 1564 GPH23_mandshurica_clone3 ATGGTTTTC-TTTAGACTATTTTGATTTTGGATGTTTCCTTAACACACAT 1516 GPH23_ananassa_clone4 ATGGTTTTC-CTTGGACTATTTAGATTTTG-ATGTTTCCTTAACACACAT 1560 GPH23_ananassa_clone3 ATGGTTTTTTCTTAGAATATTTTGATTTTG-ATGTTTCCTTGACACACAT 1503 ******** ** ** ***** ******* ********** ********

PAGE 165

165 GPH23_iinumae_clone2 TATATTTA--------------------AAGTAATTTTCCGTATCACTTA 1595 GPH23_iinumae_clone5 TATATTTA--------------------AAGTAATTTTCTGTATCACTTA 1594 GPH23_mandshurica_clone3 TATATTTCTCCATTTTCTTTTTATGTATAAGTAATTTTCTGTATCACTTA 1566 GPH23_ananassa_clone4 TATATTTCTCCATTTTCTT-------GTAAGTAATTTTCTGTATCACTTA 1603 GPH23_ananassa_clone3 TATATTTCTCCATGTTCTTCT-ATGTATAAGTAATTTTCTGTATCACTTA 1552 ******* *********** ********** GPH23_iinumae_clone2 AAAACATTTCTTACTCTTTCCAGAAGCATCTCCAAACATCTCC------1638 GPH23_iinumae_clone5 AAAACATTTCTTACTCTTTTCCAAACCATCTCCAAACATCTCC------1637 GPH23_mandshurica_clone3 AAAACATTTCTTACTCTTTCCAGAAGCATCTCTA----TCCCC------1605 GPH23_ananassa_clone4 AAAACATTTCTTACTCTTCCCAGAAACATCTCCAAACATCCCTA--AACC 1651 GPH23_ananassa_clone3 GAAACATTTCTTACTCTTTCCAGAAGCATCTCCAAACATCCCCCTAAACC 1602 ***************** ** ****** ** GPH23_iinumae_clone2 -------CTAA-ATGTCAATGTC-------AATAGATGAAAGATCAACCT 1673 GPH23_iinumae_clone5 -------CCAA-ATGTCCATGGCCA----TAATAGATGAAAGATCAACCT 1675 GPH23_mandshurica_clone3 -------CTAA-A-----------------------------------CC 1612 GPH23_ananassa_clone4 GATAGCTCTAACATGTCAATGTC-------AATAGATGAAAGATCAACCT 1694 GPH23_ananassa_clone3 AATAGCCCTAACATGTCAATGTCACATGTCAATAGATGAAAGATCAACCT 1652 ** GPH23_iinumae_clone2 AAATGGTACCATATGTCCATACATAAAAAGACCCAAAAAGA--------1714 GPH23_iinumae_clone5 AAATGGTACCATATGTCCATACATAAAAAGACCCAAAAAGA--------1716 GPH23_mandshurica_clone3 AAATACACTAACATGTCAATGTCCA-------CCCAAAAGA--------1646 GPH23_ananassa_clone4 AAATGGTACCATATGTCCATACATAAAAAGACCCAAAAAGA--------1735 GPH23_ananassa_clone3 AAATGGTACCATATGTCCATACATAAAAGGACCCAAAAAAATAAATAAAG 1702 **** ***** ** ** **** GPH23_iinumae_clone2 AA-TAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAGTAGAGAAGAAT 1763 GPH23_iinumae_clone5 AA-TAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAGTAGAGAAGAAT 1765 GPH23_mandshurica_clone3 AA-TAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAATAGAGAAGAAT 1695 GPH23_ananassa_clone4 AAATAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAGTAGAGAAGAAT 1785 GPH23_ananassa_clone3 AAATAAATATGCACCTTCATTTTTAAGCGCCAGAAAAAGTAGAGAAGAAT 1752 ** ****** ********************** ***** *********** GPH23_iinumae_clone2 ATAAGGTTTGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTC 1813 GPH23_iinumae_clone5 ATAAGGTTTGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTC 1815 GPH23_mandshurica_clone3 ATAAGGTTTGAAGTGAACAAGGGGATAAGCAGTTTAAGGTCGACTTGTTC 1745 GPH23_ananassa_clone4 ACAAGGTTTGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTC 1835 GPH23_ananassa_clone3 ATAAGGTTTGAAGTGATCAAGGGGATAAGCAGTTTAAGGTCGACTTGTTT 1802 ************** ******************************** GPH23_iinumae_clone2 GGAAACAATGCTAACCACCACCACTGCCAGTCTCAGCT---G CTCCTCCT 1860 GPH23_iinumae_clone5 GGAAACAATGCTAACCACCACCACTGCCAGTCTCAGCT---G CTCCTCCT 1862 GPH23_mandshurica_clone3 GGAAACAATGCTGACCACCACCACTGCCACTCTCAGTCTCAG CTCCTCCT 1795 GPH23_ananassa_clone4 GGAAACAATGCTAACCACCACCACTGCCACTCTCAGCT---G CTCCTCCT 1882 GPH23_ananassa_clone3 GGAAACAATGCTAACCACCACCACTGCCACTCACAGTCTCAG CTCCTCCT 1852 ************ **************** ** *** ********* GPH23_iinumae_clone2 CCT CTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACCAAACCCA 1910 GPH23_iinumae_clone5 CCT CTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACCAAACCCA 1912 GPH23_mandshurica_clone3 CCT CTACTTCCCAACTCCCACCACTCTTCCACTCTCTAACACCAAACCCA 1845 GPH23_ananassa_clone4 CCT CTGCTTCCCAACTCCCACCACTCTTCCACTCTCTATCACCAAACCCA 1932 GPH23_ananassa_clone3 CCT CTGCTTCCCAACTCCCATCGCTCTTCCACTCTCTATCACAAAACCCA 1902 ***** ************** *************** *** ******* GPH23_iinumae_clone2 ATCTCCCTCAGATTCTCCTCCACATTACAGCTAACCAAAACCAGAACCAG 1960 GPH23_iinumae_clone5 ATCTCCCTCAGATTCTCCTCCACATTACAGCTAACCAAAACCAGAACCAG 1962 GPH23_mandshurica_clone3 ATCTCCCTCAGATTCTCCTCCACATTACAGCTAACTAAAACCAGAAC--1892 GPH23_ananassa_clone4 ATCTCCCTCAGATTCTCCTCCACATTACAGCTAACCAAAACCAGAAC--1979 GPH23_ananassa_clone3 ATCTCCCTCAGATTCTCCTCCACATTAAAGCTAACCAAAACCAGAAC--1949 *************************** ******* ***********

PAGE 166

166 GPH23_iinumae_clone2 AACCAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 2010 GPH23_iinumae_clone5 AACCAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 2012 GPH23_mandshurica_clone3 ---CAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 1939 GPH23_ananassa_clone4 ---CAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 2026 GPH23_ananassa_clone3 ---CAGACCAACCCTTAAAACTCTCACTCGCCAAAAATGCCAGCTCCCTG 1996 *********************************************** GPH23_iinumae_clone2 CTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCC 2060 GPH23_iinumae_clone5 CTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCC 2062 GPH23_mandshurica_clone3 CTCTGAGAGTGTCTGCTAAATACGAAGCTTCCCCTGCCACAGCTGAGGCC 1989 GPH23_ananassa_clone4 CTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCC 2076 GPH23_ananassa_clone3 CTCTGAGAGTGTCTGCTAATTACGAAGCTGCCCCTGCCACGGCTGAGGCC 2046 ******************* ********* ********** ********* GPH23_iinumae_clone2 TCCACGGTGCCGTTGGAGATGAAGGCGTGGGTGTA 2095 GPH23_iinumae_clone5 TCCACGGTGCCGTTGGAGATGAAGGCGTGGGTGTA 2097 GPH23_mandshurica_clone3 TCCACGGTGCCGTCGGAGATGAAGGCGTGGGTGTA 2024 GPH23_ananassa_clone4 TCCACGGTGCCGTCGGAGATGAAGGCGTGGGTGTA 2111 GPH23_ananassa_clone3 TCCACAGTGCCGTCGGAGATGAAGGCGTGGGTGTA 2081 ***** ******* ********************* --------------------------------------------------------------------------------------------------------------Gene Pairs Detected Through Pred iction from Genomic Sequence GPH10 GPH10_ananassa_clone2 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50 GPH10_ananassa_clone20 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50 GPH10_ananassa_clone18 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50 GPH10_ananassa_clone19 GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGC 50 ************************************************** GPH10_ananassa_clone2 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100 GPH10_ananassa_clone20 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100 GPH10_ananassa_clone18 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100 GPH10_ananassa_clone19 CGATACCTCCTCCGCCTCCGACGACTTCGAACACAGCGGAATCGCTAGCC 100 ************************************************** GPH10_ananassa_clone2 TCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGT 150 GPH10_ananassa_clone20 TCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGT 150 GPH10_ananassa_clone18 TCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGT 150 GPH10_ananassa_clone19 TCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGT 150 ************************************************** GPH10_ananassa_clone2 GAATAGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAAC 200 GPH10_ananassa_clone20 GAATAGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAAC 200 GPH10_ananassa_clone18 GAATAGCGAACTGAGTGGTTTGGATTTGAGAAGAGGATGAAATTGGTAAC 200 GPH10_ananassa_clone19 GAATAGCGAACTGAGTGGTTTGGATTTGAGAAGAGGATGAAATTGGTAAC 200 **************** ********************** ********** GPH10_ananassa_clone2 GGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCATCTTTGAAGTG 250 GPH10_ananassa_clone20 GGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAAGTG 250 GPH10_ananassa_clone18 GGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAAGTG 250 GPH10_ananassa_clone19 GGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTTAGCTTTGAAGTG 250 ************************* *********** ********** GPH10_ananassa_clone2 GAGTGTAGGATAATAACAAACTCGTTATC--------------------279 GPH10_ananassa_clone20 GAGTGTAGGATAATAACAAACTCGTGATTAAAAGACAGGATTAATGTCAG 300 GPH10_ananassa_clone18 GAGTGTAGGATAATAACAAACTCGTTATC--------------------279 GPH10_ananassa_clone19 GAGTGTAGGATAATAACAAACTCGTTATC--------------------279 ************************* **

PAGE 167

167 GPH10_ananassa_clone2 -------------------------------------------------GPH10_ananassa_clone20 TGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTT 350 GPH10_ananassa_clone18 -------------------------------------------------GPH10_ananassa_clone19 -------------------------------------------------GPH10_ananassa_clone2 --------------------------------------TAAAAGGCAGGT 291 GPH10_ananassa_clone20 CAAACCTCACGACATATGTAGGGTGTATGAATTATTAATAAAAGACAAAT 400 GPH10_ananassa_clone18 --------------------------------------TAAAAGACAGGT 291 GPH10_ananassa_clone19 --------------------------------------TAAAAGACAGGT 291 ****** ** GPH10_ananassa_clone2 TTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGT 341 GPH10_ananassa_clone20 TTAATATCAGCCGTTAGATCATATTACGGCC-TGATCACTCGACATATGT 449 GPH10_ananassa_clone18 TTAATATCGGCCGTTAGATCACATTACGGCCCTGATCACTCGACATATGT 341 GPH10_ananassa_clone19 TTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGT 341 ******** ************ ********* ****************** GPH10_ananassa_clone2 TGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACAT----386 GPH10_ananassa_clone20 TGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACGT----494 GPH10_ananassa_clone18 TGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACATTTTTT 391 GPH10_ananassa_clone19 TGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACATTTTTT 391 *********** ******************************* GPH10_ananassa_clone2 ----------------------------------------ATATTTTATT 396 GPH10_ananassa_clone20 ----------------------------------------ATATTTTATT 504 GPH10_ananassa_clone18 TTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTT 441 GPH10_ananassa_clone19 TTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTT 441 *** ** ** GPH10_ananassa_clone2 TTTTTAAAGTAACTAAATGACTATGTACATCGTTTAACAAAAGAAACAAT 446 GPH10_ananassa_clone20 TTTTTAAAATAATTAAATAACTATTTACGTTGTTTAACAAAAGAAACAAT 554 GPH10_ananassa_clone18 TTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAAT 491 GPH10_ananassa_clone19 TTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAAT 491 ******** *** ***** ***** *** **** ************ GPH10_ananassa_clone2 TGAAGTTAAATTAAGAGCACCATAACAGCTGAGAAAGAGTACGAGAACAA 496 GPH10_ananassa_clone20 TGAAGTTAAATTAAGAGCACCGTAACAGCTGAGCAAGAGTACGAGAACAA 604 GPH10_ananassa_clone18 TGAAGTTAAATTAAGAGCACCATGACAG---------AGTACGAGAACAA 532 GPH10_ananassa_clone19 TGAAGTTAAATTAAGAGCACCATGACAG---------AGTACGAGAACAA 532 ********************* **** ************* GPH10_ananassa_clone2 AAGTATGAGCTAAAACAA--------------------ATAGAGAAAATA 526 GPH10_ananassa_clone20 AAGTATGAGCTACATCATTTG------------TTCATATAGAGAAAATA 642 GPH10_ananassa_clone18 AAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATG 582 GPH10_ananassa_clone19 AAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATA 582 ************ ** ******** GPH10_ananassa_clone2 TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTA 576 GPH10_ananassa_clone20 TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTA 692 GPH10_ananassa_clone18 TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTA 632 GPH10_ananassa_clone19 TAGAGGCGATGTTGTAGAAATAATAGAACATTAGAAAATTAAATTACCTA 632 ************************ ************************* GPH10_ananassa_clone2 AAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCA 626 GPH10_ananassa_clone20 AAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCA 742 GPH10_ananassa_clone18 AAAGCCGATGAGTAAAATAATAACAAACTCGTAACCTAAAAGCGGCTTCA 682 GPH10_ananassa_clone19 AAAGCCGATGAGTAAAATAATAACAAACTCGTAACCTAAAAGCGGCTTCA 682 ************************ ************************* GPH10_ananassa_clone2 TATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 676 GPH10_ananassa_clone20 TATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 792 GPH10_ananassa_clone18 TATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 732 GPH10_ananassa_clone19 TATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 732 ******** ** **************************************

PAGE 168

168 GPH10_ananassa_clone2 CCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACCC 726 GPH10_ananassa_clone20 CCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAATAAAACCC 842 GPH10_ananassa_clone18 CCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACCC 782 GPH10_ananassa_clone19 CCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAACAAAACCC 782 ****************************************** ******* GPH10_ananassa_clone2 ACACGACGCCGTTTTGCTCCAATCCCCC-----TTTCTTCTTCAACCCCA 771 GPH10_ananassa_clone20 ACACGACGCCGTTTTGCTCCAATCCCCC-----TTTCTTCTTCAACCCCA 887 GPH10_ananassa_clone18 ACACGACGCCGTTTTGCTCCAATCCCCCCCCCCTTTCTTCTTCAACCCCA 832 GPH10_ananassa_clone19 ACACGACGCCGTTTTGCTCCAATCCCCCCCCC-TTTCTTCTTCAACCCCA 831 **************************** ***************** GPH10_ananassa_clone2 TAGTCGCCTC---AGCTCAGTTCCATTTGTCTCAGATGCGATGGCCTCCG 818 GPH10_ananassa_clone20 TAGTCGCCTC---AGCTCAGTTCCATTTGTCTCAGATGCGATGGCCTCCG 934 GPH10_ananassa_clone18 TAGTCGCCTCCTCAGCTCAGTTCCATTTGTCTCA--TGCGATGGCTTCCG 880 GPH10_ananassa_clone19 TAGTCGCCTCCTCAGCTCAGTTCCATTTGTCTCA--TGCGATGGCTTCCG 879 ********** ********************* ********* **** GPH10_ananassa_clone2 ------------GCGACCCAATCTCCGACTACACCCAAACACATCGCATT 856 GPH10_ananassa_clone20 ------------GCGACCCAATCTCCGACTACACCCAAACACATCGCATT 972 GPH10_ananassa_clone18 ACTC GAATTC CGGCGACCCAATCTCCTCCTACACCCAAACCCATCGCATC 930 GPH10_ananassa_clone19 ACTCAAATTCCGGCGACCCAATCTCCTCCTACACCCAAACCCATCGCATC 929 ************** ************ ******** GPH10_ananassa_clone2 GTCCTTCTAATCGACCTCAACCCACTCCTCCATCTCCAAGATCCAACCCA 906 GPH10_ananassa_clone20 GTCCTTCTAATCGACCTCAACCCACTCCTCCATCTCCAAGATCCAACCCA 1022 GPH10_ananassa_clone18 GTCCTTCTAATCGACCTCAACCCACTCCTCAATCTCCAAGATCCAACCCA 980 GPH10_ananassa_clone19 GTCCTTCTAATCGACCTCAACCCACTCCTCAATCTCCAAGATCCAACCCA 979 ****************************** ******************* GPH10_ananassa_clone2 ATTCCTCACCTCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTT 956 GPH10_ananassa_clone20 ATTCCTCACCTCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTT 1072 GPH10_ananassa_clone18 ATTCCTCACCCCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTT 1030 GPH10_ananassa_clone19 ATTCCTCACCCCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTT 1029 ********** *************************************** GPH10_ananassa_clone2 CTCTCTCTTC----------GCCGTCAGGCCCTTCTTCTCGTCTCTCTCT 996 GPH10_ananassa_clone20 CTCTCTCTTCCTCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCT 1122 GPH10_ananassa_clone18 CTCTCTCTTCATCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCT 1080 GPH10_ananassa_clone19 CTCTCTCTTCATCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCT 1079 ********** ****************************** GPH10_ananassa_clone2 CCTCTCCTCTCC-----GCCTCCAAGCTCCCGTCTTCGTCTCTAACGATC 1041 GPH10_ananassa_clone20 CCTCTCCTCTCCTCTCCGCCTCCAAGCTCCCGTCTTCGTCTCTAACGATC 1172 GPH10_ananassa_clone18 CCTCTCCTCTCC-----GCCTCCAAGCTCCCGTCTTCGTCTCTAACGATC 1125 GPH10_ananassa_clone19 CCTCTCCTCTCC-----GCCTCCAAGCTCCCGTCTTCGTCTCTAACGATC 1124 ************ ********************************* GPH10_ananassa_clone2 TCTTTCAACTCGCCGGAAGACACATATCGATCCCTATCTCAAACCCTGGC 1091 GPH10_ananassa_clone20 TCTTTCAACTCGCCGGAAGACACATATCGATCCCTATCTCAAACCCTGGC 1222 GPH10_ananassa_clone18 TCTTTCAACTCGCCGGAAGACACTTATCGATCCCTATCTCAAACCCTGGC 1175 GPH10_ananassa_clone19 TCTTTCAACTCGCCGGAAGACACTTATCGATCCCTATCTCAAACCCTGGC 1174 *********************** ************************** GPH10_ananassa_clone2 GTCTCTCTCGTTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAA 1141 GPH10_ananassa_clone20 GTCTCTCTCGTTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAA 1272 GPH10_ananassa_clone18 GTCTCTCTCTTTTGACCGCAAGTTGGCCGGGTCCGATTCGCCGCGGGGAA 1225 GPH10_ananassa_clone19 GTCTCTCTCTTTTGACCGCAAGTTGGCCGGGTCCGATTCGCCGCGGGGAA 1224 ********* ******** ****** ************************ GPH10_ananassa_clone2 CGCTTGTTGCGGCTGCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAG 1191 GPH10_ananassa_clone20 CGCTTGTTGCGGCTGCGATGCGGCAGCTGGTACATGATTACGCTTGGGAG 1322 GPH10_ananassa_clone18 CGCNTGTTGCGGCGGCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAG 1275 GPH10_ananassa_clone19 CGCTTGTTGCGGCGGCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAG 1274 *** ********* ***************** ******************

PAGE 169

169 GPH10_ananassa_clone2 CAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGATACGTTTTCGAATTG 1241 GPH10_ananassa_clone20 CAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGGTACGTTTTCGAATTG 1372 GPH10_ananassa_clone18 CCGGTGATCTGCGACGCCGCGGCGGCGGAGACCGGTACGTTATCGAATTG 1325 GPH10_ananassa_clone19 CCGGTGATCTGCGACGCCGCGGCGGCGGAGACCGGTACGTTATCGAATTG 1324 ***************** ************ ****** ******** GPH10_ananassa_clone2 CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAAT 1291 GPH10_ananassa_clone20 CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTTACCGGCGTGTCAAT 1422 GPH10_ananassa_clone18 CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAAT 1375 GPH10_ananassa_clone19 CTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAAT 1374 *********************************** ************** GPH10_ananassa_clone2 TTGTGAATGAGTTCTTGAATTGTGAG------------GGTTTGGAGGAT 1329 GPH10_ananassa_clone20 TTGTGAATGAGTTCTTGAATTGTGAGTTGAATTGTGAGGGTTTGGAGGAT 1472 GPH10_ananassa_clone18 TTGTGAATGAGTTCTTGAATTGTGAG------------GGTTTGGAGGAT 1413 GPH10_ananassa_clone19 TTGTGAATGAGTTCTTGAATTGTGAG------------GGTTTGGAGGAT 1412 ************************** ************ GPH10_ananassa_clone2 TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGA 1379 GPH10_ananassa_clone20 TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGA 1522 GPH10_ananassa_clone18 TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGA 1463 GPH10_ananassa_clone19 TTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGA 1462 ************************************************** GPH10_ananassa_clone2 GGCATATGTGTATAGAGATATTCAATTGAGTCGGGTTGATGTGAGGTATG 1429 GPH10_ananassa_clone20 GGCATATGTGTATAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATG 1572 GPH10_ananassa_clone18 GGCATTTGTGTGTAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATG 1513 GPH10_ananassa_clone19 GGCATTTGTGTGTAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATG 1512 ***** ***** ******************* ****************** GPH10_ananassa_clone2 GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1479 GPH10_ananassa_clone20 GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1622 GPH10_ananassa_clone18 GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1563 GPH10_ananassa_clone19 GATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTGTTTTC 1562 ************************************************** GPH10_ananassa_clone2 GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1529 GPH10_ananassa_clone20 GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1672 GPH10_ananassa_clone18 GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1613 GPH10_ananassa_clone19 GAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGAT 1612 ************************************************** GPH10_ananassa_clone2 TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1579 GPH10_ananassa_clone20 TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1722 GPH10_ananassa_clone18 TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1663 GPH10_ananassa_clone19 TGTGCTTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTG 1662 ************************************************** GPH10_ananassa_clone2 GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAGA 1629 GPH10_ananassa_clone20 GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAGA 1772 GPH10_ananassa_clone18 GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGTTTAGA 1713 GPH10_ananassa_clone19 GGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGTTTAGA 1712 ******************************************** ***** GPH10_ananassa_clone2 GCGCATTTGAGTCTTGAGATATCGGATGTAAAGGGGATGCCTTTGGAGTG 1679 GPH10_ananassa_clone20 GCGCATTTGAGTCTTGAGATATCAGATGTAAAGGGGATGCCTTTGGAGTG 1822 GPH10_ananassa_clone18 GCGCATTTGAGTCTTGAGATATCGGATGGAAAGGGGATGCCTTTGGAGTG 1763 GPH10_ananassa_clone19 GCGCATTTGAGTCTTGAGATATCGGATGGAAAGGGGATGCCTTTGGAGTG 1762 *********************** **** ********************* GPH10_ananassa_clone2 CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1729 GPH10_ananassa_clone20 CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1872 GPH10_ananassa_clone18 CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1813 GPH10_ananassa_clone19 CAAGTTTTGTGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTA 1812 **************************************************

PAGE 170

170 GPH10_ananassa_clone2 GAGGTGATGATCGCTTGTTTTCGGTGGAAGGCATGAGCTCGCAGACAAGA 1779 GPH10_ananassa_clone20 GAGGTGATGATCGCTTGTTTTCGGTGGAAGGCATGAACTCGCAGACAAGA 1922 GPH10_ananassa_clone18 GAGGTGATGATGGCTTGTTTTCGGTGGAAGGCATGAACTCGCAGACAAGA 1863 GPH10_ananassa_clone19 GAGGTGATGATGGCTTGTTTTCGGTGGAAGGCATGAACTCGCAGACAAGA 1862 *********** ************************ ************* GPH10_ananassa_clone2 GGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAATGGAGTGTC 1829 GPH10_ananassa_clone20 GGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAATGGAGTGTC 1972 GPH10_ananassa_clone18 GGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAACGGAGTGTT 1913 GPH10_ananassa_clone19 GGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAACGGAGTGTT 1912 ***************************************** ******* GPH10_ananassa_clone2 GAAGATTCAGGTTAAGGCTTTGCAGAAGGATAGTGAGTTTGGGAAATTTA 1879 GPH10_ananassa_clone20 GAAGATTCAGGTTAAGGCTTTGCAGAAGGATAGTGAGTTTGGGAAATTTA 2022 GPH10_ananassa_clone18 GAAGATTCAGGTTAAGGCTTTGCAGAAGGATAGTGAGTTTGGGAAATTTA 1963 GPH10_ananassa_clone19 GAAGATTCAGGTTAAGGCTTTGCAGAAGGATAGTGAGTTTGGGAAATTTA 1962 ************************************************** GPH10_ananassa_clone2 AGGGGGAATTGTCGGATCTGATTCTGGTCTATGAAGTTTCAGGAAAAGAT 1929 GPH10_ananassa_clone20 AGGGGGAATTGTCGGATCTGATTCTGGTCTATGAAGTTTCAGGAAAAGAT 2072 GPH10_ananassa_clone18 AGGGGGAATTGTCGGATCCGATTCTGGTCTATGAAGTTTCAGGAAAAGAT 2013 GPH10_ananassa_clone19 AGGGGGAATTGTCGGATCCGATTCTGGTCTATGAAGTTTCAGGAAAAGAT 2012 ****************** ******************************* GPH10_ananassa_clone2 GGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCT 1979 GPH10_ananassa_clone20 GGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAGTGCT 2122 GPH10_ananassa_clone18 GGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCT 2063 GPH10_ananassa_clone19 GGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCT 2062 ********************************************* **** GPH10_ananassa_clone2 ATCA-GTGGAATTGGGTGAGTTTGTACCGAGGAAATTGCCACCTGTTTGG 2028 GPH10_ananassa_clone20 ATCAAGTGAAATTGGGTGAGTTTGTACCGAGGAAATTGCCACCTGTTTGG 2172 GPH10_ananassa_clone18 ATCAAGTGGAATTGGGTGAGTTTGTACCAAGGAAATTGCCACCTGTTTGG 2113 GPH10_ananassa_clone19 ATCA-GTGGAATTGGGTGAGTTTGTACCAAGGAAATTGCCACCTGTTTGG 2111 **** *** ******************* ********************* GPH10_ananassa_clone2 CAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTTTC 2078 GPH10_ananassa_clone20 CAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTTTC 2222 GPH10_ananassa_clone18 CAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTGTC 2163 GPH10_ananassa_clone19 CAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTGTC 2161 *********************************************** ** GPH10_ananassa_clone2 TATTTCAAATGATAGTGGTGTATCACATACTGGAATC CTTAAG CCTTTTA 2128 GPH10_ananassa_clone20 TATTTCAAATGATAGTGGTGTATCACATACTGGAATC CTTAAG CCTTTTA 2272 GPH10_ananassa_clone18 TATTTCAAATGATAGTGGTGTATCACATACTGGAATC CTTAAG CCTTTTA 2213 GPH10_ananassa_clone19 TATTTCAAATGATAGTGGTGTATCACATACTGGAATC CTTAAG CCTTTTA 2211 ************************************************** GPH10_ananassa_clone2 CAGTTTCTTCAGCTCTTATTTTTGTTATGGATGAAG GAATTC ACCCTCAT 2178 GPH10_ananassa_clone20 CAGTTTCTTCAGCTCTTATTTTTGTTATGGATGAAG GAATTC ACCCTCAT 2322 GPH10_ananassa_clone18 CAGTTTCTTCAGCTCTTATTTTTGTTATGGATGAAG GAATTC ACCCTCAT 2263 GPH10_ananassa_clone19 CAGTTTCTTCAGCTCTTATTTTTGTTATGGATGAAG GAATTC ACCCTCAT 2261 ************************************************** GPH10_ananassa_clone2 AAAAAAGGGCATGGCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAAA 2228 GPH10_ananassa_clone20 AAAAAAGGGCATGGCATTGGTGCAGAGAATAAGGGTCAGTCTCGTCCAAA 2372 GPH10_ananassa_clone18 AAAAAAGGGCATGTCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAAA 2313 GPH10_ananassa_clone19 AAAAAAGGGCATGTCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAAA 2311 ************* *********** ************************ GPH10_ananassa_clone2 GATGAAGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTG 2278 GPH10_ananassa_clone20 GATGAAGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTG 2422 GPH10_ananassa_clone18 GATGAAGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTG 2363 GPH10_ananassa_clone19 GATGAAGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTG 2361 **************************************************

PAGE 171

171 GPH10_ananassa_clone2 GGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGAGATTGAT 2328 GPH10_ananassa_clone20 GGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGAGATTGAT 2472 GPH10_ananassa_clone18 GGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGAGATTGAT 2413 GPH10_ananassa_clone19 GGTCGCAAACTGGGCCTTCACCATCTAATAAGCATTCTGCTGAGATTGAT 2411 ************************************************** GPH10_ananassa_clone2 GGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCAC 2378 GPH10_ananassa_clone20 GGAAAGAAAAAAAGTAGCGAAAGAAGTTCACATTCACTCAAAGATCTCAC 2522 GPH10_ananassa_clone18 GGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCAC 2463 GPH10_ananassa_clone19 GGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCAC 2461 ****************** ******************************* GPH10_ananassa_clone2 CTGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGG 2428 GPH10_ananassa_clone20 CCGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGG 2572 GPH10_ananassa_clone18 CTGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGG 2513 GPH10_ananassa_clone19 CTGGAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGG 2511 ************************************************ GPH10_ananassa_clone2 AAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAAATTT 2478 GPH10_ananassa_clone20 AAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAAATTT 2622 GPH10_ananassa_clone18 AAGAGGCTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAAATTT 2563 GPH10_ananassa_clone19 AAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAAAAAAGTTGAAATTT 2561 ****** ******************************************* GPH10_ananassa_clone2 CTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGA 2528 GPH10_ananassa_clone20 CTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGA 2672 GPH10_ananassa_clone18 CTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGA 2613 GPH10_ananassa_clone19 CTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGA 2611 ************************************************** GPH10_ananassa_clone2 GGAATCTAAGGTGCACCAGGAAAAACAAAAGGAGATGAGCAATAGGTTGG 2578 GPH10_ananassa_clone20 GGAGTCTAAGGTGCACCAGGAAAAACAAAAGGAGATGAGCAATAGGTTGG 2722 GPH10_ananassa_clone18 GGAGTCTAAGGTGCACCAGGAAAAACAAAAGGAGATGAGCAATAGGTTGG 2663 GPH10_ananassa_clone19 GGAGTCTAAGGTGCACCAGGAAAAACAAAAGGGGATGAGCAATAGGTTGG 2661 *** **************************** ***************** GPH10_ananassa_clone2 ATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCGTCATCTGGTTCAGCT 2628 GPH10_ananassa_clone20 ATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCGTCATCTGGTTCAGCT 2772 GPH10_ananassa_clone18 ATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCATCATCTGGTTCAGCT 2713 GPH10_ananassa_clone19 ATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCATCATCTGGTTCAGCT 2711 ********************************** *************** GPH10_ananassa_clone2 GGAGAAATTTCTTTCCCTGTGGCCTTTGGAGTACAGGATGAAGCTGCTCA 2678 GPH10_ananassa_clone20 GGAGAAATTTCTTTCCCTGTCGCCTTTGGAGTACAGGATGAAGCTGCTCA 2822 GPH10_ananassa_clone18 GGAGAAATTTCTTTCTCTGCGGCCTTTGGAGTACAGGATGAAGCTGCTCA 2763 GPH10_ananassa_clone19 GGAGAAATTTCTTTCTCTGCGGCCTTTGGAGTACAGGATGAAGCTGCTCA 2761 *************** *** ***************************** GPH10_ananassa_clone2 GGAACATAGATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATA 2728 GPH10_ananassa_clone20 GGAACATAGATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATA 2872 GPH10_ananassa_clone18 GGAACATAGATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATA 2813 GPH10_ananassa_clone19 GGAACATAGATTACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATA 2811 ************************************************** GPH10_ananassa_clone2 AGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCGCA 2778 GPH10_ananassa_clone20 AGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCACA 2922 GPH10_ananassa_clone18 AGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCGCA 2863 GPH10_ananassa_clone19 AGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTGGGGGCATTCGCA 2861 *********************************************** ** GPH10_ananassa_clone2 CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCAC 2828 GPH10_ananassa_clone20 CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCAC 2972 GPH10_ananassa_clone18 CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAGCATAGCTC 2913 GPH10_ananassa_clone19 CATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAGCATAGCTC 2911 ***************************************** ******

PAGE 172

172 GPH10_ananassa_clone2 AACAACCCCTTCAGAAGATCAAACTCCTGTAAAATATGACAATCTTGATG 2878 GPH10_ananassa_clone20 AACAACCCCTTCAGAAGATCAAACTCCTGTAAAATCTGACAATCTTGATG 3022 GPH10_ananassa_clone18 AACAACCCCTTCAGAAGATCAAACTCCTGTAAAATCTGACAATCTTGATG 2963 GPH10_ananassa_clone19 AACAGCCCCTTCAGAAGATCAAACTCCTGTAAAATCTGACAATCTTGATG 2961 **** ****************************** ************** GPH10_ananassa_clone2 ATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGAT 2928 GPH10_ananassa_clone20 ATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGAT 3072 GPH10_ananassa_clone18 ATTTGGTTACTGCTGAGCTGTTAAAACTTT-ACTCAGAGATCCCAAGGAT 3012 GPH10_ananassa_clone19 ATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGAT 3011 ****************************** ******************* GPH10_ananassa_clone2 ATGGTTGCCAGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCC 2978 GPH10_ananassa_clone20 ATGGTTGCCAGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCC 3122 GPH10_ananassa_clone18 ATGGTTGCCAGGCACAAAAGCTATGATCCATCTTCTCAAGCATCTGATCC 3062 GPH10_ananassa_clone19 ATGGTTGCCAGGCACAAAAGCTATGATCCATCTTCTCAAGCATCTGATCC 3061 *************************** ********************** GPH10_ananassa_clone2 TGGATGTGAAGGCTTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3028 GPH10_ananassa_clone20 TGGATGTGAAGGCTTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3172 GPH10_ananassa_clone18 TGGATGTGATGGCTTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3112 GPH10_ananassa_clone19 TGGATGTGATGGCTTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3111 ********* **************************************** GPH10_ananassa_clone2 TTTCTCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3078 GPH10_ananassa_clone20 TTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3222 GPH10_ananassa_clone18 TTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3162 GPH10_ananassa_clone19 TTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3161 *** ********************************************** GPH10_ananassa_clone2 ACTTTAATGCTATGTAAACTTTGCCCCTTGTTAGTGTTACACTTTTCCTT 3128 GPH10_ananassa_clone20 ACTTTAATGCTATGTAAACTTTGCCCCTTGTTACTGTTACACTTTTCCTT 3272 GPH10_ananassa_clone18 ACTTTAATGCTATGTAAACTTTGCCCCCTGTTACTGTTACACT--TCCTT 3210 GPH10_ananassa_clone19 ACTTTAATGCTATGTAAACTTTGCCCCCTGTTACTGTTACACT--TCCTT 3209 *************************** ***** ********* ***** GPH10_ananassa_clone2 CACTAGCACAAAGATATGAATTACAGATACTTTTCCGGATGGAGATTTTA 3178 GPH10_ananassa_clone20 CACTAGCACAAAGATATGAATTACAGATACTTTTCCGGATGGAGATTTTA 3322 GPH10_ananassa_clone18 CACTAGCACAAAGATATGAATTACAGATACTTTTCCGGATGGAGATTTTA 3260 GPH10_ananassa_clone19 CACTAGCACAAAGATATGAATTACAGATACTTTTCCGGATGGAGATTTTA 3259 ************************************************** GPH10_ananassa_clone2 CAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGT 3228 GPH10_ananassa_clone20 CAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGT 3372 GPH10_ananassa_clone18 CAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGT 3310 GPH10_ananassa_clone19 CAATCAGAAGTTGGGGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGT 3309 ************** *********************************** GPH10_ananassa_clone2 GAAACATATTTGCACGCTTTTGGAGACCATTCG-TGCTCGGTGTCATCTG 3277 GPH10_ananassa_clone20 GAAACATATTTGCACGCTTTTGGAGACCATTCG-TGCTCGGTGTCATCTG 3421 GPH10_ananassa_clone18 GAAACATATTTGCACGCTTTTGGAGACCATTCG-TGCTCAGTGTCATCTG 3359 GPH10_ananassa_clone19 GAAACATATTTGCACGCTTTTGGAGACCATTCGGTGCTCAGTGTCATCTG 3359 ********************************* ***** ********** GPH10_ananassa_clone2 GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3327 GPH10_ananassa_clone20 GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3471 GPH10_ananassa_clone18 GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3409 GPH10_ananassa_clone19 GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3409 ************************************************** GPH10_ananassa_clone2 TATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAAC 3377 GPH10_ananassa_clone20 TATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAAC 3521 GPH10_ananassa_clone18 TATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAAC 3459 GPH10_ananassa_clone19 TATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAAC 3459 **************************************************

PAGE 173

173 GPH10_ananassa_clone2 TATTATTTTCTAATGAAATT-GTATTCATGAACACTGAAATGGTAGATAC 3426 GPH10_ananassa_clone20 TATTATTTTCTAATGAAATTTGTATTCATGAACACTGAAATGGTAGATAC 3571 GPH10_ananassa_clone18 TATTATTTTCTAATGAAATTTGTATTCATGAACACTGAAATGGTAGATAC 3509 GPH10_ananassa_clone19 TATTATTTTCTAATGAAATTTGTATTCATGAACACTGAAATGGTAGATAC 3509 ******************** ***************************** GPH10_ananassa_clone2 TCAGTTATTTACAATGAAACTCCAATATATGTTTATGGTTTGCCTGTTAA 3476 GPH10_ananassa_clone20 TCAGTTATTTACAATGAAACTCCAATATATGTTTATGGTTTGCCTGTTAA 3621 GPH10_ananassa_clone18 TCAGTTATTTACAATGAAACTCCAGTATATGTTTATGTTTTGCCTGTTAA 3559 GPH10_ananassa_clone19 TCAGTTATTTACAATGAAACTCCAATATATGTTTATGTTTTGCCTGTTAA 3559 ************************ ************ ************ GPH10_ananassa_clone2 TGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTAT 3526 GPH10_ananassa_clone20 TGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTAT 3671 GPH10_ananassa_clone18 TGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTAT 3609 GPH10_ananassa_clone19 TGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTAT 3609 ************************************************** GPH10_ananassa_clone2 GTGATTGATTTGTATGCCCTCCCAAAAGGCCTTTGGGGGTAGTATGAAGA 3576 GPH10_ananassa_clone20 GTGATTGATTTGTATGCCCTCCCAAAAGGCCTTTGGGGGTAGTATGAAGA 3721 GPH10_ananassa_clone18 GTGATTGATTTGTATGCCCTCCCAAATGGCCTTTGGGGGTAGTAAGAAGA 3659 GPH10_ananassa_clone19 GTGATTGATTTGTATGCCCTCCCAAATGGCCTTTGGGGGTAGTAAGAAGA 3659 ************************** ***************** ***** GPH10_ananassa_clone2 AGGGAGACATTGACAGTCAAAAATATTATCTCCTTATTTTACGTACAAAA 3626 GPH10_ananassa_clone20 AGGGAGACATTGACCGTCAAAACTATTATCTCCTTATTTTACGTACAAAA 3771 GPH10_ananassa_clone18 AGGGAGACATTGACAGTCAAAAATATTATCTCCTTATTTTACGTACAAAA 3709 GPH10_ananassa_clone19 AGGGAGACATTGACAGTCAAAAATATTATCTCCTTATTTTACGTACAAAA 3709 ************** ******* *************************** GPH10_ananassa_clone2 TTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAA 3676 GPH10_ananassa_clone20 TTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAA 3821 GPH10_ananassa_clone18 TTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAA 3759 GPH10_ananassa_clone19 TTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAA 3759 ************************************************** GPH10_ananassa_clone2 AAGCTAAATACCTCCTTTGCATAATTTCATATGA CTTAAG TGACTTTCCT 3726 GPH10_ananassa_clone20 AAGCTAAATACCTCCTTTGCATAATTTCATATGA CTTAAG TGACTTTCCT 3871 GPH10_ananassa_clone18 AAGCTAAATACCTCCTTTGCATAATTTCATATGA CTTAAG TGACTTTCCT 3809 GPH10_ananassa_clone19 AAGCTAAATACCTCCTTTGCATAATTTCATATGA CTTAAG TGACTTTCCT 3809 ************************************************** GPH10_ananassa_clone2 TATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATGCATACA 3776 GPH10_ananassa_clone20 TATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATGCATACA 3921 GPH10_ananassa_clone18 TATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATTCATACA 3859 GPH10_ananassa_clone19 TATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATTCATACA 3859 ******************************************* ****** GPH10_ananassa_clone2 ACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTC 3826 GPH10_ananassa_clone20 ACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTC 3971 GPH10_ananassa_clone18 ACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTC 3909 GPH10_ananassa_clone19 ACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTC 3909 ************************************************** GPH10_ananassa_clone2 CAGACCCAAAAAAAACTGCCCAAATTTATGTGAAAACACTGCATTTATGT 3876 GPH10_ananassa_clone20 CAGACCCAAAAAAAACTGCCCAAATTTATGTGAACACACTGCATTTATGT 4021 GPH10_ananassa_clone18 CAGACCCAAAAAAAACGGCCCAAATTTATGTGAAAACACTGCATTTATGT 3959 GPH10_ananassa_clone19 CAGACCCAAAAAAAACGGCCCAAATTTATGTGAAAACACTGCATTTATGT 3959 **************** ***************** *************** GPH10_ananassa_clone2 TTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTT 3926 GPH10_ananassa_clone20 TTGAAG---TAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTT 4068 GPH10_ananassa_clone18 TTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTT 4009 GPH10_ananassa_clone19 TTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTT 4009 ****** *****************************************

PAGE 174

174 GPH10_ananassa_clone2 TCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAA 3976 GPH10_ananassa_clone20 TCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAA 4118 GPH10_ananassa_clone18 TCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAA 4059 GPH10_ananassa_clone19 TCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAA 4059 ************************************************** GPH10_ananassa_clone2 ATGGATTTGTTACTGTTTGATGATGAGGAAGAACTCCCTAATAATTTATT 4026 GPH10_ananassa_clone20 ATGGATTTGTTACTGTTTGATGATGAGGAAGAACTCCCTAATAATTTATC 4168 GPH10_ananassa_clone18 ATGGATTTGTTACTGTTTGATGATGAGGAAGAACTCCCTAATAATGTATT 4109 GPH10_ananassa_clone19 ATGGATTTGTTACTGTTTGATGATGAGGAAGAACTCCCTAATAATGTATT 4109 ********************************************* *** GPH10_ananassa_clone2 CAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATG 4076 GPH10_ananassa_clone20 CAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATG 4218 GPH10_ananassa_clone18 CAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATG 4159 GPH10_ananassa_clone19 CAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATG 4159 ************************************************** GPH10_ananassa_clone2 AGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAA 4126 GPH10_ananassa_clone20 AGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAA 4268 GPH10_ananassa_clone18 AGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAA 4209 GPH10_ananassa_clone19 AGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAA 4209 ************************************************** GPH10_ananassa_clone2 TCCCCTGATCCACAGAAACATTACAATGGAAGGCCAAGTGCTCAAGTACT 4176 GPH10_ananassa_clone20 TCCCCTGATCCACAGAAACATTACAATGGAAGGCCAAGTGCTCAAGTAGT 4318 GPH10_ananassa_clone18 TCCCCTGATCCACAGAAACATTACAATGGAAGGCCAAGTGCTCAAGTAGT 4259 GPH10_ananassa_clone19 TCCCCTGATCCACAGAAGCATTACAATGGAAGGCCAAGTGCTCAAGTAGT 4259 ***************** ****************************** GPH10_ananassa_clone2 TAAACAAGAAGAGCATGCTCGCAAGTTGATGAAAGCTCAAGAGAGTAGAG 4226 GPH10_ananassa_clone20 TAAACAAGAAGAGCATGCTCGCAAGTTGATGAAAGCTCAAGAGAGTAGAG 4368 GPH10_ananassa_clone18 TAAACAAGAAGAGCATGCTCGCAAGTTGATGGAAGCTCAAGAGAGTAGAG 4309 GPH10_ananassa_clone19 TAAACAAGAAGAGCATGCTCGCAAGTTGATGGAAGCTCAAGAGAGTAGAG 4309 ******************************* ****************** GPH10_ananassa_clone2 AGAGGGCTTGGAGAATTGCTTCTTTCACAAGTCGGGTAGCTGATTTGCAG 4276 GPH10_ananassa_clone20 AGAGGGCTAGGAGAATTGCTTCTTTCACAAGTCGGGTAGCTGATTTGCAG 4418 GPH10_ananassa_clone18 AGAGAGCTAGGAGAATTGCTTCTTTTACAAGTCGGGTAGCTGATTTGCAG 4359 GPH10_ananassa_clone19 AGAGAGCTAGGAGAATTGCTTCTTTTACAAGTCGGGTAGCTGATTTGCAG 4359 **** *** **************** ************************ GPH10_ananassa_clone2 CGAG 4280 GPH10_ananassa_clone20 CGAG 4422 GPH10_ananassa_clone18 CGAG 4363 GPH10_ananassa_clone19 CGAG 4363 **** Polymorphic fragments of GP H10: clones from octoploid versus clones from diploids. SSRs detected are magenta-colored. 10PPR1AB22_nubicola AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAA 50 10PPR1AB22_mandshurica AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAA 50 10PPR1AB22_vesca AACGGAGAAGAAGACTGTCGACATTTTAAGAGAAAGCTTTCAGCTTTGAA 50 10PPR1AB22_viridis AACGGAGAAGAAGACTGTCGACATTTCTAGAGAAAGCTTTCAGCTTTGAA 50 10PPR1AB22_nilgerrensis AACGGAGAAGAAGACTGTCGACATATCTAGAGAAAGCTTTCAGCTTTGAA 50 10PPR1AB22_ananassa_clone18 AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAA 50 10PPR1AB22_ananassa_clone20 AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAA 50 10PPR1AB22_ananassa_clone19 AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTTAGCTTTGAA 50 10PPR1AB22_ananassa_clone2 AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCATCTTTGAA 50 10PPR1AB22_iinumae AACGGAGAAGAAGACTGTCGACATTTTTAGAGAAAGCTTTCAGCTTTGAA 50 ************************ *********** *******

PAGE 175

175 10PPR1AB22_nubicola GT-----G-----TAGGATAATAACAAAGAAACTCGTTATCTGAAAGACA 90 10PPR1AB22_mandshurica GT-----G-----TAGGATAATAACAAAGAAACTCGTTATCTGAAAGACA 90 10PPR1AB22_vesca GT-----G-----TAGGATAATAACAAAGAAACTCGTTATCTGAAAGACA 90 10PPR1AB22_viridis GT-----G-----TAGGATAATAACAAAGAAACTCGTTATCTGAAAGACA 90 10PPR1AB22_nilgerrensis GT-----GGAGTGTAGGATAATAAC----AAACTCGTTATCTGAAAGACA 91 10PPR1AB22_ananassa_clone18 GT-----GGAGTGTAGGATAATAAC----AAACTCGTTATCTAAAAGACA 91 10PPR1AB22_ananassa_clone20 GT-----GGAGTGTAGGATAATAAC----AAACTCGTGAT-TAAAAGACA 90 10PPR1AB22_ananassa_clone19 GT-----GGAGTGTAGGATAATAAC----AAACTCGTTATCTAAAAGACA 91 10PPR1AB22_ananassa_clone2 GT-----GGAGTGTAGGATAATAAC----AAACTCGTTATCTAAAAGGCA 91 10PPR1AB22_iinumae CTTTGAAGTAGTGTAGGATAATAAC----AAACTCGTTATCTAAAAGACA 96 ************ ******** ** **** ** 10PPR1AB22_nubicola GGTTTAATATCAGC-------------------CGTTGGATCATA---TT 118 10PPR1AB22_mandshurica GGTTTAATATCAGC-------------------CGTTGGATCATA---TT 118 10PPR1AB22_vesca AGTTTAATATCAGC-------------------CGTTGGATCATA---TT 118 10PPR1AB22_viridis AGTTTAATACCAGC-------------------CGTTGGATCATA---TT 118 10PPR1AB22_nilgerrensis GGTTTAATATCAGC-------------------CGTTGGATTATA---TT 119 10PPR1AB22_ananassa_clone18 GGTTTAATATCGGC-------------------CGTTAGATCACA---TT 119 10PPR1AB22_ananassa_clone20 GGATTAATGTCAGTGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTT 140 10PPR1AB22_ananassa_clone19 GGTTTAATATCAGC-------------------CGTTAGATCATA---TT 119 10PPR1AB22_ananassa_clone2 GGTTTAATATCAGC-------------------CGTTAGATCATA---TT 119 10PPR1AB22_iinumae GGTTTAATATCAGC-------------------CGTTAGATCCTA---TT 124 ***** *** 10PPR1AB22_nubicola ACGGCCCTG-------ATCGCTCGACATA--------------------140 10PPR1AB22_mandshurica ACGGCCCTG-------ATCGCTCGACATA--------------------140 10PPR1AB22_vesca ACGGCCCTG-------ATCGCTCGACATA--------------------140 10PPR1AB22_viridis ACTGCCCTG-------ATCGCTCGACATA--------------------140 10PPR1AB22_nilgerrensis CCGGCCCTG-------ATCTCTCGACATA--------------------141 10PPR1AB22_ananassa_clone18 ACGGCCCTG-------ATCACTCGACATATGTTGA-TATACGCCTAACT160 10PPR1AB22_ananassa_clone20 AAGGTCATAGGTTCAAACCTCACGACATATGTAGGGTGTATGAATTATTA 190 10PPR1AB22_ananassa_clone19 ACGGCCCTG-------ATCACTCGACATATGTTGA-TATACGCCTAACT160 10PPR1AB22_ananassa_clone2 ACGGCCCTG-------ATCACT--------------------------134 10PPR1AB22_iinumae --------------------------------------------------10PPR1AB22_nubicola -------------------------------------------------A 141 10PPR1AB22_mandshurica -------------------------------------------------A 141 10PPR1AB22_vesca -------------------------------------------------A 141 10PPR1AB22_viridis -------------------------------------------------A 141 10PPR1AB22_nilgerrensis -------------------------------------------------T 142 10PPR1AB22_ananassa_clone18 --------CAAATTCGATAT-------------ATATT-----------177 10PPR1AB22_ananassa_clone20 ATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACGGCCTGATCA 240 10PPR1AB22_ananassa_clone19 --------CAAATTCGATAT-------------ATATT-----------177 10PPR1AB22_ananassa_clone2 -------------------------------------------------10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola TTCGA TATATATATATA ------TTATTTTTTTCTAAA----------AA 175 10PPR1AB22_mandshurica TTCGA TATATATATA --------TTATTTTTTTCTAAA----------AA 173 10PPR1AB22_vesca TTCGA TATATATATATATATA TTTTTTTTTTTTCTAAA----------AA 181 10PPR1AB22_viridis TTCGA TATATATATATATA ----TTATTTTTTTCTAAA----------AA 177 10PPR1AB22_nilgerrensis GTTGATATACGC---------------------CTGAC----------TC 161 10PPR1AB22_ananassa_clone18 TTCGATATACA------TTTTTTTTTTAAGTAACTAAATGACTATTCGAT 221 10PPR1AB22_ananassa_clone20 CTCGACATATGTTGATATAC-------GCCCAACTCAA-----ATTCGAT 278 10PPR1AB22_ananassa_clone19 TTCGATATACA------TTTTTTTTTTAAGTAACTAAATGACTATTCGAT 221 10PPR1AB22_ananassa_clone2 --CGACATATGTTGATATAC-------GCCCAACTCAA-----ATTCGAT 170 10PPR1AB22_iinumae -------------------------------------------------

PAGE 176

176 10PPR1AB22_nubicola AAAAAA--TCGATATACAGTATATT----TTTTTTGAATTAATTAAATAA 219 10PPR1AB22_mandshurica AAAAAAAATCGATATACAGTATATT---TTTTTTTGAATTAATTAAATGA 220 10PPR1AB22_vesca AAAAAA--TCGATATACAGTATATT----TTTTTTGAATTAATTAAATGA 225 10PPR1AB22_viridis AAATAAA-TCGATATACAGTATATT---TTTTTTTGAAGTAATTAAATGA 223 10PPR1AB22_nilgerrensis AAAT----TCTATATACA------------TTTTCGAAAGAATT--TTTG 193 10PPR1AB22_ananassa_clone18 ATATATTTTCGATATACA-TTT------TTTTTTTAAAGTAACTAAATGA 264 10PPR1AB22_ananassa_clone20 ATATATTTTCGATATACG-TATATTTTATTTTTTTAAAATAATTAAATAA 327 10PPR1AB22_ananassa_clone19 ATATATTTTCGATATACA-TTT------TTTTTTTAAAGTAACTAAATGA 264 10PPR1AB22_ananassa_clone2 ATATATTTTCGATATACA-TATATTTTATTTTTTTAAAGTAACTAAATGA 219 10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola GTATTTAGATCGCTTAAAAAGA-TAAACAATTGAAGTTGGTTTAGAAGCA 268 10PPR1AB22_mandshurica GTATTTAGATCGCTTAAAAAAAATAAACAATCGAAGTTGAATTAGGAGCA 270 10PPR1AB22_vesca GTATTTAGATCGCTTAAAAAGA-TAAACAATCGAAATTGGTTTAAGAACA 274 10PPR1AB22_viridis TTATTTAAATCGCTTAAAAAGA-TAAACAA--GAAGTTGGTTTAGGAGCA 270 10PPR1AB22_nilgerrensis TTGTTGAAGTAACT--AAATGACTATACGATTGAAGATAGATTAAGAGAA 241 10PPR1AB22_ananassa_clone18 CTATTTACGTCGGTTAATAAAA-GAAACAATTGAAGTTAAATTAAGAGCA 313 10PPR1AB22_ananassa_clone20 CTATTTACGTTGTTTAACAAAA-GAAACAATTGAAGTTAAATTAAGAGCA 376 10PPR1AB22_ananassa_clone19 CTATTTACGTCGGTTAATAAAA-GAAACAATTGAAGTTAAATTAAGAGCA 313 10PPR1AB22_ananassa_clone2 CTATGTACATCGTTTAACAAAA-GAAACAATTGAAGTTAAATTAAGAGCA 268 10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola TCATAGGAGC------AAGAGTACGAGAACAAAAGTATGAGCTACACTGT 312 10PPR1AB22_mandshurica CCATAGGAGC------AAGAGTATGAGAACAAAAGTATGAGCTACATTGT 314 10PPR1AB22_vesca CCATAGGAGC------AAGAGTATGAGAACAAAAGTATGAGCTACACTGT 318 10PPR1AB22_viridis CCATAGGAGC------AAGAGTATGAGAACAAAAGTATGAGCCACACTGT 314 10PPR1AB22_nilgerrensis ACATAGCAACTGAGTAAAAAGTATGAGAACAAAAGTATGAGCTACATTGT 291 10PPR1AB22_ananassa_clone18 CCATGACAG----------AGTACGAGAACAAAAGTATGAGCTACATTGT 353 10PPR1AB22_ananassa_clone20 CCGTAACAGCTGAGC-AAGAGTACGAGAACAAAAGTATGAGCTACATCAT 425 10PPR1AB22_ananassa_clone19 CCATGACAG----------AGTACGAGAACAAAAGTATGAGCTACATTGT 353 10PPR1AB22_ananassa_clone2 CCATAACAGCTGAGA-AAGAGTACGAGAACAAAAGTATGAGCTAAAACAA 317 10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola TTGCTCGTCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATA 362 10PPR1AB22_mandshurica TTGCTCGTCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATA 364 10PPR1AB22_vesca TTGCTCATCGGTTTATTTATATGGAGAAAATATCAAGGTGATGTTGTATA 368 10PPR1AB22_viridis TTGCTCTTCGGTTTGTTTATACAGAGAAAATATAAAAGTGATGTTGTAGA 364 10PPR1AB22_nilgerrensis TTGCTCCTCGGTCTGTTTATATGGAGAAAATATAAATGTGATGTTGTAAA 341 10PPR1AB22_ananassa_clone18 TTGCTCGTCGGTTTGTTCATATGGAGAAAATGTAGAGGCGATGTTGTAGA 403 10PPR1AB22_ananassa_clone20 ------------TTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAGA 463 10PPR1AB22_ananassa_clone19 TTGCTCGTCGGTTTGTTCATATGGAGAAAATATAGAGGCGATGTTGTAGA 403 10PPR1AB22_ananassa_clone2 --------------------ATAGAGAAAATATAGAGGCGATGTTGTAGA 347 10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola AACAATTAAACATTACAAAA--TCAAATTACTTAACAATGAACCATCTTC 410 10PPR1AB22_mandshurica AACAATTAAACATTACAAAA--TCAAATTACTTAACAATAAACCATCTTC 412 10PPR1AB22_vesca AACAATTAAACATTACAAAA--TCAAATTACCTAACAATGAACCATTTTC 416 10PPR1AB22_viridis AACAATTGAACACTAAAAAA--TCAAATTACCTAACAACGAACCATCTTC 412 10PPR1AB22_nilgerrensis AATAATTGAACATTAAAAAAAATTAAATTATCTAATAACGAATCATCTTT 391 10PPR1AB22_ananassa_clone18 AATAATTGAACATTAGAAAA--TTAAATTACCTAA--------------436 10PPR1AB22_ananassa_clone20 AATAATTGAACATTAGAAAA--TTAAATTACCTAA--------------496 10PPR1AB22_ananassa_clone19 AATAATAGAACATTAGAAAA--TTAAATTACCTAA--------------436 10PPR1AB22_ananassa_clone2 AATAATTGAACATTAGAAAA--TTAAATTACCTAA--------------380 10PPR1AB22_iinumae -------------------------------------------------

PAGE 177

177 10PPR1AB22_nubicola AGACATGT----AAAATCAGAAAGTTAAAAGGTTCGAGTCGCATATGAGT 456 10PPR1AB22_mandshurica AGACATGT----AAAATCAAAAAGTTAAAAGGTTCGAGTCGCATATGAGT 458 10PPR1AB22_vesca AGACATGT----AAAATCATAAAATTAAAAGGTTCGAGTCGCATATGAGT 462 10PPR1AB22_viridis AGACATAC----AAGATCAGAAAGTTAAGAGGTTCGAGTCGCACATGAGT 458 10PPR1AB22_nilgerrensis AGACGTACGTACAAAATCAGAGAGTTAAGAGATTCGAGTTGC-------433 10PPR1AB22_ananassa_clone18 -------------------------------------------------10PPR1AB22_ananassa_clone20 -------------------------------------------------10PPR1AB22_ananassa_clone19 -------------------------------------------------10PPR1AB22_ananassa_clone2 -------------------------------------------------10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola TTGTCGAGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGT 506 10PPR1AB22_mandshurica TTGTCGAGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGT 508 10PPR1AB22_vesca TTGTCGAGCTGATCAAATACCACAGTTTACTTGACTGAACAAACTTACGT 512 10PPR1AB22_viridis TTCTTGAGGCGATCAAATACCACAGTTTACTTGACTCAACAACTTTACGC 508 10PPR1AB22_nilgerrensis ------------TCAAATACCATATTTTACTTGACTTAACAAT------464 10PPR1AB22_ananassa_clone18 -------------------------------------------------10PPR1AB22_ananassa_clone20 -------------------------------------------------10PPR1AB22_ananassa_clone19 -------------------------------------------------10PPR1AB22_ananassa_clone2 -------------------------------------------------10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola A-ACGAGTCAAACGAGCTAAAAACGAGTCGAATAAAAATCGGGCACCATC 555 10PPR1AB22_mandshurica A-ACGAGTCAAACGAGCTAAAAACGAGTCGAATAAAAATCGGGCACCATC 557 10PPR1AB22_vesca A-ACGAGTCAAACGAGCTAAAAACGAGTCGAATAAAAATCGGGCACCATC 561 10PPR1AB22_viridis ATACGAGTCAAACGAGCTAAAAACGAGTCGAATAAAAATCGGGCACCATC 558 10PPR1AB22_nilgerrensis --ACGAGTCAAACGAGCTAAAAACGAGTCGATTAAA-------------498 10PPR1AB22_ananassa_clone18 -------------------------------------------------10PPR1AB22_ananassa_clone20 -------------------------------------------------10PPR1AB22_ananassa_clone19 -------------------------------------------------10PPR1AB22_ananassa_clone2 -------------------------------------------------10PPR1AB22_iinumae -------------------------------------------------10PPR1AB22_nubicola TATATCGAGACTATGTAAGAGCCGAGGAGTAAAA--TAATAACAAACTCG 603 10PPR1AB22_mandshurica AATATCGAGACTATGTAAAAGCCGAGGAGTAAAA--TAATAACAAACTCG 605 10PPR1AB22_vesca AATATCGAGACTATGTAAGAGCCGAGGAGTAAAA--TAATAACAAACTCG 609 10PPR1AB22_viridis AATATCGAGACTATGTAAGAGCCGAGGAGTAAAAAATAATAACAAACTCG 608 10PPR1AB22_nilgerrensis -------------TCTAAGAGCCGAGGAGTAAAG--TAATAACAAACTCG 533 10PPR1AB22_ananassa_clone18 ------------------AAGCCGATGAGTAAAA--TAATAACAAACTCG 466 10PPR1AB22_ananassa_clone20 ------------------AAGCCGATGAGTAAAA--TAATAACGAACTCG 526 10PPR1AB22_ananassa_clone19 ------------------AAGCCGATGAGTAAAA--TAATAACAAACTCG 466 10PPR1AB22_ananassa_clone2 ------------------AAGCCGATGAGTAAAA--TAATAACGAACTCG 410 10PPR1AB22_iinumae ----------------AAGAGCCGAGGAGTAAAA--TAATAACAAAGTCG 156 ***** ******* ******* ** *** 10PPR1AB22_nubicola TTATCTAAAAGACAGGTTTAATATCAGCCCTTGGACCATATGTACGGGTG 653 10PPR1AB22_mandshurica TTATCTAAAAGACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTG 655 10PPR1AB22_vesca TTATCTAAAAGACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTG 659 10PPR1AB22_viridis TTATCTAAAAGACAGGTTTAATATCAGCCGTTGGACCATATGTACAGGTG 658 10PPR1AB22_nilgerrensis TTATCTAAAATACAGGTTTAATATCAGCCGTTGGATCATATATACAGGTG 583 10PPR1AB22_ananassa_clone18 TAACCTAAAAGC--GGCTTCATATCATCCACTGGATCATATATGCGGGTG 514 10PPR1AB22_ananassa_clone20 TAACCTAAAAGC--GGCTTCATATCATCCGCTTGATCATATATGCGGGTG 574 10PPR1AB22_ananassa_clone19 TAACCTAAAAGC--GGCTTCATATCATCCACTGGATCATATATGCGGGTG 514 10PPR1AB22_ananassa_clone2 TAACCTAAAAGC--GGCTTCATATCATCCGCTTGATCATATATGCGGGTG 458 10PPR1AB22_iinumae TAACCTAAAAGC--GGCTTCATATCATCTACTGGATCATATATGCGGGTG 204 ****** ** ** ****** ** ***** ****

PAGE 178

178 10PPR1AB22_nubicola TGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCCTTCC-AATTTTCAT 702 10PPR1AB22_mandshurica TGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCATTCCCAATTTTCAT 705 10PPR1AB22_vesca TGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCATTCCCAATTTTCAT 709 10PPR1AB22_viridis TGATTCGAAAACCGA-GTTAACCCGCCAAACCCTCCTTCCCAATTTT-AT 706 10PPR1AB22_nilgerrensis TGATTCGAAAACCGAAGTTAACCCGCCAAACCCTCATTCCCAATTTTCAT 633 10PPR1AB22_ananassa_clone18 TGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCAT 564 10PPR1AB22_ananassa_clone20 TGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCAT 624 10PPR1AB22_ananassa_clone19 TGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCAT 564 10PPR1AB22_ananassa_clone2 TGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCAT 508 10PPR1AB22_iinumae TGATTCGAAAACCAAAGTTAACCCGCCAAA--------CCCAATTTTCAT 246 ************* ************** ** ****** ** 10PPR1AB22_nubicola TTCCCACCAAAAACAAAACC 722 10PPR1AB22_mandshurica T-CCCACCAAAAACAAAACC 724 10PPR1AB22_vesca T-CCCACCAAAAACAAAACC 728 10PPR1AB22_viridis TTCCCACCAAAAACAAAACC 726 10PPR1AB22_nilgerrensis T-CCCACCAAAAACAAAACC 652 10PPR1AB22_ananassa_clone18 TTCCCACCAAAAACAAAACC 584 10PPR1AB22_ananassa_clone20 TTCCCACCAAAAATAAAAC643 10PPR1AB22_ananassa_clone19 TTCCCACCAAAAACAAAACC 584 10PPR1AB22_ananassa_clone2 TTCCCACCAAAAACAAAACC 528 10PPR1AB22_iinumae TTCCCACCAAAAACAAAACC 266 *********** ***** --------------------------------------------------------------------------------------------------------------11D02 11D02_viridis GAGCTGCTGTGTGA-CCAAATGGGTACAGAAGAAGCCNGTTTGCCAAACCTACCCATGAT 59 11D02_nubicola GAGCTGCTGTGTGA-CCAAATGG-TACAGA-GAAGCCNGTTTGCCAAACCTACCCATGAT 57 11D02_vesca GAGCTGCTGTGTGAACCAAATGG-TACAGA-GAAGCCG-TTTGCCAAACCTACCCATGAT 57 11D02_iinumae GAGCTGCTGTGTGAACCAAATGG-TACAGA-GAAGCCG-TTTGCCAAACCTACCCATGAT 57 ************** ******** ****** ****** ********************* 11D02_viridis CCAATCAA-TGCATAAACTTTAAGAACTCAAATACCAAACGATCAAACATAATGACTGAA 118 11D02_nubicola CCAATCAAATGCATAAACTTTAAGAACTCAAATACCAAACGATCAAACATAATGACTGAA 117 11D02_vesca CCAATCAAATGCATAAACTTTAAGAACTCAAATACCAAACGATCAAACATAATGACTGAA 117 11D02_iinumae CCAATCAAATGCATAAACTTTAAGAACTCAAATACCAAACGATCAAACATAATGACTGCA 117 ******** ************************************************* 11D02_viridis ATGAACAAAAATCAAATGAGCAAAGACTAAATGAGAAAACAGACCTCTTTGTAAACTGGG 178 11D02_nubicola ATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAAAACAGACCTCTTTGTAAACTGGG 177 11D02_vesca ATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAAAACAGACCTCTTTGTAAACTGGG 177 11D02_iinumae ATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAAAACAGACCTCTTTGTAAACTGGG 177 ****************** ***************************************** 11D02_viridis TTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCTGCCATTCCTGTT 238 11D02_nubicola TTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCAGCCATTCCTGTT 237 11D02_vesca TTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCAGCCATTCCTGTT 237 11D02_iinumae TTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCTGCCATTCCTGTT 237 *********************************************** ************ 11D02_viridis TATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCA----AACACAA 294 11D02_nubicola TATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAA 297 11D02_vesca TATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAA 297 11D02_iinumae TATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAA 297 ************************************************* ******* 11D02_viridis TTTGATACAGAAGTCTTCACACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAA 354 11D02_nubicola TTTCATACAGAAGTCTTCCCACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAA 357 11D02_vesca TTTCATACAGAAGTCTTCCCACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAA 357 11D02_iinumae TTTGATACAGAAGTCTTCACACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAA 357 *** ************** *****************************************

PAGE 179

179 11D02_viridis TGATAACTTTTCAATAGTATAAGGAGAAGATGAGGACAGTACCAGAGACTGCAGCTACTT 414 11D02_nubicola TGATAACTTTTCAATAGTATAAGAAGGAGATGAGGACAGTACCAGAGACTGCAGCTACTT 417 11D02_vesca TGATAACTTTTCAATAGTATAAGAAGGAGATGAGGACAGTACCAGAGACTGCAGCTACTT 417 11D02_iinumae TGATAACTTTTCAATACTATAAGAAGGAGATGAGGACAGTACCAGAGACTGCAGCTACTT 417 **************** ****** ** ********************************* 11D02_viridis TGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCCTAACTTGGTTTCCTTTCAGTGT 474 11D02_nubicola TGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTGGTTTCCTTTCAGCCT 477 11D02_vesca TGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTGGTTTCCTTTCAGTCT 477 11D02_iinumae TGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCTTAACTTGGTTTCC--------468 ******* ***************************** ********** 11D02_viridis CTTCGACCTTCTTCTAAAACGACGGAGTCGGTGAAACTGTGCAAGTCTTCTTGTGA---530 11D02_nubicola CTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCA 537 11D02_vesca CTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCA 537 11D02_iinumae --------------------GACGGAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCA 508 **** ******** ********************** 11D02_viridis ATTTTCTTTTCTAGGTGATTTTTTTTTTCTTTTATAATTAATTTGGTTTTATTTTTCCAA 590 11D02_nubicola ATTTTCTTTTCTAGGTGATTTTTTTT--CTTTTATAATTAATTTGGTTTTATTTTTCCAA 595 11D02_vesca ATTTTCTTTTCTAGGTGATTTTTTTT--CTTTTATAATTAATTTGGTTTTATTTTTCCAA 595 11D02_iinumae ATTTTCTTTTCTAGGTGTTTTTTTTT--CTTTTATAATTAATTTGGTTTTATTTTTCCAA 566 ***************** ******** ******************************** 11D02_viridis ATAATACCTGAAAGACTTTTTTTTTTTTTTTTTGATAGAAATACCTAAAAGACTTCATAA 650 11D02_nubicola ATAATACCTGAAAGACTTTTTTTTC--------GATAG---------------------625 11D02_vesca ATAATACCTGAAAGACTTTTTTTTT-------CGATAGGA-------------------628 11D02_iinumae ATAATACCTGAAAGACTTTTTTTTTTT-----CGATAGAAATACCTGTTAAGACT----616 ************************ ***** 11D02_viridis AAGCTGTTAAGGCTTCATTTAGGATTGCAGTAATTTTTTTTGGACAGTATTACGGGACAC 710 11D02_nubicola ----------------------GATTGCAGTAATTTTTTTTGGACAGTATTACGGGACAC 663 11D02_vesca ------------------------TTGCAGTAATTTTTTTTGGACAGTATTACGGGACAC 664 11D02_iinumae -------TAAGACTTCATTTAGTATTGCAGTAATTTTTTT-GGACAGTATTACGGGACAC 668 **************** ******************* 11D02_viridis TGTGACAGCTT--GAGTTTGAATCTTAGGTGGGATGATTTAAGTATCTTAGTTGAATGGA 768 11D02_nubicola TGTGACAGCTTTAGAGTTTGAATCTTAGGTTGGATGATTTAAGTATCTTAGTTGAATGGA 723 11D02_vesca TGTGACAGCTTTAGAGTTTGAATCTTAGGTTGGATGATTTAAGTATCTTAGTTGAATGGA 724 11D02_iinumae TG--ACAGCTTTAGAGTTTGAATCTTAGGTTGGATGATTTAAGTATCTTAGTTGAACGGA 726 ** ******* ***************** ************************* *** 11D02_viridis TGTTATGACATATTGGTGATTAGTATTAGAGTTATGAGA------AAATAAAATGAAAAT 822 11D02_nubicola TGTTATGACATATTGGTCATTAGTATTAGAGTTATGAGAAAGAGAAAATAAAATGAAAAT 783 11D02_vesca TGTTATGACATATTGGTGATTAGTATTAGAGTAATGAGAAAGAGAAAATAAAATGAAAAT 784 11D02_iinumae TGTTATGACATATTGGTACTTAGTATTAGAGTTATGAGAA----AAGAAAAAATGAAAAT 782 ***************** ************* ****** *********** 11D02_viridis ACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAAAGCAATAGATTGACAA-G 881 11D02_nubicola ACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAATGCAATAGATTGACAAAG 843 11D02_vesca ACAGTACTGGCAATAAACACAATACGGTGGAGCAATCAACAATGCAATAGATTGACAAAG 844 11D02_iinumae ACAGTACTGGCAATAAACACAATTCGGAGGAGCAATCAACAATGCAATAGATTGGCAA-G 841 *********************** *** ************** *********** *** 11D02_viridis AAATGAAGACCTAAAAAAAACCATTGCATTAATGCAATAGTGTTGATTTTCCAATCTCTC 941 11D02_nubicola AAATGAAGACCTAAAAAAA-CCATTGCATTAATGCAATAGTGTTGATATTCCAATCTCTC 902 11D02_vesca AAATGAAGACCTAAAAAAA-CCATTGCATTAATGCAATAGTGTTGATATTCCAATCTCTC 903 11D02_iinumae AAATGAAGACCTAAAAAAA-CCATTGCATTAATGCAATAGTGTCGATTTTCCAATCTCTC 900 ******************* *********************** *** ************ 11D02_viridis CTGAATAGTATTACAACTCTCCTGGACAAGTCATAACTGTGGGGGGTAATGGTGTAAGCA 1001 11D02_nubicola CTGAATAGTATTACAACTCTCCTGGACAAGTCGTAACTGTGGGGGGTAATGGTGTAAACA 962 11D02_vesca CTGAATAGTATTACAACTCTCCTGGACAAGTCATAACTGTGGGGGGTAATGGTGTAAACA 963 11D02_iinumae CTGAATAGTATTACAACTCTCCTGGACAAGTCATACCTGTGGGGGGTAATGGTGTAAACA 960 ******************************** ** ********************* **

PAGE 180

180 11D02_viridis AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACATAGCACCCCA 1061 11D02_nubicola AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACATAGCACCCCA 1022 11D02_vesca AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACATAGCACCCCA 1023 11D02_iinumae AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACGTAGCACCCCC 1020 ************************************************* ********* 11D02_viridis TAAATCATATCAGATGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAAACCT 1121 11D02_nubicola TAAATCATATCAGGTGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAAACCT 1082 11D02_vesca TATATCATATCAGATGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAAACCT 1083 11D02_iinumae TAAATCATATCAGATGGGGTTAATACTACCCAGGTGTGACATATTTGTACAGTTAAACCT 1080 ** ********** ********** *********************************** 11D02_viridis AATTTTGTCTAAAGAATGCTAAAATCGAA--CTCCCAAGCAACCAAATCTTCTGTTCCCC 1179 11D02_nubicola AATTTTGTCTAAAGAATGCTAAAATCGAACACTCCCAAGCAACCGAATCTTCTGTTCCCC 1142 11D02_vesca AATTTTGTCTAAAGAATGCTAAAATCGAA--CTCCCAAGCAACCGAATCTTCTGTTCCCC 1141 11D02_iinumae AATTTTGTCTAAAGAATGCTAAAATCGAA--CTCCCAAGCAACCGAATCTTCTGTTCCTC 1138 ***************************** ************* ************* 11D02_viridis TGCTTTAGTATGTTGTGATTATGCCTCTGCTTCCCCAGCAGCATGAATCCGCTCGTCTGG 1239 11D02_nubicola TGCTTTAGTATGTTGTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGG 1202 11D02_vesca TGCTTTAGTATGTTGTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGG 1201 11D02_iinumae TGCTTTAGTATGTTGTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGG 1198 ***************** ********* ******************************** 11D02_viridis AGTTACAGCATGAAGCAGTTCGTCTCT---------------------------TGTTGC 1272 11D02_nubicola AGTTACAGCATGAAGCAGCTCGTCTCT---------------------------TGTTGC 1235 11D02_vesca AGTTACAGCATGAAGCAGCTCATCTCT---------------------------TGTTGC 1234 11D02_iinumae AGTTACAGCATGAAGCAGCTCGTCTCTAGTTGCAGCATGAGGTAGCTCGTCTCTTGTTGC 1258 ****************** ** ***** ****** 11D02_viridis AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCGTCTGGCATTGCAGCATG 1332 11D02_nubicola AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATG 1295 11D02_vesca AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATG 1294 11D02_iinumae AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCGTCTGGCATTGCAAGATG 1318 ****************************************** ************ *** 11D02_viridis AAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCTAG 1392 11D02_nubicola AAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1355 11D02_vesca AAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAG 1354 11D02_iinumae AAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAG 1378 **** 11D02_viridis GTCCCAACACTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1452 11D02_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1415 11D02_vesca GTCCCAACACTTACCAGGTAGGTTAGTCTCTTCTGCGTCGAGTAACCATGCGGGCACCTG 1414 11D02_iinumae GTCCCAACACTTACCAGGTAGGTTAGTCTCTTCTGCGTCGAGTAACCATGCGGGTACCTG 1438 11D02_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1512 11D02_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1475 11D02_vesca GTGAGAAAAGCGTAACATCTCTCTTCTCGGAATCCATAGAATGGCGCTTCTGTCCGTATC 1474 11D02_iinumae GTGGGAAAAGCGTAACATCTCTCTTCTCGGAATCCATAGAATGGCGCTTCTGTCCGTATC 1498 11D02_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1572 11D02_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1535 11D02_vesca AGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGGTGAGACACAAACACCAAT 1534 11D02_iinumae AGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGGTGAGACACAA-CACCAAT 1557 11D02_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1632 11D02_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1595 11D02_vesca CTCTTCAGAGTAATCATCAAGAACCTCCACCATTTGATATTGGTGCCTCACTTCATCTGG 1594 11D02_iinumae CTCTTCAGAGTAATCATCAAGAACCTCCACCATTTGATAT-GGTGCCTCACTTCATCTGG 1616

PAGE 181

181 11D02_viridis NNNNNNNN 1640 11D02_nubicola NNNNNNNN 1603 11D02_vesca AGTTGAAC 1602 11D02_iinumae AGTNNNNN 1624 --------------------------------------------------------------------------------------------------------------17O22 17O22_vesca AAAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCT 60 17O22_mandshurica -AAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCT 59 17O22_viridis TAAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCT 60 17O22_nubicola -AAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCT 59 17O22_iinumae AAAATGGGTTG-ACGAGTTCGTGAACA-ACACTTTACGACCCAAAGCGTCCAATACTTCT 58 ********** ************** *** ************************* ** 17O22_vesca TAATTTGACAACAGACATAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACT 120 17O22_mandshurica TAATTTGACAACAGACATAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACT 119 17O22_viridis TAATCTGACAACGGACATAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACT 120 17O22_nubicola TAATTTGACAACGGACATAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACT 119 17O22_iinumae TAATTTGACAACGGACATAGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACT 118 **** ******* *********************************************** 17O22_vesca AGAGACTGCATTTCTTATAAAGGCAATGGAATCGTAGAGACTGCATTTCTTACTCAGTAC 180 17O22_mandshurica AGAGACTGCATTTCTTATAAAGGCAATGGAATCGTAGAGACTGCATTTCTTACTCAGTAC 179 17O22_viridis AGAGACTGCATTTCTTATAAAGGCAATGGAATCGTAGAGACTGCATTTCTTACTCAGTAC 180 17O22_nubicola AGAGACTGCATTTCTTATAAAGGCAATGGAATCGTAGAGACTGCATTTCTTACTCAGTAC 179 17O22_iinumae AGAGACTGCTTTTCTTATAAAAGCAATGGAATCGTAGAGACTGCATATCTTACTCAGCAC 178 ********* *********** ************************ ********** ** 17O22_vesca TGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTCCCGAAATT 240 17O22_mandshurica TGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTCCCGAAATT 239 17O22_viridis TGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTCCCGAAATT 240 17O22_nubicola TGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTCCCGAAATT 239 17O22_iinumae TGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTATAACTTCCCGAAATT 238 ******************************************** *************** 17O22_vesca CAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATC 300 17O22_mandshurica CAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACC--ACATTCATTCGATC 297 17O22_viridis CAACAGCCATCAGAGTTCATCTGCCAATCAAGGCAAATATGACTCTACATTCATTCGATC 300 17O22_nubicola CAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATC 299 17O22_iinumae CAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATC 298 ****************************** ************ ************** 17O22_vesca CCCTTATCACTGTAGGGCTTCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATA 360 17O22_mandshurica CCCTTATCACTGTAGGGCTTCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATT 357 17O22_viridis CCCTTATCACTGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATT 360 17O22_nubicola CCCTTATCACTGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATT 359 17O22_iinumae CCCTTATCACTGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATT 358 ******************* *************************************** 17O22_vesca GTAGCCTAGTTTAGTTTCTTATGCTGAAGCAAAATATGTAATCACCTACGCTACAGAATA 420 17O22_mandshurica GTAGCCTAGTTTAGTTTCTTATGCTGAAGCAAAATATGTAATCACCTAGGCTACAGAATA 417 17O22_viridis GTAGCCTAGTCTAGTTTCTTATGCTGAAGCAAAATATGTAATCACCTAGGCTACAGAATA 420 17O22_nubicola GTAGCCTAGTTTAGTTTCTTATGCTGAAGCAAAATATGTAATCACCTAGGCTACAGAATA 419 17O22_iinumae GTAGCCTAGTTTAGTTTCTTATGCTGAAGCAAAATATGTAATCACCTAGGCTACAGAAGA 418 ********** ************************************* ********* 17O22_vesca GTGTT-ACTTGTTACCGGACATGTTCACAATCTTTGAAGATGAAGAACGGTACCAGTTAC 479 17O22_mandshurica GTGTT-ACTTGTTACCGGACATGTTCACAATCTTTGAAGATGAAGAACGGTACCAGTTAC 476 17O22_viridis GTGTT-ACTTGTTACCGGACATGTTCACAATCTTTGAAGATAAAGAACGGTACCAGTTAC 479 17O22_nubicola GTGTTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATGAAGAACGGTACCAGTTAC 479 17O22_iinumae GTGTT-ACTTATTACCGGACATGTTCACAATCTTTGAAGATGAAGAACGGTACCAGTTAC 477 ***** **** ****************************** ******************

PAGE 182

182 17O22_vesca CCAACATAATCATAGTTATTTTGGCCTATTGATATTTTGATTAACGTGTAATTGATCGCT 539 17O22_mandshurica CCAACATAATCATAGTTATTTTGGCCTATTGATATTTTGATTAATGTGTAATTGATCGCT 536 17O22_viridis CCAACATAATCATACTTGTTTTGGCCTATTGATATTTTGATTAATATGTAATTGATCGCT 539 17O22_nubicola CCAACATAATCATAGTTATTTTGGCCTA-----ATTTTGATTAATATGTAATTGATCGCT 534 17O22_iinumae CCAACATAATCATAGTTATTTCGGAATATTGATATTTTGATTAATATGTAATTGATCGCT 537 ************** ** *** ** ** *********** ************** 17O22_vesca ACTTGAATGATGTATATTAT-----GAATGGCACTATTTAATATTTTGGGCTGCTACCTA 594 17O22_mandshurica ACTTGAATGATGTATATTAT-----GAATGGCACTATTTAATATTTTGTGCTGCTACCTA 591 17O22_viridis ACTGGAATGATGTATATTATATTATGAATGGCACTATTTAATATTTTGGGCTGCTACCTA 599 17O22_nubicola ACTTGAATGATGTATATTAT-----GAATGGCACTATTTAATATTTTGGGCTGCTACCTA 589 17O22_iinumae ACTTGACTGATGTATATTAT-----GAATGTCACTATTTAATATTTTGGGCTGCTACCTA 592 *** ** ************* ***** ***************** *********** 17O22_vesca CTCTTCAACAAACTCTAATTAATTAACCAAACATCAGTGTCACAAGTCACACCAACCTAG 654 17O22_mandshurica CTCTTCAACAAACTCTAATTAATTAACCAAACATCAGTGTCACAAGTCACACCAACCTAG 651 17O22_viridis CTCTTCAACAAACTCTAATTAATTAACCAAACATCAGTGTCACAGGTCACACCAACCTAG 659 17O22_nubicola CTCTACAACAAACTCTAATTAATTAACCAAACATCAGTGTCACAAGTCACACCAACCTAA 649 17O22_iinumae CTCTTCAACAAACTTTAATTAATTAACCAAACATCAGTGTCACAAGTCACACCAACCTAG 652 **** ********* ***************************** ************** 17O22_vesca TTAAACTTTCCATTATAAGTAGCTTTCCCAATAACCTACCTCCCAAAAATAGTTACTTTA 714 17O22_mandshurica TTAAACTTTCCATTATAAGTAGCTTTCCCAATAACCTACCTCCCAAAAATAGTTACTTTA 711 17O22_viridis TTAAACTTTCCATTATAAGTAGCTTTCCCAATAACCTACCTCCCAAAAATAGTTACTTTA 719 17O22_nubicola TTAAACTTTCCATTACAAGTAGCTTTCCCAATAACCTACCTCCCAAAAATAGTTACTTTA 709 17O22_iinumae TTAAACTTTCCATTATAAGTAGCTTTCCCAATAACATACCTCCCAAAAATAGTTACTTTA 712 *************** ******************* ************************ 17O22_vesca AA-GCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTTG 773 17O22_mandshurica AA-GCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTTG 770 17O22_viridis AAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTTG 779 17O22_nubicola AA-GCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTGCCTATAATTCTGGATTTG 768 17O22_iinumae AA-GCTGGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTTG 771 ** *** ********************************** ****************** 17O22_vesca TTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTCTGGTCAT 833 17O22_mandshurica TTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCGT 830 17O22_viridis TTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCAT 839 17O22_nubicola TTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCAT 828 17O22_iinumae TTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTCGGTCAT 831 **************************************************** **** 17O22_vesca CAGTGTCAAACTAGAAGAACCGTGAATTGCGAC--------------------------866 17O22_mandshurica CAGTGTCAAACTAGAAGAACCGTGAATTGCGACTATACCAGGATGCCTTTGGTCACTTAC 890 17O22_viridis CAGTGTCAAACTAGAAGAACTGTGAATTGCGACTACACCAGGATGCCTTTGGTCACTTAC 899 17O22_nubicola CAGTGTCAAACTAGAAGAACCGTGAATTGCGACTATACCAGGATGCCTTTGGTCACTTAC 888 17O22_iinumae CAGTGTCAAACAAGAAGAACCGTGAATTGCGACTATACCAGGATGCCTTTGGCCACTTGC 891 *********** ******** ************ 17O22_vesca -------------------CCCTCAGAATGTCAAAATGAGATCACTGTGATTCCTTTTAA 907 17O22_mandshurica CAACCTCAAGAAAAGGAC-CCCTCAGAATGTCACAATGAGATCACTGTGATTCCTTTTAA 949 17O22_viridis CAACCTCAAGAAAAGGACCCCCTCAGAATGTCAAAATGAGATCACTGTGATTCCTTTTAA 959 17O22_nubicola CAACCTCAAGAA----------------TGTCAAAATGAGATCTCTGTAATTCCTTTTAA 932 17O22_iinumae CAACCTCAAGAAAAGGAC-CCCTCAGAATGTCAAGATGAGATCACTGTGATTCCTTTTAA 950 ***** ******** **** ***********

PAGE 183

183 17O22_vesca AATTTTAACAGCGATTCTTCTACAAAAGATG-GACTAAATTCCACCTTGTACTGTACAAA 966 17O22_mandshurica AATTTTAACAGTGATTCTTCTACAAAAGATAAGACTAAATTCCACCTTGTACTGTACAAA 1009 17O22_viridis AATTTTAACAGTGATTCTTCTACAAAAGA-----CTAAATTCCACTTTGTACTGTACAAA 1014 17O22_nubicola AATTTTAACAGTGATTCTTCTACAAAAGATG-GACTAAATTCCACCTTGTACTGTACAAA 991 17O22_iinumae AATTTTGACAGTGATTCTTCTACAAAAGATG-GACCAAATTCCACCTTGTACTGTACAAA 1009 ****** **** ***************** ********* ************** 17O22_vesca AAACGAGTTTGAGTAGTGGGAATCGTTCCAATAT-ATTTCTGCTCTGTTTACCAATTGCC 1025 17O22_mandshurica AAACGAGTTTGAGTAGTGGGAATCGTTCCAATAT-ATTTCTGCTCTGTTTACCAAGTGCC 1068 17O22_viridis AAACGAGTTTGAGTAGTGGGAATCGTTCCAATAT-ATTTCTGCTCTGTTTACCAATTGCC 1073 17O22_nubicola AAACGAGTTTGAGTAGTGGGAATCGTTCCAATATTATTTCTGCTCTGTTTACCAATTGCC 1051 17O22_iinumae AAACGAGTTTGAGCAGTGGGAATCGTTCCAATAT-ATTTCTGCTCTGTTTACCAATTGCC 1068 ************* ******************** ******************** **** 17O22_vesca AGGATGATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAGAA-TGAGAAGA 1084 17O22_mandshurica AGGATGATACAAACATCTAAACTCTACAGGAACCATCTTCTAGCAAAAAAA-TGAGAAGA 1127 17O22_viridis AGGATGATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAA-TGAGAAGA 1132 17O22_nubicola AGGATGATTCAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAA-TGAGAAGA 1110 17O22_iinumae AGGATGATACAAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAAATGAGAAGA 1128 ******** ************************* *********** ** ******** 17O22_vesca AAGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1144 17O22_mandshurica AAGAACTCTACAAGAATC-AAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1186 17O22_viridis AAGAACTCTACAAGAATCCAAAGTGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1192 17O22_nubicola AGGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1170 17O22_iinumae AAGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTAAGACTAGACATGAAC 1188 **************** **** ************************************ 17O22_vesca AAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGA 1204 17O22_mandshurica AAATTTGCTGCAGCCTCCACTGAGGAGCATCTCCAGCAAGAACAAAAGAATCAAACCAGA 1246 17O22_viridis AAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGA 1252 17O22_nubicola AAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGTACAAAAGAATCAAACCAGA 1230 17O22_iinumae AAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACAAAAGAATCAAACCAGA 1248 *********************** **** *********** ******************* 17O22_vesca TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1264 17O22_mandshurica TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1306 17O22_viridis TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1312 17O22_nubicola TAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1290 17O22_iinumae TAAAATGGAAAATCTCCTCTCACGTCGGAACAATATCATTGATTTCAGATTTTGTCTCAG 1308 ************************* ********************************** 17O22_vesca ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTG 1324 17O22_mandshurica ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTG 1366 17O22_viridis ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTG 1372 17O22_nubicola ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGAAGGATTCAGAAAATTTG 1350 17O22_iinumae ATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTG 1368 ******************************************* **************** 17O22_vesca CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1374 17O22_mandshurica CTACAAAAGCCCATAACTTGTAAGGCATCATCGAAGTATTGTGAGGAAACCC 1418 17O22_viridis CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1422 17O22_nubicola CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1400 17O22_iinumae CTACAAAAGCCCATAACTTGTAAA-CATCATCGAAGT-TTGTGAGGAAACCC 1418 *********************** ************ ************** --------------------------------------------------------------------------------------------------------------27F10 27F10_vesca CCTGCAGGGTTTTTCATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 59 27F10_mandshurica CCTGCAGGGCTTTTTATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 59 27F10_nubicola CCTGCGGG--TTTTTATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 57 27F10_iinumae CCTGCAGG-TTTTTCATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 58 27F10_ananassa CCTGCAGG-TTTTT-ATCATGTAAGGACCTCCATTGT-CAGTAGCTTTATGCATATCATC 57 27F10_viridis --TGCGGG-TTTTTCATCATGTAAGGACCTCCATTGTTCGGTAGCTTTATGCATATCATC 57 *** ** **** ********************** ********************

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184 27F10_vesca TTCATCACAACAGCTGAAGCAGCTCATG-ATTCCTTTAAACACACACAAAAAAA---CCC 115 27F10_mandshurica TTCATCACAACAGCTGAAGCAGCTCATG-ATTCCTTTAAACACACACAAAAAAA---CCC 115 27F10_nubicola TTCATCACAACAGCTGAAGCAGCTCATG-ATTCCTTTAAACACACACAAAAAAAA--CCC 114 27F10_iinumae TTCATCACAACAGCTGAAGCAGCTCATG-ATTCCTTTAAACACACA--AAAAAAA--CCC 113 27F10_ananassa TTCATCACAACAGCGGAAGCAGCTCATGGACTCCTTTAAACACACA--AAAAAAA--CCC 113 27F10_viridis TTCATCACAACAGCTGAAGCAGCTCATG-ATTCCTTTAAACACACAAAAAAAAAACACCC 116 ************** ************* *************** ****** *** 27F10_vesca ACAGTCAAAATGAGGAAATGAACAATACCCAAGTCATGAACACACAAAATTCAGTAAAAA 175 27F10_mandshurica ACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGAACACACAAAATTCAGTAAAAA 175 27F10_nubicola ACAATCAAAATGAGAAAATGAACAATACCCAAGTCATGAACACACAAAATTCAGTAAAAA 174 27F10_iinumae ACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGAACGCACAAAATTCAGTAAAAA 173 27F10_ananassa ACGATCAAAATGAGGAAATGAACAATACCTAAGTCATGAACACACAAAATTCAGTAAAAA 173 27F10_viridis ATAATCAAAATGAGGAAATGAACAATACCCTAGTCATGAACACACAAAATTCAGTAAAAA 176 ********** ************** ********** ****************** 27F10_vesca AGTAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 235 27F10_mandshurica AGTAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 235 27F10_nubicola AG-AAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 233 27F10_iinumae AGAAAAAAGGGATCCGCTTCAATACAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 233 27F10_ananassa AGAAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCACCCCTTTGGAGACAAAT 233 27F10_viridis AGAAAAAAGGGATCCGCTTCAAGCCAATCCCATCAAACTTGCAGACCTTTGGAGACAAAT 236 ** ******************* ******************* *************** 27F10_vesca TTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATG 295 27F10_mandshurica TTCGTTGCTTAATGTAATAAGCAACAAAAAAATCAGCTCGGCTGGATCAAAGCCCAGATG 295 27F10_nubicola TTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATG 293 27F10_iinumae TTCGTTGCTTAATGTAATAAGCAACAAAAA-TCCAGCTCAGCTGGATCAAAGCCCAGATG 292 27F10_ananassa TTCGTTGCTTAATGTAATAAGCAACAAAAA-TTCAGCTCAGCTGGATCAAAGCCCAGATG 292 27F10_viridis TTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATG 296 ****************************** ****** ******************** 27F10_vesca AAAAAGATTAAAACTTTAAACAAGAAAATAAAGATCAGAGAAAGAAAATATGATGGGTAG 355 27F10_mandshurica AAAAAGATTAAAACTTTAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGTAG 355 27F10_nubicola AAAAAGATTAAAACTTCAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGTAG 353 27F10_iinumae AAAAAGATTAAAACTTTACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGATGGGTAG 352 27F10_ananassa AAAAAGATTAAAACTTTACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGGTGGGTAG 352 27F10_viridis AAAAAGATTAAAACTTTAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGNAG 356 **************** ************* ****** *********** **** ** 27F10_vesca ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCAGTGAAGGACTGA 415 27F10_mandshurica ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCAGTGAAGGACTGA 415 27F10_nubicola ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCAGTGAAGGACTGA 413 27F10_iinumae ATCGGGAGAGATAAAATTACCAGAATCTGAAGTGGGGGAAGTGAATCAGTGAAGGACTGA 412 27F10_ananassa ATCGGGAGAGATAAAATTACCAGAATCTGAAGTGGGGGAAGTGAATCAGTGAAGGACTGA 412 27F10_viridis ATCGGGAGAGATAAAATTACCTGAATCTGAAGTGGGGGAAGTGAGTCCGTGAAGAAGTGA 416 ********************* ********************** ** ****** *** 27F10_vesca GTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG-AAGGATGCGCGG 474 27F10_mandshurica GTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG-AAGGATGCGCGG 474 27F10_nubicola GTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG-AAGGATGCGCGG 472 27F10_iinumae GTTGCTGGAGTCTTGGGAGATCTGAG-------CTCTAAAGCCGGCG-AAGGATGCGCGG 464 27F10_ananassa GTTGCTGGAGTCGTGGAAGATCTGAG-------C-------CCGGCG-AAGGATGCGCGG 457 27F10_viridis GTTGGTGGANTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCGCAAGGATGCCCGG 476 **** **** ** *** ********* ****** ******** *** 27F10_vesca CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAGATGAGAATA 534 27F10_mandshurica CGCAGGATAGGAGGGAACAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATA 534 27F10_nubicola CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATA 532 27F10_iinumae CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATA 524 27F10_ananassa CGCAGGATCGGAGGGAAAAGGGTGCGTAGGATAACCCAACCAATGAACCAAATGAGAACA 517 27F10_viridis CGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCACTCCANGAACCANATGACAATG 536 ******** ******** ******************** ****** **** **

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185 27F10_vesca CGCTAGTGATTTTGATTATGAATTCTATAAATTCTATAAAAA-TTTATTTCATTTCTTAA 593 27F10_mandshurica CGCTAGTGATTTTGATTATGAATTCTATAAATTCTATAAAAA-TTTATTTCATTTCTTAA 593 27F10_nubicola CGCTAGTGATTTTGATTATGAATTCTATAAATTCTACAAAAAATTTATTTCATTTCTTAA 592 27F10_iinumae CGCTAGTGATTTTGATTATGAATTCTATAAATTCTACAAAAA-TTTATTTCATTTCTTAA 583 27F10_ananassa CGCTAGTGATTTTGATTATGAATTCTATAAATTCTACAAAAA-TTTATTTCATTTCTTAA 576 27F10_viridis CNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 596 27F10_vesca TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACTCTTCTGTGCTTCAGT------TTTG 647 27F10_mandshurica TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACTCTTCTGTGCTTCAGT------TTTG 647 27F10_nubicola TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACTCTTCTGTGCTTCAGT------TTTG 646 27F10_iinumae TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACTCTTCTGTGCTTCAGTCATGACTTTG 643 27F10_ananassa TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACACTTCTGTGCCTCAGTCATGACTTTG 636 27F10_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 656 27F10_vesca ACCATTTACTTTTATAACCTCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTC706 27F10_mandshurica ACCATTTATTTTTATATCCTCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTC706 27F10_nubicola ACCATTTACATTTATAACCCCGGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTC705 27F10_iinumae ACCATTTACTTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTCT 703 27F10_ananassa GCCATTTACTTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTCT 696 27F10_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 716 27F10_vesca -----------AAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 755 27F10_mandshurica -----------AAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 755 27F10_nubicola -----------AAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 754 27F10_iinumae GGTTCGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 763 27F10_ananassa GGTTCGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 756 27F10_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 776 27F10_vesca TCCCGGTAACAGTAACTTTATCGACAAAACGCTTCTTATTTTATTTTATTTTTTTTGGCG 815 27F10_mandshurica TCCCGGTA-CAGTAACTTTATCGTTTTACCGCTAGTATGTCTCTGTCAGTACTCT--GTC 812 27F10_nubicola TCCCGGTAACAGTAACTTTATCGTTTTACCGCTAGTATGTCTCTGTCT---------GTC 805 27F10_iinumae TCCGGGTAACAGTAACTTTATCGTTTTACCGCTAGCATGTCTCTGTCTGTC-----GACA 818 27F10_ananassa TCCCGGCAACAGTAACTTTATCGTTTTACCGCTAGTATGTCTCTGTCT-----------804 27F10_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 836 27F10_vesca AGCAAAACGCTTCTTATTTGTTTTGGGTCTGTACGCTTTTGGGTCTTATTTGTCAAGTTT 875 27F10_mandshurica GACATAACGCTTCTTATTTGTTTTGGGTCTCTACGCTTTTGGGTCTTATTTGTCAAGTTT 872 27F10_nubicola GACATGACGCTTCTTATTTCTTTTGGGTCTCTACGCTTTTGGGTCTTATTTGTCAAGTTT 865 27F10_iinumae TATATAACGCTTCTTATTTGTTTTGGGTCTCTACGCTTTTGGGTCTTATTTGTCAAGTTT 878 27F10_ananassa --------------TATTTGTTTTGGGTCCCTACGCTTTTGGGTCTTATTTGTCAAGTTT 850 27F10_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 896 27F10_vesca CAATCACTAGCAGGA--------AGACTTGCGTAT------------------------902 27F10_mandshurica CAATCACTAGCAGGT--------AGACTTGCGTAT------------------------899 27F10_nubicola CAATCACTAGCAGGA--------AGACTTGCGTAT------------------------892 27F10_iinumae CAATCACTTGAAACT--------------------------------------------893 27F10_ananassa CAATCACTTGAAACATAGCAGGAAGACTTGCATAT------------------------885 27F10_viridis NNNNNNNNNNNNNNN--------NNNNNNNNNTATGCAAAAATACACTCATATTTATGTA 948 27F10_vesca -----------------------------------------------------------27F10_mandshurica -----------------------------------------------------------27F10_nubicola -----------------------------------------------------------27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------27F10_viridis GAAAACGAGAATTGAACCTCTAACCTCTTACAAACAACCTATGAAATGTATAATATATGT 1008

PAGE 186

186 27F10_vesca -----------------------------------------------------------27F10_mandshurica -----------------------------------------------------------27F10_nubicola -----------------------------------------------------------27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------27F10_viridis AAAGACGATTAAAATATATGTATAATATATAATATATGTATTGTTTTATATTTATAACAT 1068 27F10_vesca -----------------------------------------------------------27F10_mandshurica -----------------------------------------------------------27F10_nubicola -----------------------------------------------------------27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------27F10_viridis ACTATAGATATAAATACAATTAAAATATAAAATGTTCAAATTTTAGCAAGAGGTATGTTT 1128 27F10_vesca -----------------------------------------------------------27F10_mandshurica -----------------------------------------------------------27F10_nubicola -----------------------------------------------------------27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------27F10_viridis CGAAACCATGACTGCTCTGATGGAAAATATGACCACTTACGATCAAAACAAAGCTATCAT 1188 27F10_vesca -----------------------------------------------------------27F10_mandshurica -----------------------------------------------------------27F10_nubicola -----------------------------------------------------------27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------27F10_viridis TGCATTATATTTGTGAAAAAAATTATATTTATCACTTCATTTTTTGGGCCACAATCTAAG 1248 27F10_vesca -----------------------------------------------------------27F10_mandshurica -----------------------------------------------------------27F10_nubicola -----------------------------------------------------------27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------27F10_viridis TTTAGTAGAGGCCTATTACCAACCGTACCAACTAAGTCGGTATACCAACATCGATGGTTG 1308 27F10_vesca -----------------------------------------------------------27F10_mandshurica -----------------------------------------------------------27F10_nubicola -----------------------------------------------------------27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------27F10_viridis GTTTTGATAGAGGATTTTGCCTACCAATCATAAGTTGGTTGGTACATGATATTGGTAAAT 1368 27F10_vesca -----------------------------------------------------------A 903 27F10_mandshurica -----------------------------------------------------------A 900 27F10_nubicola -----------------------------------------------------------A 893 27F10_iinumae -----------------------------------------------------------27F10_ananassa -----------------------------------------------------------A 886 27F10_viridis AAAGTCGGTATATCTACCAATGCCAGCCCTACTTGAAACTTAGCCGGAAGACTTCATATA 1428 27F10_vesca ATTGAAATAGCCACTATCTATACT--CTATATGCAAACACAA---GAGTAGAGAAGGAGA 958 27F10_mandshurica ATTGAAATAGCCACTATCTATACT--CTATATGCAAACACAA---GAG-AGAGAAGGAGA 954 27F10_nubicola ATTGAAATAGCCATTATCTATACT--CTATATGCAAACACAA---GAG-AGAGAAGGAGA 947 27F10_iinumae ATTGAAATAGCTGAAATACACACTTACTATATGCAAACACAA---GGG-AGAG-AGGAGA 948 27F10_ananassa ATTGAAATAGCTGCAATACACACTTGCTATATGCAAACACAACAAGAG-AGAGGAGGAGA 945 27F10_viridis ATTGAAATAGCTGAGATACACACTTGCTATATGCAAACACAA---GAGTAGAGAAGGAGA 1485 *********** ** *** **************** **** ******

PAGE 187

187 27F10_vesca ACCAGAATACATTTCCA 975 27F10_mandshurica ACCAGAATACATTTCCA 971 27F10_nubicola ACCAGAAT---CATCCA 961 27F10_iinumae ACCAGATC---ATTCTA 962 27F10_ananassa ACCAGAAT---CATCCA 959 27F10_viridis ACCAGAATACATTTCCA 1502 ****** ** --------------------------------------------------------------------------------------------------------------29G10 29G10_vesca TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGAGCTTTGGAGAGGTAGGAAGAGTTTATT 60 29G10_mandshurica TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGAGCTTTGGAGAGGTAGGAAGAGTTTATT 60 29G10_nubicola TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGGGCTTTGGAGAGGTAGGAAGAGTTGATT 60 29G10_nilgerrensis TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGGGCTTTGGAGAGTTAGGAAGAGTTGATT 60 ******************************** *********** *********** *** 29G10_vesca TCTAGAGGGAGGCTACCCATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAG--GAA 118 29G10_mandshurica TCTAGAGGGAGGCTACCCATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAG--GAA 118 29G10_nubicola TCTAGAGGGAGGCTACCCATTTGAAGTAGAGATTG-ACTAAAAACAACTTGAAAGAGGAA 119 29G10_nilgerrensis TCTAGTGGGAGGCTACCCATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAG--GAA 118 ***** ***************************** ******************* *** 29G10_vesca GATGGGGAGGATAAATAAAAAGGATAGAAACTGCTCAAGTGCTTAACAATGGTTGTAGAC 178 29G10_mandshurica GATGGGGAGGATAAATAAAAAGGATAGAAACTGCTCAAGTGCTTAACAATGGTTGTAGAC 178 29G10_nubicola GATGGGG-------------AGGATAGAAACTGCTCAAGTGCTTAACAATGGTTGTAGAC 166 29G10_nilgerrensis GATGGGGAGGATAAATAAAAAGGATAGAAACTGCTCAAGTGCTTAACAATGGTTGTGGAC 178 ******* ************************************ *** 29G10_vesca GAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGATGAAGAC 238 29G10_mandshurica GAGTTGTGTCTTGCTGCATATATTGAAGAGATCATATAGAGGTGCATGAAGGATGAAGAC 238 29G10_nubicola GAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGATGAAGAC 226 29G10_nilgerrensis GAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGATGAAGAC 238 ******************************** *************** *********** 29G10_vesca GCCGTATCTTAAATTTTGATTTGGTTCTTCTCA-------CACACCAGAGATTGAGTTCG 291 29G10_mandshurica ACCGTATCTTAAATTTTGATTTGGTTCTTCTCAGTTCTCACACACCAGAGATTGAGTTCG 298 29G10_nubicola ACCGTATCTTAAATTTTGATTTGGTTCTTCTCA-------CACACCAGAGATTGAGTTCG 279 29G10_nilgerrensis ACCGTATCTTAAATTTTGATTTGGTTCTTCTCA-------CACACCAGAGATTGAGTTCG 291 ******************************** ******************** 29G10_vesca GATCATCGGATCCGAAAAATCAAGTCCTTGTGTATAAAAGCACGTTACGGAGTGATCCCA 351 29G10_mandshurica GATCATCGGATCCGAAAAATCAAGTCCTTGTGTATAAAAGCACGTTACGGAGTGATCCCA 358 29G10_nubicola GATCATCGGATTCGAAAAATCAAGTCCTTGTGTATAAAAGCACGTTACGGAGTGATCCCA 339 29G10_nilgerrensis GATAATCGGATTCGAAAAATCAAGTCCTTGTGTGTAAAAGCACGTTGCGGAGTGATCCCA 351 *** ******* ********************* ************ ************* 29G10_vesca CTCATCAATAAGTTATCGGACTTAATTATTGTCACGGTGGACCACGTCAGTCTGGCATAT 411 29G10_mandshurica CTCATCAATAAGTTATCGGACTTAATTATTGTCACGGTGGACCACGTCAGTCTGGCATAT 418 29G10_nubicola CTCATCAATAAGTTATCGGACTTAATTATTGTCACGGTGGACCATGTCAGTCTGGCATAT 399 29G10_nilgerrensis CTCATCAATAAGTTATCGGACTTAATTATTGTCACGGTGGACCACGTCAGTCTGGCATAT 411 ******************************************** *************** 29G10_vesca CGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCATATCAGACCCAAGCCAT 471 29G10_mandshurica CGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCATATCAGACCGAAGCCAT 478 29G10_nubicola CGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCATATCAGACCGAAGCCAT 459 29G10_nilgerrensis CTATCATCACTCTCAATCTTGTCGATCATCAATTTGGC-TACATATCAGACCGAAGCCAT 470 ********** ************************* *********** ******* 29G10_vesca TACTTGCTTCTATGAACGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATT 531 29G10_mandshurica TACTTGCTTCTATGAACGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATT 538 29G10_nubicola TACTTGCTTCTATGAATGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATT 519 29G10_nilgerrensis TACTTGCTTCTATGAATGTATTTATATCATTACTAATCACCCAGAATTATGGATAATATT 530 **************** ************** ****************************

PAGE 188

188 29G10_vesca TCTTATTCACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGAC-AGTTCATT 590 29G10_mandshurica TCTTATTCACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGAC-AGTTCATT 597 29G10_nubicola TCTTATTCACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGAC-AGTTCATT 578 29G10_nilgerrensis TCTTATTCACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGACCAGTTCATT 590 *************************************************** ******** 29G10_vesca GAATTTCAGGAATCCACAATTGGGTGCTGCCTTCTTCT 628 29G10_mandshurica GAATTTCAGGAATCCACAATTGGGTGCTGCCTTCTTCT 635 29G10_nubicola GAATTTCAGGAATCCACAATTGGGTGCTGCCTCT---612 29G10_nilgerrensis GAATTTCAGGAATCCACAATTGGGTGCTGCCTTCTTCT 628 ******************************** --------------------------------------------------------------------------------------------------------------32L07 32L07_vesca GAGTTGAAAAACGGGTCGAATCCCGGCACCACCGTCCGCGTCGCGTAGGACTTGAATCCT 60 32L07_viridis GAGTTGAAAAACGGGTCGAATCCCGGCACCACCGTCCGCGTCGCGTAGGACTTGAATCCT 60 ************************************************************ 32L07_vesca TCCAAGGTCACCTCCTTGATGTACATAGCTGCCCTCGCCGGAGAGGTGCGGACGCTAATC 120 32L07_viridis TCCAAGGTCACCTCCTTGATGTACATAGCTGCGCTCGCCGGAGAGGTGTGGACGCTAATC 120 ******************************** *************** *********** 32L07_vesca GGAAGCCGATTTTGGAGAGATTTAGTGTCGGTGATAGATCGGAACCCTAGAAATCTGAGC 180 32L07_viridis GGTAGCCGATTTTGAAGAGATTTAGGGTCGGTGATAGATCGGAACCCTAGAAA------173 ** *********** ********** *************************** 32L07_vesca TTCTGGTTTTTGCTTTCGGAAGTTGAGAGTCTGAAATGACATGGTTCGAATTTCTTTTTG 240 32L07_viridis -----------------------------------------------------------32L07_vesca TTGTTTTCCGCTTTTTTGGTGGGTTCGAATTTTTAGACCAAGGCGGGAGATATTTGGGCC 300 32L07_viridis -----------------------------------------------------------32L07_vesca AGTGATTTATATCTTGGGCTCACTCTGGGACTCATGTCTTTGGGCCTCGTCGACCTCGAG 360 32L07_viridis -----------------------------------------------------------32L07_vesca GTGCTCATGAAGTCCGGCCGTCCTCAGGGTCGGAAACACCGCGGTACTACTGACTACTGT 420 32L07_viridis -----------------------------------------------------------32L07_vesca GTCATCGCTTTAGAATTTCATTAATTGGCTTTGCGAGCTATAAATAATTGTGATTTGGTT 480 32L07_viridis -----------------------------------------------------------32L07_vesca TGAATTTAGGTAAGTTTTAGTATTAGTATTTATCAACGGGAATTGCGGAGATGAGAAAAG 540 32L07_viridis -----------------------------------------------------------32L07_vesca TTGAGGTTGATTTGGGGGAGTGTGGTGTTGTTAGTTAGTTGAATTATTAGAAACGAAAAA 600 32L07_viridis -----------------------------------------------------------32L07_vesca ATAACAGAAGAATATAAATGTGGATGGATTATTGGATTAAGATTTGATTCAACGGAAGAA 660 32L07_viridis -----------------------------------------------------------32L07_vesca GGAGGCGTGGTGTGTGTTTTGATAGTCTAATTTGAACTGTTTTGCTTCTGACAGCTAAAA 720 32L07_viridis -----------------------------------------------------------32L07_vesca TCTATCCGGTGGTGAAAAATCAGCATCGGCTACTATGTACACTTTTAATCGGCAACGCAT 780 32L07_viridis -----------------------------------------------------------

PAGE 189

189 32L07_vesca TAGCGATGGAGGTGACTTGTCTAATTTACTAAGTTTATTTAGGTTGTTACTGGTACATTT 840 32L07_viridis -----------------------------------------------------------32L07_vesca TATGTGTTTATTGCCGTGGATGTAGTTTGTATGGGCCAGTTGACCAGCAGTTTCAAATGG 900 32L07_viridis -----------------------------------------------------------32L07_vesca CAGGCCAATAGGGCCAACCTAGATTGTAGTTGAATTTTGGGAAGGAAAAAAAAAAGCAAA 960 32L07_viridis -----------------------------------------------------------32L07_vesca CCAAAAGACATCACCACGAGCCACTTTGGCCTATCTATATATATTACTTCCTTGCTTAAT 1020 32L07_viridis -----ATACGTCACCACGAGCCACTTTGGCCTATCTATGT----TACTTCCTTGCTTAAT 224 ** **************************** **************** 32L07_vesca GTGTTGCTCAATTGCTAAACAATATCATCAATGTCTAAAATAACGCGCCTCAAGGCTAAG 1080 32L07_viridis GTGTTGCTCAATTGCTCAACAATATCATCAATGTCTAAAATAACGCGCCTCGAGGCTAAG 284 **************** ********************************** ******** 32L07_vesca GCAAGGGAAGGCGT-GCCTTAGGACGACCTCTGAAAGACATTTGATATCAAAGGTGTGAT 1139 32L07_viridis GCAAGGGAAGGCGTCACCTTAGGACGACCTCTGAAAGACATTTGATATCAAAGGTGTGAT 344 ************** ******************************************** 32L07_vesca TGAGGCGCGCGATCAAGACGACGAGGTCAAGGTGCCTAATACAACATCAGGTTATAGGTT 1199 32L07_viridis TGAGGCGCGCGATCAAGACGACGAGGTCAAGGTGCCTAATACAACATCAGGTTATAGATT 404 ********************************************************* ** 32L07_vesca TGAATCTCACTTTGAGAAATGTGATGGTTTGAACGGTTAAATCTATTGTCTTTTTATATT 1259 32L07_viridis TGAATCTCACTTTGAGAAATGTGATGGTTTGAACGGTTAAATCTATTGTCTCTTTATATT 464 *************************************************** ******** 32L07_vesca GTATGGGCGGTAAAATTAAATGTTAAACTTCGGTAAATTGTCAAATGTTTAATAGTATAA 1319 32L07_viridis GTATGGGTGGTAAAATTAAATGTTAAATTTCGGTAAATTGTCAAATGTTTAATAGTATAA 524 ******* ******************* ******************************** 32L07_vesca GAATCTACATATAGTAGGTGTAAAATAGATACCGAAATGATAATATTTTGTGAATAACGT 1379 32L07_viridis GAATCTACATATAGTAGGTGTAAAATAGATACCGAAATGATAATATGTTGTGAATAATAT 584 ********************************************** ********** 32L07_vesca ACGTCATATGATTTAATATTAAGACTTTGTACGATTTAACGTTACACATTAAAATTGTAG 1439 32L07_viridis ACGTCATATGGTTTAATATTAAGACTTTGTGCGATTTAATGCTACACATTAAAATTGTAG 644 ********** ******************* ******** ****************** 32L07_vesca ATAAAAAGTTTATATCATCATCAACATCGATGTTCGAATAAATTTTATAACGTTCAATGC 1499 32L07_viridis ATAAAAAATTTATATCATTATCATCATCGATGTTCGAATAAATTTTATAACGTTCAATGC 704 ******* ********** **** ************************************ 32L07_vesca GGTACAAATCTCCCAATGACTATTATCGAGTACAACGTCCATATCCGACACATGATATAG 1559 32L07_viridis GGTACAAATCTCCCAATGACTATAATCGAGTACAACGTCCATATCGGACACATGATATAG 764 *********************** ********************* ************** 32L07_vesca GCTATCAAATTATCAAAACCCTTTGATCCGATTCTGTAGCTTTGACGACTATAAGCTTAG 1619 32L07_viridis GCTATCAAATTATCAAAACCCTTTGATCCGATTCTGTAGCTTTGACGACTATAAGCTTAG 824 ************************************************************ 32L07_vesca TTAAGTTTAGTAGGACTCACCGCAATTTCGCACTAGTAGGACAAAAAGATGGTAAGATTC 1679 32L07_viridis TTAAGTTTAGTAGGACTCACCGCAATTTCGCACTAGTAGGACAGAAAGATGGTAAGATTC 884 ******************************************* **************** 32L07_vesca CTTTCATTTTTCTTCTTTACTATCCTTCTTTTCCTCAATTTTTCCCTAGAATCCTACAAC 1739 32L07_viridis CTTTTATTTTTCTTCTTTACTATCCTTCTTTTCCTCATTTTTTCCCTAGAATCCTACAAC 944 **** ******************************** ********************** 32L07_vesca AAGAAAGGACTTTGGCCCCTTGTGCTCCTTTATCATCTTAAAAGCATCACCACCATCCCC 1799 32L07_viridis AAGAAAGGACTTTGGCCCCTTGTGCTCCTTTATCATCTTAAAAGCATCACCACCATCCCC 1004 ************************************************************

PAGE 190

190 32L07_vesca TATATAGATGCATATTCACTATCAAGCTACCCAAGTATGCAAATTTATAGCATCTCATTA 1859 32L07_viridis TATTTAGATGCATATTCACTATCAAGCTACCCAAGTATGCAAATTAATAGCATCTCATTA 1064 *** ***************************************** ************** 32L07_vesca TCTTGTTTCCTCTAGCTATTCTACTCAATGCATATCAACAACCTGACCCAGTTCTCCTAT 1919 32L07_viridis TCTTGTTTCCTCTAGCTATTCTACTCAATGCATATCAACAACCTGACCCAGTTCTCCTAT 1124 ************************************************************ 32L07_vesca AATTGCTGGCAGATAGTAATACCAATTACTCCAGAATCTTCACACCCAGAACTTGAAATT 1979 32L07_viridis AATTGCTGGCAGATAGTAATACCAATTACTCCAGAATCTTCACACCCAGAACTTGAAATT 1184 ************************************************************ 32L07_vesca ACACGACCTCAATACTCCAAACAGTAC---------AAAACAACCCAGATGATCAAAACA 2030 32L07_viridis ACACGACCTCAATACTCCAAACAGTACTGTCAGTACAAAACAACCCAGATGATCAAAACA 1244 *************************** ************************ 32L07_vesca CATAACATTCTTTATTTCATCTTATTGGGAAAATCTCTATATCTATTATCTTCATTATTC 2090 32L07_viridis CTTAAAATTCTTTATTTCATCTTATTG---------CTATCTCTATCATCTTCATTATTC 1295 *** ********************* **** ***** ************* 32L07_vesca AATTTTTCTACACTGCATGCTATACATGTTACAAAAGAGAAAGAAAAGACACTAGTCCAT 2150 32L07_viridis AATTTTTCTACACTGCATGCTATACATGTTACAAAAGAGAAACAAAAGACACTAGCCCAT 1355 ****************************************** ************ **** 32L07_vesca ATCACATAGGCCATGTCCTTCCCAATTCTAACCCAACAATTCAAGGACCACACCCATGAG 2210 32L07_viridis ATCACATAGGCCATGTCCTTCCCAATTCTAACCCAACAATTCAAGGACCACACCCATGA1414 *********************************************************** 32L07_vesca TAGTGGCACTGAATCACTGAATCGTCGCCTTCACAACTACACTACCTATCCAACCCAGAC 2270 32L07_viridis --GTGGCACTGAATCACTGAATCGTCACCTTCACAACCACACTACCTATCCAACCCAGAC 1472 ************************ ********** ********************** 32L07_vesca TCAACACAGATGAAAATTCACAGCAGCTAAGAATATAGTACTAGTTTTGCTCTATCTTTT 2330 32L07_viridis -----ACAGATGAAAATTCACAGCAGCTAAGAATATAGTACCAATTTTGCTCTATCTTTC 1527 ************************************ *************** 32L07_vesca TTCTTTACCAAAACAAAAAAAACCCTGTAGTAACCAATATAACCGCTAACAGCTTTTCCC 2390 32L07_viridis TTCTTTACCAAAACAAAAAAGATCCTGTAGTAACTAATATAACAGCTAACAGCTTTTCCC 1587 ******************** *********** ******** **************** 32L07_vesca ATCCTGCCCATAACAGCTTTTCCCCTGCAGTATGGGAAACCCTTATCTAAAACCCCCCGA 2450 32L07_viridis ATCCTGCCCATAACAGCTTTTCCCCTGCAGTATGGGAAACCCTGATCTAAA-TCCCCCGA 1646 ******************************************* ******* ******* 32L07_vesca TTTATAGTAACAAAAAAATAAATAAAATAATTTACTTTCCTCATTTACCATTTTACCCTC 2510 32L07_viridis TTTATAGTAACAAAAAAATAAATAAAATAATTTGCTTTCCTCATTTACCATTTTACCCTC 1706 ********************************* ************************** 32L07_vesca ATCTTCTCCTTCATTGCCACTTGAACCCCCACTCTCCATGCTCCTTGAACCTTCTCAACA 2570 32L07_viridis ATCTTCTCCTTCATTGCCACTTGAACCCCCACTCTCCATGCTCCTTGAACCTTCTCAACA 1766 ************************************************************ 32L07_vesca CCCTTTCTAGGGCAATGTCAAAAGCGTCTTTTACCGTCTCCAACCCCTCCTGCGGTTTCG 2630 32L07_viridis CCCTTTCTAGGGCAATGTCAAAAGCGTCTTTTACCGTCTCCAACCCCTCCTGCGGTTTCG 1826 ************************************************************ 32L07_vesca CGTACAGAAAATTCGGTATGTAATCGATAACTTTCTCCCGCATTTTCCT 2679 32L07_viridis CGTACAGAAAATTCGGTATGTAATCGATAACTTTCTCCCGCATTTTCCT 1875 ************************************************* --------------------------------------------------------------------------------------------------------------

PAGE 191

191 34D20 34D20_vesca GCAGAAAGAAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTC 60 34D20_mandshurica NNNNNGAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGAGCAGTTC 60 34D20_nilgerrensis NGCAGAAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTC 60 34D20_iinumae NNNNNNNNNAACTGATGTGCTTTCCGGAGGGACTGACAGTAGAAAAGGACAGTGCAGTTC 60 34D20_ananassa NNNNNNNNNAACTGATGCGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTC 60 34D20_viridis NNNGAGAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTC 60 34D20_nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 60 34D20_vesca AGGGGATAAAGGAAGTATTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCT 120 34D20_mandshurica AGGGGATAAAGGAAGTATTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCT 120 34D20_nilgerrensis AGGGGATAAAGGAAGTATTA-TGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCTCT 119 34D20_iinumae AGGGGATAAAGGAAGTATTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCT 120 34D20_ananassa ATGGGATAAAGGAAGTATTAATGTTAGGCATCCTAGACGGCATCTGGTTTTGGAGTCCCT 120 34D20_viridis AGGAGATAAAGGAAGTATTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCT 120 34D20_nubicola NNNNNNNNNNNNNNNNNNNNATGTT-GGCATCC-AGACGGCATCTGGTTTTGGAGTCCCT 118 **** ******* *********************** ** 34D20_vesca CTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGAATTTGATTTCTACAAGGTGAGCAA 180 34D20_mandshurica CTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGAATTTGATTTCTACAAGGTGAGCAA 180 34D20_nilgerrensis CTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGAATTTGATTTCTACAAGGTGAGCAA 179 34D20_iinumae CTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGAATTTGATTTCTACAAGGTGAGCAA 180 34D20_ananassa CTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGAATTTGATTTCTACAAGGTGATCAA 180 34D20_viridis CTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGAATTTGATTTCTACAAGGTGAGCAA 180 34D20_nubicola CTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGAATTTGATTTCTACAAGGTTAGCAA 178 ****************************************************** *** 34D20_vesca CATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGTTATGTGAAT 240 34D20_mandshurica CATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGTTATGTGAAT 240 34D20_nilgerrensis CATGCCGGCAAACTAGTTATATTTTGTTTTTCTTACTATTACAGTGTGTGTTATGTGAAT 239 34D20_iinumae CATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGTTATGTGAAT 240 34D20_ananassa CATGCCTGCACACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGTTATGTGAAT 240 34D20_viridis CATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGTTATGTGAAT 240 34D20_nubicola CA-GCCTGCAAACTAGATATATTTTGTTTTTCTAACTATTACAGTGTGTATTATGTGAAT 237 ** *** *** ***** **************** *************** ********** 34D20_vesca CATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGA 300 34D20_mandshurica CATCTGCAAATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGA 300 34D20_nilgerrensis CATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGA 299 34D20_iinumae CATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGA 300 34D20_ananassa CATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACCAGGAAAGA 300 34D20_viridis CATCTGCATATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGA 300 34D20_nubicola CATCTGCATATTATCTATATCTAACTCTATGGTATAATGATTCAGAACTACAAGGAAAGA 297 ******** ***************************** ************ ******** 34D20_vesca TTATCGGCGAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCT 360 34D20_mandshurica TTATCGGCGAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCT 360 34D20_nilgerrensis TTATCGGCGAGAAGGTGTTTTGCATGCAAGCAGCAGCAAATGCTATGGGCCAATTTCCCT 359 34D20_iinumae TTATCGGCGAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCT 360 34D20_ananassa TTATCGGCAAGAAGGTGTTTTGCATGCATGCAGCACAAAATGCTATGGGCCAATTTCCCT 360 34D20_viridis TTATCGGCGAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCT 360 34D20_nubicola TTATCGGCGAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCT 357 ******** ******************* ****** *********************** 34D20_vesca TGCAAACACTTGCTATGGTGTAATGACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTT 420 34D20_mandshurica TGCAAACACTTGCTATGGTGTAATGACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTT 420 34D20_nilgerrensis TGCAAACACTTGCTATGGTGTAATGACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTT 419 34D20_iinumae TGCAAACACTTGCTATGGTGTAATGACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTT 420 34D20_ananassa TGCAAACACTTGCTATGGTGTAATGACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTT 420 34D20_viridis TGCAAACACTTGCTATGGTGTAATGACTGCAAGTTACGGGCAATAGGGGCTTCGGGTCTT 420 34D20_nubicola TGCAAACACTTGCTATGGTGTAATGACTGCAAGTTGCGGGCAATAGGGGCTTCAGGCCTT 417 *********************************** ***************** ** ***

PAGE 192

192 34D20_vesca TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480 34D20_mandshurica TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480 34D20_nilgerrensis TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 479 34D20_iinumae TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480 34D20_ananassa TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480 34D20_viridis TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480 34D20_nubicola TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 477 ************************************************************ 34D20_vesca TTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGAACTGCA 540 34D20_mandshurica TTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGAACTGCA 540 34D20_nilgerrensis TTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCATAGGAGAACTGCA 539 34D20_iinumae TTAACTAGTAACTGAGGAATCCAATTGCACTCTGTGTTTAC-ATGCACAGGAGAACTGCA 539 34D20_ananassa TTAACTAGTAACTGAGGAATACAATTGCACTCTGTGTTTTCCATGCACAGGAGAACTGCA 540 34D20_viridis TTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTTCATGCACAGGAGAACTGCA 540 34D20_nubicola TTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGTTTTCCATGCACAGGAGAACTGCA 537 **** *************** ******** ****** ** ***** ************ 34D20_vesca GGTGAACCGTATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGT 600 34D20_mandshurica GGTGAACCGTATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGT 600 34D20_nilgerrensis GGTGAACCATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGT 599 34D20_iinumae GGTGAACTATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGT 599 34D20_ananassa GGTGAACTATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGT 600 34D20_viridis GGTGAACCATATGTCTATATAGATATGTCGAATGTTAGATAGGATACATAGTATGAGGGT 600 34D20_nubicola GGTGAACTTTATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTGGGT 597 ******* ********************* ************************ **** 34D20_vesca GTGGATGAACTATACGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 660 34D20_mandshurica GTGGATGAACTATACGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 660 34D20_nilgerrensis GTGGATGAACTATACGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 659 34D20_iinumae GTGGATGAACTATAAGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 659 34D20_ananassa GTGTATGAACTATAAGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 660 34D20_viridis GTGGATGAACTATACGTAGAGCACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 660 34D20_nubicola GTGGATGAACTATACGTAGAACACCCAGAAAACCAGAGAAAGTAAAGAGGAACTGCAGGT 657 *** ********** ***** **************** ****************** *** 34D20_vesca TGGGTATGAATCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 720 34D20_mandshurica TGGGTATGAATCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 720 34D20_nilgerrensis TGGGTATGAGTCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 719 34D20_iinumae TGGGCATGAGTCTCCCTCCCGGCCACACTAGACCAC-----------------------695 34D20_ananassa TGGGCATGAGTCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 720 34D20_viridis TGGGCATGAATCTCCCTCCCGGCCACAGTAGACCACACTTTTGAACTGGCGGATTCCATC 720 34D20_nubicola TGGGCATGAATCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 717 **** **** ***************** ******** 34D20_vesca CGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCTCCTAGCCATA 780 34D20_mandshurica CGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCTCCTAGCCATA 780 34D20_nilgerrensis CGGCCTAGATTTTGTGCCGACTATCACAATAGTGTA----AGTTGGTCCTCCTAGCTATA 775 34D20_iinumae ----CTAGATTTTGTGCCGACTATCACAATAGTGAA----AGTTGGTCCTCCTAGCTATA 747 34D20_ananassa CGGCCTAGATCTTGTGCCGACTATCACAATAGTGTA----AGTTGGTCCTCCTAGCTATA 776 34D20_viridis CGGCCTAGATTTTGTGCCGACTATCACAATAGTGTA----AGTTGGTCCTCCTAGCTATA 776 34D20_nubicola CGGCGTAGATTTTGTGCCGACTATCACAATAGTGTA----AGTTGGTCCTCCTAGCTATA 773 ***** *********************** **************** *** 34D20_vesca GTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATATGA 840 34D20_mandshurica GTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATATGA 840 34D20_nilgerrensis GTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGA 835 34D20_iinumae GTTTCTAGTACTATTCTACTGATATCATGTTTCGTCTCAGCTTTTGACAATGGAATATGA 807 34D20_ananassa GTTTCTAGTACTATTCTACTGATATCATGTTTCGTCTCAGCTTTTGACAATGGAATATGA 836 34D20_viridis GTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGA 836 34D20_nubicola GTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGA 833 ****************************** *************************

PAGE 193

193 34D20_vesca TGAATTTGGAATGAATACAAAAACTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAA 900 34D20_mandshurica TGAATTTGGAATGAATACAAAGACTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAA 900 34D20_nilgerrensis TGAATATGGAAC-------AAAGCTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAA 888 34D20_iinumae TGAATATGGAATGA--ACAAAACCTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAA 865 34D20_ananassa TGGATATGGAATGA--ACAAAACCTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAA 894 34D20_viridis TGAATATGGAATGA--ACAAAAGCTGCTTTGTCCATCTTTTAGCATTTTCTGAAACCCAA 894 34D20_nubicola TGAATATGGAATGA--ACAAAAGCTGCTTTGTCCATCTGTTAGCATTTTCTGAAACCCAA 891 ** ** ***** ** *************** ********************* 34D20_vesca AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 960 34D20_mandshurica AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 960 34D20_nilgerrensis AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 948 34D20_iinumae AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 925 34D20_ananassa AAGATGGGTACATGTTTGCTTATTTTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 954 34D20_viridis AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 954 34D20_nubicola AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 951 ************************ *********************************** 34D20_vesca ATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGTATAGTTGTATTGA 1020 34D20_mandshurica ATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGTATAGTTGTATTGA 1020 34D20_nilgerrensis ATGTCTATGCATTCTGCTGATTTAGGAATAAGGATTGCAGTACTTGTATAGTTGTATTGA 1008 34D20_iinumae ATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCACTACTTGTGTAGTTGTATTGA 985 34D20_ananassa ATGTTTATGCATTCTGCTGATTTAGGATTTAGGATTGCACTACTTGTATAGTTGTATTGA 1014 34D20_viridis ATGTTTATGTATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGTATAGTTGTATTGA 1014 34D20_nubicola ATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGTATAGTT--ATTGA 1009 **** **** ***************** ********* ******* ***** ***** 34D20_vesca TCTGATATAACATAAATTTAATGAATCTAATAGACATTTTT-CCTAGTTAACAGAGGATA 1079 34D20_mandshurica TCTGATATAACATAAATTTAATGAATCTAATAGACATTTTT-CCTAGTTAACAGAGGATA 1079 34D20_nilgerrensis TCTGATATAACATAAATTTAATGAATCTAATATAAATTTTT-CCTAGTTAAG-------1059 34D20_iinumae TCT------------------------------AAATTTTT-CCTAGTTAACAGAGGATA 1014 34D20_ananassa TCT------------------------------AAATTTTT-CCTAGTTAACAGAGGATA 1043 34D20_viridis TCTGATATAACTCCCATT-AATGAATCTAATATAAATTTTTTCCTAGTTAACAGAGGATA 1073 34D20_nubicola TCTAATATAACATAAATTTAATGAATCTAATATAAATTTTT-CCTAGTTAAG-------1060 *** ****** ********* 34D20_vesca GGTCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACAC 1139 34D20_mandshurica GGCCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACAC 1139 34D20_nilgerrensis -----CCAAAAGACCTTATCCTACAAGGAAACAGAAACGTACAATTAACGCATTATACAC 1114 34D20_iinumae GGTCTCCGGCTGACGTTATCCTACAAGGAAACAGAAACGTACAATTAACGGATT---CAC 1071 34D20_ananassa GGACTCCGGCTGACCTTATCCTACAAGGAAACAGAAACGTACAATTAACGGATT---CAC 1100 34D20_viridis GGTCTCCGGCTGACCTTATCCAACAAGGAAACAGAAACATACAATTAACGCATTATACAC 1133 34D20_nubicola -----TCAAAAGACCTTATCCTACAAGGAAACAGAAACGTACAATTAACGCATTATACAC 1115 *** ****** ********* ****** *********** *** *** 34D20_vesca AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCT 1199 34D20_mandshurica AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCT 1199 34D20_nilgerrensis AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT-------------------1154 34D20_iinumae AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT-------------------1111 34D20_ananassa AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT-------------------1140 34D20_viridis AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT-------------------1173 34D20_nubicola AAGACCGGTCTATATAAGACATCAAAATCTCTTTATCTGT-------------------1155 ***** ************ ******* ************* 34D20_vesca CTTTATCTGTTTCATACTTTCAT--TGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1257 34D20_mandshurica CTTTATCTGTTTCATACTTTCAT--TGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1257 34D20_nilgerrensis ------------------TTCAT--TGATCATATTGTCTAGTACTGGAAGAGTTATATTT 1194 34D20_iinumae ------------------TTCAT--TGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1151 34D20_ananassa ------------------TTCAT--TGATCATATTGTCCAGTACTGGAAGAGCTATATTT 1180 34D20_viridis ------------------TTCAT--TGATCATATTGTCTAGTACTGGAAGAGCTATATTC 1213 34D20_nubicola ------------------TTCATATTGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1197 ***** ************* ************* ******

PAGE 194

194 34D20_vesca ATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCCTT 1317 34D20_mandshurica ATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCTTT 1317 34D20_nilgerrensis ATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCCTT 1254 34D20_iinumae ATCTGATAACAGAAAGTGCTTACTTGCTGGTTCCTACTCATTATGGATCCGAAGGTCCTT 1211 34D20_ananassa ATCTGATAACAGAAAGTGCGTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCCTT 1240 34D20_viridis ATCTGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCCTT 1273 34D20_nubicola ATCTGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCCTT 1257 *** *************** ************* ****** **************** ** 34D20_vesca AGTTACAATGGTGTTGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTTGTTAC 1377 34D20_mandshurica AGTTACAATGGTGTTGACTTGAGCATGAGCGACTTGGATCTTCTTAGAGTCCCTTGTTAC 1377 34D20_nilgerrensis AGTTACAATGGTGTTGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTTGTTAC 1314 34D20_iinumae AGTTACAAAGGTGTTGACCTGAGCATGAGCGACTAGGATATTCTTAGAGGACCTTATTAC 1271 34D20_ananassa AGTTCCCAAGGTGTTGACCTGAGAATGAGCGACTTGGATCTTCTTAGAGGCCCTTATTAC 1300 34D20_viridis AGTTACAATGGTGTTGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTTATTAC 1333 34D20_nubicola AGTTACAAAGGTGTAGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTTGTTAC 1317 **** ***** *** **** ********** **** ********* **** **** 34D20_vesca TTAACCGATAGCATCATTTGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGAT 1437 34D20_mandshurica TTAACCGATAGCATCATTCGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGAT 1437 34D20_nilgerrensis TTAACCGATAGCATCATT-----------CACTTATCTTACTTCCCATTACGATGATGAT 1363 34D20_iinumae TTAACCGATAGCATCATTCGATTCTATTTCACTTATCTTACTTCCCATTATGATGATGAT 1331 34D20_ananassa TTAACCGATAGCATCATTCAATTCTATTTCACTTATCTTACTTCCCATTATGATGATGAT 1360 34D20_viridis TTAACCGATAGCATCATTCGATTCTATTTCACTAATCTTAGTTCCCATTATGATGATGAT 1393 34D20_nubicola TTAACCGATAGCATCATTCGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGAT 1377 ****************** **** ****** ********* ********* 34D20_vesca ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGAT 1497 34D20_mandshurica ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGAT 1497 34D20_nilgerrensis ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCTGGATCCTGAGGAT 1423 34D20_iinumae ATCCTTCTGGTTTCCCCTAATATCTCTGATCTCCTGGTAAATTCTCCGGATCCCGAGGAT 1391 34D20_ananassa ATCCTTCTGGTTTCCCCTAATATCTCTGATTTTCTGGTAAATTCTCCGGATCCCGAGGAT 1420 34D20_viridis ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGAT 1453 34D20_nubicola ATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGAT 1437 ****************************** ************* ****** ****** 34D20_vesca GAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATCTTCGCA 1557 34D20_mandshurica GAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATCTTCGCA 1557 34D20_nilgerrensis GAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATCTTCGCA 1483 34D20_iinumae GAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATCTTCGCA 1451 34D20_ananassa GAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATCTTCGCA 1480 34D20_viridis GAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATATTTGCA 1513 34D20_nubicola GAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGAAAAGTTGTGATCTTTGCA 1497 ***************************************** *********** ** *** 34D20_vesca GTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG 1617 34D20_mandshurica GTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG 1617 34D20_nilgerrensis GTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG 1543 34D20_iinumae GTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG 1511 34D20_ananassa GTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG 1540 34D20_viridis GTGAATGATAACGAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG 1573 34D20_nubicola GTGAATGATAACGAAGATCCGAGTCGAAGCGACGGTGGAAACCATTGGAGCTTGCTGGTG 1557 ************ ********************** ************************ 34D20_vesca TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTG 1677 34D20_mandshurica TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTG 1677 34D20_nilgerrensis TATTTCAGAAAATCAAACGCATTCGTACACTACGACAGCTTGGGGGGTAACAATAGTTTG 1603 34D20_iinumae TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTG 1571 34D20_ananassa TATTTCAGAAAATCAAACGCATTGGTACATTACGACAGCTTGGGGGGTAACAATAGTTTG 1600 34D20_viridis TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTG 1633 34D20_nubicola TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTG 1617 *********************** ***** ******************************

PAGE 195

195 34D20_vesca GAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1737 34D20_mandshurica GAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1737 34D20_nilgerrensis GATGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCAGCTCCAGCAACACAAGCA 1663 34D20_iinumae GAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1631 34D20_ananassa GATGCTAGGAAAATGTATACAGCATTCAAGAAACTTGTGGCAGTTCCAGCAACACAAGCA 1660 34D20_viridis GAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1693 34D20_nubicola GAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1677 ** ******************* ****************** **************** 34D20_vesca CCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGACAC 1797 34D20_mandshurica CCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGACAC 1797 34D20_nilgerrensis CCAA---CTCCATCTGGGACTAGTAGTTTGGTTACCAACAACAGTTCTACAATGCGACAC 1720 34D20_iinumae CCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGACAC 1691 34D20_ananassa CCAA---CTCCAGCTGGGAGTAGTAGTTTGGTTACCAACAACAGTTCTACAATGGGACAC 1717 34D20_viridis CCAATAACTCCAGCTGGGACTAGTAGTTTGGTTACCAACAACAGTTCTACAATGAGACAC 1753 34D20_nubicola CCAATAACTCCAGCTGGGACTAGTAGTTTGGTTACCGACAACAGTTCTACAATGAGACAC 1737 *** ***** ****** *********** **** ***************** ***** 34D20_vesca GAGTGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTT 1857 34D20_mandshurica GAGTGCCACTCTACGCAGTCGGGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTT 1857 34D20_nilgerrensis GAGTGCTACTCTACGCGGTCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTT 1780 34D20_iinumae GAGTGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTT 1751 34D20_ananassa GAGTGCTACTCTACGCAGTCGCGGCGGTTTATAGACCATACCAAGATAATGCTTCGGGTT 1777 34D20_viridis GAGTGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTT 1813 34D20_nubicola GAGTGCCACTCTACGCAGTCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTT 1797 ****** ********* **** **** ********* ********* ******* ***** 34D20_vesca TGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCAT 1917 34D20_mandshurica TGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCAT 1917 34D20_nilgerrensis TGGGGTTTTGTTGTCAATTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCAT 1840 34D20_iinumae TGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCAT 1811 34D20_ananassa GGGGGTTTTGTTGTCAAATACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCAT 1837 34D20_viridis TGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCAT 1873 34D20_nubicola TGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCAT 1857 **************** ****************************************** 34D20_vesca TATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTA---ATCATGGA 1974 34D20_mandshurica TATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTA---ATCATGGA 1974 34D20_nilgerrensis TATC---CTTCGAAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTAGTAATCATGGA 1897 34D20_iinumae TATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTA---ATCATGGA 1868 34D20_ananassa CATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTA---ATCATGGA 1894 34D20_viridis TATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAAATCACGAATGGTA---ATCATGGA 1930 34D20_nubicola TATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAAATCACCAATAGTA---ATCATGGA 1914 *** ***** ******* ************** ***** *** *** ******** 34D20_vesca GATGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 2018 34D20_mandshurica GATGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 2018 34D20_nilgerrensis GATGATGAAGAGCGTAATGATGATGATGTTAATGAACCTTGGTATAGAGAAG-AGACCGT 1956 34D20_iinumae GATGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 1912 34D20_ananassa GATGATGAAGAGCTTAATGATGATGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 1953 34D20_viridis GGTGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 1974 34D20_nubicola GATGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAGGAGACTCT 1959 ****** ***************************** **** 34D20_vesca TATGCCTCAGCAGACGAATTTTTACGACTGCG 2050 34D20_mandshurica TATGCCTCAGCAGACGAATTTT-ACGAGTGCG 2049 34D20_nilgerrensis TATGCCTCAGCAGACGAATTT--ACGACTNNN 1986 34D20_iinumae T-TGCCTCAGCAGACGAATTT--ACGACTNNN 1941 34D20_ananassa TATGCCTCGGCAGACGAATTTT-ACGACTGCG 1984 34D20_viridis TATGCCTCAGCAGACGAATTT--ACGAC-GCG 2003 34D20_nubicola TATGCCTCAGCAGACGAATT---ACGAC-GCA 1987 ****** *********** **** --------------------------------------------------------------------------------------------------------------

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196 40M11 40M11_vesca CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGA 60 40M11_mandshurica CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGA 60 ************************************************************ 40M11_vesca TGCATCAGAACAGTATGTGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTC 120 40M11_mandshurica TGCATCAGAACAGTATGTGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTC 120 ************************************************************ 40M11_vesca ATTAATCTGCAAACAATAAGATTTTTTAGGCAAAGCAGAACTATGAGTTCCCCAAACTAA 180 40M11_mandshurica ATTAATCTGCAAACAATAAGATTTTTTAGGCAAAGCGGAACTATGAGTTCCCCAAACTAA 180 ************************************ *********************** 40M11_vesca TAGCTTTCAAACAAGTAGAGGAGCACATTTACTAAAGATACCTTTGCCTGCTGCTCTTCA 240 40M11_mandshurica TAGCTTTCAAACAAGTAGAGGAGCACATTTACTAAAGATACCTTTGCCTGCTGCTCTTCA 240 ************************************************************ 40M11_vesca CTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTT 300 40M11_mandshurica CTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTT 300 ************************************************************ 40M11_vesca TTGAGGGGAAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACA 360 40M11_mandshurica TTGAGGGGAAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACA 360 ************************************************************ 40M11_vesca GTTCCCTTCGGTCTCCCATCACCATCAAATACAATAAATTTCAATATATTTAACAAAAAA 420 40M11_mandshurica GTTCCCTTCGGTCTCCCATCACCATCAAATACAATAAATTTCAATATATTTAACAAAAAA 420 ************************************************************ 40M11_vesca ATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATTGTTCAATATATCATTTGA 480 40M11_mandshurica ATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATTGTTCAATATATCATTTGA 480 ************************************************************ 40M11_vesca AATTAACAACTTTTATTCTTCTAGTCAACCACATTTCGCAGCTACTTGTTTAACTCATAA 540 40M11_mandshurica AATTAACAACTTTTATTCTTCTAGTCAACCACATTTTGCAGCTACTTGTTTAACTCATAA 540 ************************************ *********************** 40M11_vesca ACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTT 600 40M11_mandshurica ACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTT 600 ************************************************************ 40M11_vesca CACAACGAATCCAACACAAAAGGATCAAAAAAACCATCCAAAACTCATGCACAACATAAT 660 40M11_mandshurica CACAACGAATCCAACACAAAAGGATCAAAAAAACCATCCAAAACTCATGCACAACATAAT 660 ************************************************************ 40M11_vesca CAACCAAATATTTTAACCACAAAAACAAGCACAATTCTCCAAAGTACAAAAAGAAATGGG 720 40M11_mandshurica CAACCAAATATTTTAACCACAAAAACAAGCACAATTCTCCAAAGTACAAAAAGAAATGGG 720 ************************************************************ 40M11_vesca CTTTAGACACCAGGAAGGCATATCAAACCGGCCCACACACGTTAAAGGGATACAAAGATC 780 40M11_mandshurica CTTTAGACACCAGGAAGGCATATCAAACCGGCCCACACACGTTAAAGGGATACAAAGATC 780 ************************************************************ 40M11_vesca TCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAG 840 40M11_mandshurica TCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAG 840 ************************************************************ 40M11_vesca CTCCTCAGATGTCGGAGAGACCCATCTGAACCCAAGTCAACTGCACTGTTACAGCAACTA 900 40M11_mandshurica CTCCTCAGATGTCGGAGAGACCCATCTGAACCCAAGTCAACTGCNNNNNNNNNNNNNNNN 800 ******************************************** 40M11_vesca CAAAACGCAAAGATA GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAGA TGAG 960 40M11_mandshurica NNNNNNNNNNNNNNNNNNNNNNNN-----------------------------------924

PAGE 197

197 40M11_vesca TCACCCGACTGATCAAAGTCGTCCAAAGAATTCTCAAACTGTGGTTTCATAGGCCG 3116 40M11_mandshurica NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN ~3000 --------------------------------------------------------------------------------------------------------------63F17 63F17_vesca CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGG-TCCTACCAACCTCATCA 59 63F17_mandshurica CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGGTCCTACCAACCTCATCA 60 63F17_viridis CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGGTCCTACCAACCTCATCA 60 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 60 63F17_vesca TACATGGGCAAGAAATCATTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTAGA 119 63F17_mandshurica TACATGGGCAAGAAATCATTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTAGA 120 63F17_viridis TACATGGGCAAGAAATCATTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTATA 120 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 120 63F17_vesca TGCAGGTTTTGAATTATTAGAGTCTATAAAGGGACATAGTTACAACTGTTTGTATGCTTT 179 63F17_mandshurica TGCAGGTTTTGAATTATTAGAGTCTATAAAGGGACATAGTTACAACTGTTTGTATGCTTT 180 63F17_viridis TGCCGGTTTTGAATTATTAGAGTCTATAAAGGGAC-TAGTTACAACTGTTT------TTT 173 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 180 63F17_vesca TCCA--TTTTTTTTATTTTTTTATTTTTTGAGAATGTATGCTTTTTCACTTGTATGGCCT 237 63F17_mandshurica TCCA--TTTTTTT-ATTTTTTTATTTTTTGAGAATGTATGCTTTTTCACTTATATGGCCT 237 63F17_viridis TCCACTTTTTTTTTTTTTTTTTTTTTTTTGAGAATGTATGCTTTT-CACTTATATGGCCT 232 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 240 63F17_vesca GAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGC 297 63F17_mandshurica GAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGC 297 63F17_viridis GAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGC 292 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 300 63F17_vesca ACACAGGAACCGTTGAGGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAG 357 63F17_mandshurica ACACAGGAACCGTTGAGGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAG 357 63F17_viridis ACACAGGAACCGTTGAGGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAG 352 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 360 63F17_vesca TTAGAGCAATGGGTGAGATGAACTGGAAACAATTTGCTGCTGAGGAGGTTACAGAGATGA 417 63F17_mandshurica TTAGAGCAATGGGTGAGATGAACTGGAAACAATTTGCTGCTGAGGAGGTTACAGAGATGA 417 63F17_viridis TTAGAGCAATGGGTGAGATGAACTGGAGACAATTTGCTGCTGAGGAGGTTACAGAGATGA 412 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 420 63F17_vesca GGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCAAAGTCACATCCCTTC 477 63F17_mandshurica GGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCAAAGTCACATCCCTTC 477 63F17_viridis GGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCAAAGTCACATCCCTTC 472 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 480 63F17_vesca CTGGATGTGAGAGTTTCCCCGATGCAGGAGGAAATATAACCGGTTCTTTCCTTGGCATTC 537 63F17_mandshurica CTGGATGTGAGAGTTTCCCCGATGCAGGAGGAAGTATAACCGGTTCTTTCCTTGGCATTC 537 63F17_viridis CTGGATGTGAGAGTTTCCCCGATGCAGGAGGAAATATAACCGGTTCTTTCCTTGGCATTC 532 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 540 63F17_vesca AAGAAAATTTGACAATTTGATCACCAGTTCAGTTTTATAGAAGAACTCAGTTAGTACAGT 597 63F17_mandshurica AAGAAAATTTGACAATTTGATCACCAGTTCAATTTTATAGAAGAACTCAGTTAGTACAGT 597 63F17_viridis AAGAAAATTTGACAATTTGATCACCAGTTCAGTTTTATTGAAGAACTCGGT----ACAGT 588 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 600

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198 63F17_vesca TTTGAAACGTTTTTTTGTTGTATTTAGCAAACCCATAGGAGGATAGGGTTTTCTTTTATT 657 63F17_mandshurica ATTGAAACGTTTTTTCGTTGTATTTAGCAAACCCATAGGAGGATAGGGTTTTCTTTTATT 657 63F17_viridis TTTGAAACGTTTTTTTGTTGTATTTAGCAAACGCATAGGAGGATAGGGTTTTCTTTTATT 648 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 660 63F17_vesca CAACAGGGATATAGGCGCTTTTAGGGTTTCTTTTCCTATTCAATTTCGTTCTTTGGTAGA 717 63F17_mandshurica CAACAGGGATATAGGCGCTTTTAGGGTTTCTTCTCCTATTCAATTTCATTCTTTGGTAGA 717 63F17_viridis CAACAGGGATATAGGCGCTTTTAGGGTTTCTTTTCCTATTGAATTTCGTTCTTTGGTATA 708 63F17Rrc_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTCAATTTCATTCTGGGATAGA 720 ** ****** **** ** 63F17_vesca CCAAGTCGCTTCTTTGGCATTCAAGGAAACCTGAGCATTTGATCTGCCTGTCATCACATC 777 63F17_mandshurica CCAAGTCGCTTCTTTGGCATTCAAGGAAACCTGAGCATTTGATCTGCCTGTCATCACATC 777 63F17_viridis CCAAGTCCCTTCTTTGGCATTCAAAGAAACCTAAGCATTTGATCTGCCTGTCATCACGTC 768 63F17Rrc_ananassa CCAAGTCACTTTTTTGGCGTTCAAGGAAACCTGAGCATTTGATCTGCCGGTCATCACATC 780 ******* *** ****** ***** ******* *************** ******** ** 63F17_vesca CAGAGTTGCAGATTGTTTAGAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAA 837 63F17_mandshurica CAGAGTTGCAGATTGTTTAGAGAAGAATTCCAAT----TCCTTTTGTACAGTTTGGTTAA 833 63F17_viridis TAGAGTTGCAGATTGTTTAGAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAA 828 63F17Rrc_ananassa CAGAGTTGCAGATTGTTTATAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAA 840 ****************** ************** ********************** 63F17_vesca CTTTTGGTATTCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGAT 897 63F17_mandshurica CTTTTGGTGTTCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGAT 893 63F17_viridis CTTCTGGTGTTCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGAT 888 63F17Rrc_ananassa CTTTTGGTGTTCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGAT 900 *** **** *************************************************** 63F17_vesca ATGCAGGTAACATCTCTGACTATGCATCTTTGCTTTTTCTTCTTTTTTTGAGAACAAGGC 957 63F17_mandshurica ATGCAGGTAACATCTCTGACTATGCATCTTTGCTTTTTCTTCTATTTTTGAGAACAAGGC 953 63F17_viridis ATGCAGGTAACATCTCTGACTATGCATCTTTGCTTTTTCTTCTGTTTTTGAGAACAAGGC 948 63F17Rrc_ananassa ATGCAGGTAACATCTCTGACTATGCATCTTTGCTTTTTCTTCTTTTTTTGAGAATAAGGC 960 ******************************************* ********** ***** 63F17_vesca ATCTTGTTTATGTGTAGCCAACTTGAAGCACTGTATTTAAATAATGCTAAAACAGTGTTA 1017 63F17_mandshurica ATCTTGTTTATGTGTGGCCAACTTGAAGCACTGTATTTAAATAATGCTAAAACAGTGTTA 1013 63F17_viridis ATCTTGTTTATTTGTGGCCAACTTGAAGCACTGTATTTAAATAATGCTAAGACCGTGTCA 1008 63F17Rrc_ananassa ATCTTGTTTATGTGTAGCCAACTTGAAGCACTGTATTTAAATAATGCTAAAACAGTGTTA 1020 *********** *** ********************************** ** **** 63F17_vesca ATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGAACTTGATAAGAA 1077 63F17_mandshurica ATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAAC------TTGATAAGAA 1067 63F17_viridis ATTTTGTTACAAAAGTCTAGGCAACAATGAACTTGAACTT------------GATAAGAA 1056 63F17Rrc_ananassa ATTTTGTTATAAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGAACTTGATAAGAA 1080 ********* ****** *********************** ******** 63F17_vesca AATAGATCCCAGAGATGGTCTATCTACTACCCTTGACTACACAAGTTACTCATTCTTTAC 1137 63F17_mandshurica AATAGATCCTAGAGATGGTCTATCTACTACTCTTGACTACACAAGTTACTCATTCTTTAC 1127 63F17_viridis AATAGATCCTAGAGATGGTCTATCTACTACCCTTGACTACACAAGTTGCTCATTCTTTAC 1116 63F17Rrc_ananassa AATAGATCCCAGAGATGGTCAATCTACTACCCTTGACTACACAAGTTACTCATTCTTTAC 1140 ********* ********** ********* **************** ************ 63F17_vesca ATGTGAAATGGCTATCCAGAGCAGTCGTA--TTTCATGAGATATTAACAAGCTTTGGACG 1195 63F17_mandshurica ATGTGAAATGGCTATCCAGAGCAGTCGTAAATTTCATGAGATATTAACAAGCTTTGGACG 1187 63F17_viridis ATGTGAAATGGCTATCCAGAGCAGTCGTAAATTTCATGAGATATTAACAAGCTTTGGACG 1176 63F17Rrc_ananassa ATGTGAAATGGCTATCCAGAGCAGTCGTA--TTTCATGAGATATTAACAAGCTTTGGACG 1198 ***************************** ***************************** 63F17_vesca TCCAGTTGCCAGGTTCATCTCTACTCGGAGGCCAAGTCGAGCAAGGGCAGGCACATCAAC 1255 63F17_mandshurica TCCAGTTGCCACGTTCATCTCTACTCGGAGGCCAAGTCGAGCAAGGGCAGGCACATCAAC 1247 63F17_viridis TCCAGTTGCCAGGTTCATGTCTACTCGGAGACCAAGT-GAGCAAGGGCAGGCACATCAAC 1235 63F17Rrc_ananassa TCCAGTTGCCAGGTTCATCTCTACTCGGAGGCCAAGTCGAGCAAGGGCAGGCACATCAAC 1258 *********** ****** *********** ****** **********************

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199 63F17_vesca AGACCCCTTAA 1266 63F17_mandshurica AGACCC----1253 63F17_viridis AGACCCCTAAA 1246 63F17Rrc_ananassa AGACCCTCAA1268 ****** --------------------------------------------------------------------------------------------------------------72E18 72E18_vesca GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAATCCGGCCTAAGCATTAATATC 60 72E18_mandshurica GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAGACCCGGCCTAAACATTAACATC 60 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 60 72E18_viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTAATATC 60 72E18_iinumae GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAACCCGGCCTAAACATTAACATC 60 72E18_ananassa GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAACCCGGCCTAAACATTAACATC 60 72E18_vesca AAA-TCAGTCCTTGAGATTCGACATGCATAAAAAAGACAATAAAGGGTACAAAAACAACC 119 72E18_mandshurica AAAATCAGTCCTTGAGATTCAACATGCATAACAAAGACAATAAAGGGTACAAAAACAACC 120 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 120 72E18_viridis AAAATCAGTCCTTGAGATTCAACATGCATAACAAAGACAATAAACGGTACAAAAACAACC 120 72E18_iinumae AAAATCAGTCCTTGAGATTCGACATGCATAAAAAAGACAATAAAGGGTACAAAAACAACC 120 72E18_ananassa AAAATCAGTCCTTGAGATTCAACATGCATAAAAAAGACAATAACGGGTACAAAAACAACC 120 72E18_vesca ACTCAAACAATCACAACATAATATCATTCAATACCTTGACCATTCCGGTTCCATTATCAC 179 72E18_mandshurica ACTCAAACAATCACAACATAATATCATTCAATACCTTGACCATTCCGGTTCCATTATCAC 180 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 180 72E18_viridis ACTCAAACAATCACAACATAATATCATTCAATACCTTGACCATTCCGGTTCCATTATCAC 180 72E18_iinumae ACTCAAACAATCACAACATAATAGCATCCAATACCTTGACCATTCCGGTTCCATTATCAC 180 72E18_ananassa ACTCAAACAATCACAACATAATATCATTCAATACCTTGACCATTCCGGTTCCATTATCAC 180 72E18_vesca ACACAAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAACATACACCAC 239 72E18_mandshurica ACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAACATACACCAC 240 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 240 72E18_viridis ACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAACATACACCAC 240 72E18_iinumae ACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCTACATACACCAC 240 72E18_ananassa ACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAACATACATCAC 240 72E18_vesca AATCAATGACAACAATGCCTCATTCACACAACAAAGAAATAGACATTCAAAAACAAAACA 299 72E18_mandshurica AATCAA--------ATGCTACATTCACACAACAAAGAAATAGACATTCAAAGACAAAACA 292 72E18_nilgerrensis NNNNNN--------NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 292 72E18_viridis AATCAATGACAACAATGCCTCATTCACACAACAAAGAAATAGACATTCAAAAACAAAACA 300 72E18_iinumae AATCAATGACACCAATGCCTCATCGACACAACAAACAAATAGACATTCAAAAACAAAACA 300 72E18_ananassa AATCAATGACAACAATGCCTCA-----------AACAAATAGACATTCAAAAACAAAACA 289 72E18_vesca CAATACACACTACTAATGTG---------------------------------------319 72E18_mandshurica CAA-ACACACTACTAACGTG---------------------------------------311 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNN---------------------------------------312 72E18_viridis CAATACACACTACTAATGTG---------------------------------------320 72E18_iinumae CAATACACGATGCTAACATTTCCCTAATCTCTCCTCCATCAACTAAAATCTCCATTCCAA 360 72E18_ananassa CAATACACAATGCTAACATTTCCCTAATATCTCCTCCATCAACTAAAATCTCCATGCCAG 349 72E18_vesca -------------GCACAGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACAT 366 72E18_mandshurica -------------GCACGGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACAT 358 72E18_nilgerrensis -------------NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 359 72E18_viridis -------------GCACAGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACAT 367 72E18_iinumae ATCACACACTACTACACAGAAACCAAAGCATGATTCAAAACAAACCAAGAACATCTACAT 420 72E18_ananassa ATCAC---------CACAGAAACCAAAGCATGATTCAAAACAAACCAAGAACATCTACAT 400

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200 72E18_vesca AGTTCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTA 426 72E18_mandshurica AGTTCTCTCACAATAGTAAAGAAACGATCGTTGACAATCAAAAGGCATCGAAAGCTAGTA 418 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 419 72E18_viridis AGTTCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTA 427 72E18_iinumae AGTCCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTA 480 72E18_ananassa AGTTCTCTCACAATAGTAAAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTA 460 72E18_vesca AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTACATGCATAAAACCCTC 486 72E18_mandshurica AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTATATACATAAAACCCTC 478 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 479 72E18_viridis AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTACATGCATAAAACCCTC 487 72E18_iinumae AAGAAACGATCTTTCAGATGGGAAATACCCAAATTTGATTGCTATATACATAAAACCCTC 540 72E18_ananassa AAGAAACGATCTTTCAGATGGGAAATGCCCAAATTTGATTACTATATACATAAAACTCCC 520 72E18_vesca AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAAAA-TTCAA 545 72E18_mandshurica AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAAAAATTCAA 538 72E18_nilgerrensis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 539 72E18_viridis AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAAAAAATTCA 547 72E18_iinumae AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATTAAAAAAAAATCAA 600 72E18_ananassa AAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACAG-AAAAAAAATTCAA 579 72E18_vesca GAAAA-AAA GAGAGAGA --AAATTACAGATTTAAAGCGACGAACAA-TGAAAAGGAATGA 601 72E18_mandshurica GAAAA-AAA GAGAGAGA --AAATTACAGATCTAAAGCGACGAACAG-TGAGAAGGAATGA 594 72E18_nilgerrensis NNAAA-AAA GAGAGAGAGA ---TTACAGATCTAN-GCGACGAACAA-TGAGAAGGAATGA 593 72E18_viridis AGAAATAAA GAGAGAGA --AAATTACAGATCTAAAGTGACGAACAA-TGAGAAGGAATGA 604 72E18_iinumae GAAAAAAAGAA GAGAGA --AAATTACAGATCTAAAGCGACGAACAAATGAGAAGGAATGA 658 72E18_ananassa GAAAAAAAAAA GAGAGAGA AAATTACAGATCTAAAGCGACGAACAA-TGAGAAGGAATGA 638 *** ** ** **** ******** ** ******** *** ********* 72E18_vesca GAGGCAAAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGG GAGAGA 661 72E18_mandshurica GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGG GAGAGA 654 72E18_nilgerrensis GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGT-------GA 645 72E18_viridis GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGG-GAGA 662 72E18_iinumae GAGACAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGAATGAGAGT-------GAGAGA 710 72E18_ananassa GAGGCAGAGAGAAGAGATGAGGAAGTTGACCTTTGTGA----------GTGAGG GAGAGA 688 *** ** ******************************* **** *** *** 72E18_vesca GAGAGAGAGA TCGACGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 721 72E18_mandshurica GAGAGAGAGA TCGACGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 714 72E18_nilgerrensis GAGAGAGAGA TCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 706 72E18_viridis GAGAGAGAGA TCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 722 72E18_iinumae GAGAGAGAGA TCGAAGACGAGGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 771 72E18_ananassa GAGAGAGAGA TCGAAGACGAAGCTGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 749 ************* ***** ** ************************************ 72E18_vesca GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 781 72E18_mandshurica GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 774 72E18_nilgerrensis GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 765 72E18_viridis GCGAA-GAATTGNACCNNNATANAGGAGTGNGATTGACNAGTTATCTCNGCNGNTTGATT 781 72E18_iinumae GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 830 72E18_ananassa GCGAAAGAATTGGAGCAAAATAAAGGAGTGGGATTGACGAGTAATCTCAGCCGTTTGATT 808 ***** ****** *** ******* ******* *** ***** ** ****** 72E18_vesca TATGGACCGCGTCTATTGAGCCCTTGTGGGG-CCATTACAGCTCCTTCCGCTGTTCCAGT 840 72E18_mandshurica TATGGACCGCGTCTATTGCGCCCTTGTGGGG-CCATTACAGCTCCTTCCGCTGTTCCAGT 833 72E18_nilgerrensis TATGGACCGCGTCCATTGCGCCCTTGTGGGG-CCATTACAGCTCCTTCCGCTGTTCCAGT 824 72E18_viridis TATGGACCGCGTCCATTGTGCCNTTGTGGGG-CCATNACNGCTCCTNCCNCTGTNCCNGC 840 72E18_iinumae TATGGACCGCGTCCATTGCGCCCTTGTGGGGGCCATTACAGCTCCTTCCGCTGTTCCAGT 890 72E18_ananassa TATGGACCGCGTCCGTTGCGCCCTTGTGGGG-CCATTGCAGCTCCTTCCGCTGTTCCAGT 867 ************* *** *** ******** **** ****** ** **** **

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201 72E18_vesca CATTTTTTTCTCCACCTTCTTTACCTTTTTGCCCCTCAGTCCCTT-CCCTTTTCTCCCAA 899 72E18_mandshurica CATTTTTTTCTCCACCTTCTTCACCTTTTTGCCCCTCAGTCCCTT-CCCTTTTCTCCCAA 892 72E18_nilgerrensis CATTTTTTTCTCCACCTTCTG------------CCCCATTCCCTT-CCCTTTTCTCCCAA 871 72E18_viridis CATNTTTATCTCCACCTTCTNCACNTTTNTGCCNCNCNTGNCCNT-CCCTTTTCTCCCNN 899 72E18_iinumae CATTTTT--CTCCACCTTCTTCACCTTTCTGCCCCTCGTTCCCTTTCCCTCTTCTCCCAA 948 72E18_ananassa CATTTTT--CTCCACCTTC----------------------------------------884 *** *** ********** 72E18_vesca TTCTT---------------------------------------------TCTCAACTCT 914 72E18_mandshurica TTCTT---------------------------------------------TCTCAACTCT 907 72E18_nilgerrensis TTCTT---------------------------------------------TCTCAACTCT 886 72E18_viridis TTCTN---------------------------------------------NCTCANATCT 914 72E18_iinumae CTCTTCTTAGCCTAATTGCATTTTCATTTTAGTGCTTAGATCAATATGATTNNNNNNNNN 1008 72E18_ananassa -----------------------------------------------------------72E18_vesca TCTTAAACCTAATTGCATTTCCCTAATTGCATTTTCATTTTAGTGCTTAGATCAATATGA 974 72E18_mandshurica TCTTAAACCTAATTGCATTTCCCTAATTGCATTTTCATTTTAGTGCTGAGATCAATATGA 967 72E18_nilgerrensis TCTTAAACCTAAT--------------TGCATTTTCATTTTANTGCTTANATCAATATGA 932 72E18_viridis TCTNAANGCTNATNGCNTTTNCCNNANNGCATTTGNATTTNNGNGCTTCNATCAATNCGN 974 72E18_iinumae NNNNNNNNNNNNNN------------NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1056 72E18_ananassa ACCTTTTTGCCCCT------------CATTCCCTTTCCCTCTTCTCCGATCCCAACTTTC 932 72E18_vesca TTAAGAAGCTTCATTTTGTCAACACAAGGCAACAAGGACA--CAGGGGAGCATTTCGATC 1032 72E18_mandshurica TTAAGAAGCTTCATTTTGTCAACACAAGGCAACAAGGACA--CAAGGGAGCATGTCGATC 1025 72E18_nilgerrensis TTAAGAAGCTCCATTTTGTCAACACAAGGCAACAAGGACN--TANGGGAGCATGTCGATC 990 72E18_viridis NNNNAANGCTNCGTNTTGTCNTNNCANGGNTNCAAGGANCCTNNGGGANGCNNGTTGATC 1034 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1116 72E18_ananassa TCAACTCTTCTTAACCCACCCAGTTGCATTTTCATTGTAGTGCTTAAATCAATATGGTTA 992 72E18_vesca ATCGTTCCAGTCATTTTCGTATATAAT-TTGGGCTTGAAATGGTTGATC-GGTCGTAAAA 1090 72E18_mandshurica ATCGTTCCAGTCATTTTCGTATATAAT-TTGGGCTTGAAATGGTTAATC-AATCGTAAAA 1083 72E18_nilgerrensis ATCGTTCCGGTCACTTTCGTATATAAT-TTGGACTTAAAATGGTTGATC-GATCGTAAAA 1048 72E18_viridis GTCAANTNCGGCCTGGTNATANANAATATNNGACNTNAAATGGTTGATTGNNTCGTTAAA 1094 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1176 72E18_ananassa AGAAGCTTCATTTTGTCAACACAAGGCAACAAGGACATAGGGGAGCATGTGGATGATCGT 1052 72E18_vesca TTTAAAATGACGTTTTGATATGATATCTATAAAGA-GGACATAATTTACTTTGTATGTCA 1149 72E18_mandshurica TTTAAAATGACGTTTTGATATGATATCTATAAGGA-GGACATAATTTACTTTATATGTCA 1142 72E18_nilgerrensis TTTGAAATGACGTTTTGGTATGATATTTGTAAAGA-GGACATAATTTACT---------1097 72E18_viridis TTNGAAATAANNNNNNNNNNNNNNNNNNNNNNAAAAGGNCNTAATTTANTNNGTANNTCA 1154 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN----1231 72E18_ananassa TTTTAAACTACGTTTTGGTATGATATCGTAAGGAGGACATAATTTACTTTGTATG----1107 72E18_vesca GGTTTTAATATGGAA----CGAAGAGTATGGGTGAAA-GTGTC-ATCCCACACATTTTAA 1203 72E18_mandshurica GGTTTTAATACGGAA----GGAAGAGTATGGGTGAAA-GTGTC-ATCCCACACATTTTAA 1196 72E18_nilgerrensis ---------ATGGAA----CGAAGAGTATAGGTGAAA-GTGTC-ATCCCACACATTTTAA 1142 72E18_viridis AGGTTTTAANTAGGGAAATGGAGGAGTGNGGGTGAAAAGTGTCCATCCCACACGTTTTAA 1214 72E18_iinumae ---------NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1282 72E18_ananassa ---------TCNGGTTTTTAATACGGAATGAAGAGTGTGGGAGAAAGTGTCATCCCACAC 1158 72E18_vesca AAGAGCTGTAATGTAGGG-TAATGAGCA-CAACTGCAAGCTGCATCC-TATAA-TGGATC 1259 72E18_mandshurica AAGAGCTGTAATGTAGGG-TAATGAGCA-CAACTGCAAGCTGCATCC-TATAA-TGGATC 1252 72E18_nilgerrensis AAGAGCTTTAATGTAGGGGTAATGAGCA-CAACTACAAGCTGCATCC-TATAA-GGGATC 1199 72E18_viridis ANGAGCNTTAANGNAGGG-NAANGNGCAGNAACNGCAAGCTGCATNCCTNTAATNGGATC 1273 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1342 72E18_ananassa ATTCATTTTAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1218

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202 72E18_vesca AATCAGAACATTAA-ACAACGTAAAGAGGAAGGTATTTGCTTTACACAACCTTATAAAAT 1318 72E18_mandshurica AATCAGAACAATAA-ACAACGTAAAGAGGAAGGTATTTGCTTTACACAACCTTATAAAAT 1311 72E18_nilgerrensis AATCAGAACATTAA-ACAACGTAAAGAGGAAGGTATTTGCTTTACACAACCTTATAAAAT 1258 72E18_viridis AATCAGAACATTAAGNCAACGTAAAGAGGAAGGTATTTGCTTTACACAACCTTATAAAAT 1333 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1402 72E18_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1278 72E18_vesca GATGAGGATCTACTC-AAAATCCAGACTACCATGGTTGGCAAAATTAGATCCTCACTGTA 1377 72E18_mandshurica GATGAGGATCTACTC-AAAATCCAGACTACCATGGTTGGCAAAATTAGATCCTCACTGTA 1370 72E18_nilgerrensis GATGAGGATCTACTC-AAAATCCAGACTACCATGGTTGGCAAAATTAGATCCTGCCTGTA 1317 72E18_viridis GATGNGGATNTACTCCAAAGTGAGGACTACCATGGTCGGCAAAATTAGTTCCTGACTGTA 1393 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1462 72E18_ananassa NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAAAATCCAGACTACC 1338 72E18_vesca ACCAGCTAGGCATTGGTAATGCATAATAGCTATAGCTAACTATAGGTGGGAGACTCATCA 1437 72E18_mandshurica ACCAGCTAGGCATTGGTAATGCATAATAGCTATAGCTAACTATAGGTGGGAGACTCATCA 1430 72E18_nilgerrensis ACCAGCTAGGCATTGGCAATGCATAATAGCTAGAGCTAACCATAGGTGGGAGACTCATCA 1377 72E18_viridis ACCAGCTAGGCATNGGCAATGCATAATAGCTATAGCTGACTATAGGTGGGAGACTCATCA 1453 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1522 72E18_ananassa GTGGTGGGCAAAATTAGATCCTCACCTATAGTAGGGTAACTATAGGTGGGAGACTNGTCA 1398 72E18_vesca TTGAGATCATAGAAAAACAAAGATGAAAGAAAGAAATGAAGAAACAAGCAACAGCTATTC 1497 72E18_mandshurica TTGAGATCATAGAAAAACAGAGATGAAAGAAAGAAATGATGAAACAAGCAACAGCTATTC 1490 72E18_nilgerrensis TTGAGATCATAGGAAAA------------AAAATGATGA--AAACAAGCAACAGTTATTC 1423 72E18_viridis TTGAGATCAGAGAAAAACNAAGATGAAAGAAAGAAATGATGAAACAAG-AATAGTTATTC 1512 72E18_iinumae NNNNNNNNNNNNNNNNN---------------NNNNNNNNNNNNNNNNNNNNNNNNNNNN 1567 72E18_ananassa TTGTGATCATAGAAAAA---------------GAAATGATGAAACAAGCAACAGTTACTC 1443 72E18_vesca GAAAGCAAGTACAGAAGGGATTGTTCATGAAGTGTTCACCAAGTCACAGCTTAGGGCATT 1557 72E18_mandshurica GAAAGCAAGTACAGAAGGGATTGTTCATGAAGTGTTCACCAAGTCACAGCTTAGGGCATT 1550 72E18_nilgerrensis GAAAGCAAGTACAGAAGGGATTGTTCATTAAGTGTTCACCAAGTCACAGCTTAGGGCATT 1483 72E18_viridis GAAAGCAAGTACAGAAGGGATTGTTCATGAAGTGTTCACCAAGTCACAGCTTAGGGCATT 1572 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1627 72E18_ananassa GAAAGCAGGTACAGAAGGGATTGTTCATGAAGTGTTCACCAAGTCNCAGCTTAGGGCATT 1503 72E18_vesca CTTAGAAGTAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATATTAA 1617 72E18_mandshurica CTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATATTAA 1610 72E18_nilgerrensis CTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATATTTA 1543 72E18_viridis CTTAGAAGCAACAAGCTTGCCAACTTCCATTTACTTGTTTCAAGTTCATGNTGATATTAN 1632 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1687 72E18_ananassa CTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCAGGGTGACATTAC 1563 72E18_vesca CCATCCAACGAAAACATCCAAAGGTACTGTGACAGAAAGCTCAAGGGGATATCTGTGTTT 1677 72E18_mandshurica CCATCCAACGAAAACATCCAAAGGTACTGTGACAGAAAGCTCAAGGGGATATCTGTGTTT 1670 72E18_nilgerrensis -----------------------GTCATGT-------------------------TGTTT 1555 72E18_viridis CCATCCAACGAAAACATCCAAAGGTNCTGTGACTGAAAGCTCAAGGGGATATCNGTGTTT 1692 72E18_iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGAAAGCTCAATGGGATATCTGTGTTT 1747 72E18_ananassa CCNTCCAACGAAAACATCCAAAGGTACTGTGACTGAAAGCCCAAGGGGATATCCGTGTTT 1623 ***** 72E18_vesca AAAGCCACAACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTG 1737 72E18_mandshurica AAAGCCACAACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTG 1730 72E18_nilgerrensis AAAGCCACAACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTG 1615 72E18_viridis AAAGCCACTACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTG 1752 72E18_iinumae AAAGCCACAACATGACTAAATATAATTGCTCCCAATTTCTAAAGTTACATTCGTTTTGTA 1807 72E18_ananassa AAAGCCACAACATGACTAAATATAATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTG 1683 ******** ********************* ****************************

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203 72E18_vesca CAAATGACAAAACAGTTCAAATTGACTGCATAA-ATAGATTACTCTTGTATAGATCAACA 1796 72E18_mandshurica CAAATGACAAAACAGTTCAAATTGACTGCATAA-ATAGAT-ACTCTTGTATAGATCAACA 1788 72E18_nilgerrensis CAGATGACAAAACAGTTCAAATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACA 1675 72E18_viridis CAAATGACAAAACAGTTCTAATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACA 1812 72E18_iinumae CAAATGACAAAACAGTTCAAATTGACTGCATAA-ATAGATTACTCTTGTATAGATCAACA 1866 72E18_ananassa CAAATGACAAAACAGTTCAAATTGACTGCATAAGATAGATTACTCTTGTATAGATCAACA 1743 ** *************** ************** ****** ******************* 72E18_vesca AGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAAT 1856 72E18_mandshurica AGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAAT 1848 72E18_nilgerrensis AGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAACACTAACATAAATATTGAAAT 1735 72E18_viridis AGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAAAACTAACATCAATATTGAAAT 1872 72E18_iinumae AGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAAC 1926 72E18_ananassa AGCAAATCTCCAAATTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAAT 1803 ************* ************************* ******** ********** 72E18_vesca TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTG 1916 72E18_mandshurica TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTG 1908 72E18_nilgerrensis TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCATAGGAAATGGAATTG 1795 72E18_viridis TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGAGCTATCGTAGGAAATGGAATTG 1932 72E18_iinumae TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTG 1986 72E18_ananassa TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTG 1863 ************************************** ***** *************** 72E18_vesca AACACTTGACACCAAAGAGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGG 1976 72E18_mandshurica AACACTTGACACCAAAGAGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGG 1968 72E18_nilgerrensis AACACTTGACACCAAAGAGAACAACGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGG 1855 72E18_viridis AACACTTGACACCAAAGAGAACAACGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGG 1992 72E18_iinumae AACACTTGACACCAAAGAGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGG 2046 72E18_ananassa AACACTCGACACCAAAGAGAACAACAAAGGTAGCCTCACCAATCACTTCTACAAGAATGG 1923 ****** ***************** *********** ********************** 72E18_vesca GGGTAGAATCACCCATCGACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTG 2036 72E18_mandshurica GGGTAGAATCACCCATCGACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTG 2028 72E18_nilgerrensis GGGTAGAATCACCCATCTACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTG 1915 72E18_viridis AGGTAGAATCACCCATCGACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTG 2052 72E18_iinumae GGGTAGAATCACCCATCGACGTGGATACTTGGGTCTTCCGTCCTTCCCATCAAATAGCTG 2106 72E18_ananassa GGGTAGAATCACCCATCGACGTGGATACTTGGGTCGTCCGTCCTTCCCATCAAATAGCTG 1983 **************** ***************** ************************ 72E18_vesca GACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACT 2096 72E18_mandshurica GACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACT 2088 72E18_nilgerrensis GACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACT 1975 72E18_viridis GACATGGCAGGGTGTCACAAAAGATCAATATTGCATGCAAAGAGCTTCTACATACAAACT 2112 72E18_iinumae GACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACT 2166 72E18_ananassa GACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTACATACAAACT 2043 ************************************* ********************** 72E18_vesca CAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAG 2156 72E18_mandshurica CAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAG 2148 72E18_nilgerrensis CATATGGATATGTTCTGGCGATTGCAGAATATAATTATGTATACAAATATGCATGTACAG 2035 72E18_viridis CAAATGGAGATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAG 2172 72E18_iinumae CAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAG 2226 72E18_ananassa CAAATGGATATGTTCTGGCGCTCGTAGAATATAATTATGTATACAAATATGCATGTACAG 2103 ** ***** *********** *********************************** 72E18_vesca AGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAA 2216 72E18_mandshurica AGCTTCCACATACAAACTCATATGTATACTTGTAAATTTATGCAATTTAATTCCAATAAA 2208 72E18_nilgerrensis AGCTTCTACATACAAACTCATACGAATACTTGTAAATTTAGGCAATTTAATTCCAATAAA 2095 72E18_viridis AGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAA 2232 72E18_iinumae AGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAA 2286 72E18_ananassa AGCTTCCACATACAAACTCATATGAATACTTGTAAATTTATGCAATTTAATTCCAATAAA 2163 ****** *************** *************** *******************

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204 72E18_vesca GGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAA---CAGACAAAACATTTAAGCA 2273 72E18_mandshurica GGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAAA--CAGACAAAACATTTAAGCA 2266 72E18_nilgerrensis GGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAA-----GACAAAACATTTAAGCA 2150 72E18_viridis GGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAAAAAAAGACAAAACATTTAAGCA 2292 72E18_iinumae GGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAAAAAC---AGACAAAACATTTAAGCA 2343 72E18_ananassa GGTGAGTTTAAATAGACCAAGATGTTAGCTAAAAA--AC---AGACAAAACATTTAAGCA 2218 *********************************** ***************** 72E18_vesca AAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAG 2333 72E18_mandshurica AAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAG 2326 72E18_nilgerrensis AAAGAAGAGCAGTAGAAGTTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAATG 2210 72E18_viridis AAAGAAGAGCAGTAGAAGTTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAG 2352 72E18_iinumae AAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAG 2403 72E18_ananassa AAAGAAGAGCAGTAGAAGTTATTAAGATACCAAACAACATATTTGGGTTGGAGGACAAAG 2278 ****************** *************************************** 72E18_vesca TAGTATAGAGGAGTGTACCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCAT 2393 72E18_mandshurica TAGTATAGAGGAGTGTTCCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCAT 2386 72E18_nilgerrensis TAGCATAGAGGAGTGTACCTTCTTTAAACGGCGGTGCTTTCCTAGGGCCCAGTTGGTCAT 2270 72E18_viridis TCGTATAGAGGAGTGTACCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCAT 2412 72E18_iinumae TAGTATAGAGGAGTGTACCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCAT 2463 72E18_ananassa TAGTATAGAGGAGTGTACCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCAT 2338 ************ ******************* ************** ******** 72E18_vesca GATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAA 2453 72E18_mandshurica GATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAA 2446 72E18_nilgerrensis TATAGAAGCAGCAACTGCAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAA 2330 72E18_viridis GATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACTGTCTGTGTTGCAATATTAAA 2472 72E18_iinumae GATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAA 2523 72E18_ananassa GATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAA 2398 *************** ********************** ******************** 72E18_vesca ACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAA 2513 72E18_mandshurica ACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAA 2506 72E18_nilgerrensis ACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAA 2390 72E18_viridis ACCCAACCACTGATAAATCTCAGTTGTGTAATTTGCACATGTCACAATATTGAATAGAAA 2532 72E18_iinumae ACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAA 2583 72E18_ananassa ACCCAACCACTGATAAATCTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAA 2458 ************************ *********************************** 72E18_vesca ACCACGTGGTATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAAT 2573 72E18_mandshurica ACCACGTGGTATTTGATAGCCTCCACTTCCATCAAGACTTCTCAGATTCCTCAGTAGAAT 2566 72E18_nilgerrensis ACCACGAGGTATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAAT 2450 72E18_viridis ACCACGTGGTATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAAT 2592 72E18_iinumae ACCACGTGGTATTTGATAGCCTCCACTTCCATCAGGACTTCGCAGATTCCTCAGTAGAAT 2643 72E18_ananassa ACCACGTGGTATTTGATAGCCTCCGCTTCCATCAGGACTTCGCAGATTCCTCAGTAGAAT 2518 ****** ***************** ********* ****** ****************** 72E18_vesca ATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA 2622 72E18_mandshurica ATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA 2615 72E18_nilgerrensis ATGGCAATACAAGTTCGCGATTTGATTTATTATCCCAAAACCAAACCCA 2499 72E18_viridis ATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA 2641 72E18_iinumae ATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA 2692 72E18_ananassa ATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA 2567 ********* ******** ****************************** --------------------------------------------------------------------------------------------------------------

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205 LIST OF REFERENCES Abdulova G, Ananiev E, Grozdanov P (2002) Isolation and purific ation of nuclear DNA from excised cotyledons of Cucurbita pepo L.(zucchini). Bulg. J. Plant Physiol. 28: 3-11 Ahmadi H, Bringhurst RS, Voth V (1990) Modes of inheritance of photoperiodism in Fragaria J. Amer. Soc. Hort. Sci. 115: 146 Akiyama Y, Yamamoto Y, Ohmido N, Oshima M, Fukui K (2001) Estimation of the nuclear DNA content of strawberries ( Fragaria spp.) compared with Arabidopsis thaliana by using dual-stem flow cytometry. Cytologia 66: 431-436 Albani M, Battey NH, Wilkinson MJ (2004) The development of ISSR-derived SCAR markers around the Seasonal Flowering Locus (SFL) in Fragaria vesca Theoretical & Applied Genetics 109: 571-579 Aljanabi SM, Forget L, Dookun A (1999) An improved rapid prot ocol for the isolation of polysaccharide and polyphenol-free suga rcane DNA. Plant Mol. Biol. Rep. 17: 1-8 Anonymous (1980) IEEE Transactions on Magnetics 16: 387-490 Anonymous (1998) Montreal Protocol on Substances that Deplete the Ozone Layer. United Nations Environmental Program (UNEP). Anonymous (2001) Journal of Magnetism and Magnetic Materials 225: 1-314 Antonius K, Ahokas H (1996) Flow cytometric determ ination of polyploidy level in spontaneous clones of strawberries. Hereditas 124: 285 Arnau G, Lallemand J, Bourgoin M (2003) Fast and reliable stra wberry cultivar identificaion using inter simple sequence repeat (ISSR) amplification. Euphytica 129: 69-79 Arulsekar S, Bringhurst RS, Voth V (1981) Inheritance of PGI phosphoglucoisomerase and LAP leucine aminopeptidase isozymes in oct oploid cultivated strawberry. J Amer Soc Hort Sci 106: 679-683 Ashley MV, Wilk JA, Styan SMN, Craft KJ, Jones KL, Feldheim KA, Lewers KS, Ashman TL (2003) High variability and disomic segregat ion of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae). Theor Appl Genet. 107 Barakat A, Matassi G, Bernardi G (1998) Distribution of genes in the genome of Arabidopsis thaliana and its implications for the genome orga nization of plants. Proc Natl Acad Sci U S A 95: 10044 Bedbrook J, Gerlach W, Thompson R, Flavell RB (1980) Emergent Techniques. University of Minnesota Press, Minneapolis

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220 BIOGRAPHICAL SKETCH Denise Cristina Manfrim Tombolato was bor n to Vadir and Marlene Tombolato on March 20, 1976, in Campinas, So Paulo, Brazil. She rece ived her bachelors degree in agronomic engineering from the Escola Superior de Agricultu ra Luiz de Queiroz, at the University of So Paulo in 1998. While an undergradu ate student in Brazil, Denise was granted the FAPESP and CNPq fellowships to investigate molecular mark ers for disease resistance in maize, working under the supervision of Dr. Luiz Eduardo Aran ha Camargo at the Plant Pathology Department. During the last year of her underg raduate studies, she was introduced to University of Floridas professor Dr. Richard D. Berger, who was on sabba tical studies at ESALQ. Denise was invited to spend one semester in Dr. Bergers laborat ory, carrying out investig ation on plant disease epidemiology for the completion of her degree. Dr. David Pete Weingartner granted her a re search assistantship from 1999 to 2002, when she earned her masters degree from the Plant Pat hology Department at the University of Florida. In 2002, she started her doctorate program at the Horticultural Sc iences Department, where she attended the majority of the courses offered to the Plant Molecular and Cellular Biology program. Upon graduation, she will work as a genetic technologist for Ball Helix, the biotechnology research branch of Ball Horticul tural Company in West Chicago. She is very excited about the upcoming changes in her life!