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Structural Genomics of Fragaria--Wild and Cultivated Strawberries

Permanent Link: http://ufdc.ufl.edu/UFE0021284/00001

Material Information

Title: Structural Genomics of Fragaria--Wild and Cultivated Strawberries
Physical Description: 1 online resource (220 p.)
Language: english
Creator: Tombolato, Denise C
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 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
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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.
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.
Statement of Responsibility: by Denise C Tombolato.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Folta, Kevin M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021284:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021284/00001

Material Information

Title: Structural Genomics of Fragaria--Wild and Cultivated Strawberries
Physical Description: 1 online resource (220 p.)
Language: english
Creator: Tombolato, Denise C
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 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
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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.
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.
Statement of Responsibility: by Denise C Tombolato.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Folta, Kevin M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021284:00001


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55415688f6c6e965c2e9637f9ab40b9fef6182b3







STRUCTURAL GENOMICS OF Fragaria-WILD AND CULTIVATED STRAWBERRIES


By

DENISE CRISTINA MANFRIM TOMBOLATO
















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































2007 by Denise Cristina Manfrim Tombolato






























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 Lery, in "History of a Voyage to the Land of Brazil, Otherwise Called America", 1578









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.









Berger, Terry A. Davoli, D. Pete Weingartner, 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: BalSa Terzic', Sylvia

Morais de Sousa, Gisele, Jens, and Gabriel Schoene, Mark D. Skowronski, Luciana C. B.

Manfrim Bchir, Gustavo Ramirez, Juliana and Gustavo Astua, Aaron Hert, Botond Balogh,

Abby Guerra, Ahu Demir, Petr6nio Pinheiro, Ilka V. Araujo, Maggie Kellogg, Maria Beatriz

Padua, 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, Claudia Riegel, Valerie Rodriguez-Garcia Schweigert,

Lisa Olsen, Jared Greenberg, Wendy Gonzalez, and David Adato. Every one of them made my

life in Gainesville a more enjoyable experience.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S .................................................................. ........... ............. .....

LIST OF TA BLES .............. ......... ........................................................... 8

LIST OF FIGURES .................................. .. ... ... ...............9

A B S T R A C T ......... ........................ ........................................... ................ 1 1

CHAPTER

1 STRAWBERRY AND THE GENOMICS ERA........................................... ............... 12

In tro d u ctio n ............... ........................................ ................... ................ 12
M olecular M arkers for Straw berry .......................................................... ............... 13
T he G enom ics E ra ............................................................................ 14

2 DNA EXTRACTION FROM RECALCITRANT SPECIES............... ................16

In tro d u ctio n ................. ....... ............................... ................... ................ 16
T he D N A E xtraction Procedure ............................................................. ....................16
D N A Extraction from Plants ......................................................................... 19
M material and M methods ............... ............................ ............ ..... ......... .......... 21
R e su lts .................................................................................. ................ 2 4
Components of the "Strawberry Protocol"...................................................................26
O ptim ization of the C TA B Protocol...........................................................................27
Leaf tissue state ................................... ................................ ......... 27
Incubation temperature and duration........................ ............................28
Tissue-to-buffer ratio.................... ................... ..... ....... 28
Tissue m aceration m ethod......... ................ ................................... ............... 30
D iscu ssio n ............. .................. .................. ...................................................... 3 1

3 PRIMARY ANALYSES OF Fragaria GENE distribution ........................................ 42

In tro d u ctio n .......................................... ...... ....................... ................ 4 2
M materials and M methods .................................. ... .. ...... ..... .. ............45
Results ......... ...... ......... ................................48
Expressed Sequence Tags (ESTs) ...........................................................................49
Sim ple Sequence Repeats (SSRs) ............................................................................49
D iscu ssio n ................... ...................4...................9..........

4 GENE-PAIR HAPLOTYPES: NOVEL MOLECULAR MARKERS FOR
INVESTIGATION OF THE Fragaria x aananssa OCTOPLOID GENOME...................... 55

Introduction ......... ... ........... ......................................... ............................55









M materials and M methods ...................................... .. ......... ....... ...... 58
R e su lts ................... ...................6...................2..........
G P H 5 ..........................................................................6 3
G P H 2 3 .........................................................................6 4
G P H 1 0 ....................................................................................................................... 6 4
72E 18 ...... ........ ............................................................................. ......6 5
D iscu ssion ...... ........ .............................................................................. .....66

5 GENE-PAIR HAPLOTYPES: FUNCTIONAL AND TRANSFERABLE MARKERS
AS NOVEL ADDITIONS TO THE DIPLOID Fragaria GENETIC LINKAGE
R E FER EN C E M A P ................................... ........................................................ 82

Intro du action ........... ..... ......................................................................... 82
M materials an d M eth o d s ...........................................................................................................8 5
Results ........... ......... ......... ....................................88
D iscu ssio n ........................................................................................... 8 9
C onclu sions.......... ..........................................................9 1

APPENDIX

A DNA EXTRACTION PROTOCOLS ............... ..... ..................... 98

DNA Extraction from Leaves ........... ... ... ......... ...... ... .........98
DNA Extraction from Isolated Nuclei .................... ...... ....... ............ .........101
Modifications of Murray and Thompson 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
H APLOTYPES ................................................... ........ ..................................... 115

D SEQUENCES GENERATED DURING CHARACTERIZATION OF "GENENPAIR
H A PL O TY PE S". ........................................................................................................... 117

E GENE-PAIR HAPLOTYPE INDIVIDUAL LOCI ALIGNMENTS .............. .............. 153

Gene Pairs D detected by M icrocolinearity ....................................................................... 153
Gene Pairs Detected Through Prediction from Genomic Sequence...............................166

LIST OF REFEREN CES ......................................................... ............... ............. 205

BIOGRAPHICAL SKETCH ................... ..................................... 220









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 maceration methods and incubation temperatures on
DNA yield and purity.................... ........................ ...... 38

3-1 Number of simple sequence repeats (with a minimum of 5 repeats) observed in
Fragaria vesca genom ic sequence.......................................................... ............... 54

3-2 Different types of dinucleotide and trinucleotide repeats observed in Fragaria vesca
genom ic sequence ........................................... ........................... 54

4-1 PCR primers designed for amplification of micro-colinearity-inferred putative
intergenic fragm ents........ ...................................................................... .......... ....... 72

4-2 PCR primers that allowed amplicon generation. .................................... .................73

4-3 Overview of insertions and deletions detected through alignment of all sequenced
clon es .......................................................... ...................................80

5-1 PCR primer pairs and amplification conditions used in this study...............................94

5-2 Fragment sizes of parental amplicons digested with restriction enzymes ......................95









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









5-1 Fosmid 40M11 with primers designed on exons of FGENESH-predicted genic
re g io n s ............................... ............. .... ........................ ................ 9 4

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









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.









CHAPTER 1
STRAWBERRY AND THE GENOMICS ERA

Introduction

The cultivated strawberry, Fragaria x ananassa Duch, belongs to the family Rosaceae as

do the also economically important crops rose, apple, 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. x ananssa parentage was proposed by Antoine Nicolas Duchesne, whose

father worked at the Court of Louis XV (Darrow, 1966). F. x annassa was first observed in

several countries in Europe since the 1750's 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 two routes (Wilhelm and Sagen, 1974): to France by

the explorer Jacques Cartier during his first expedition to the Quebec Canadian Province in 1534;

and to England, by Thomas Hariot, who visited the "New Found Land of Virginia" in 1588.

Later, in 1714, F. chiloensis was taken to France by the engineer Amedee Francois Frezier.

During his mission to study the defense fortifications of Chile and Peru, Frezier noticed the

large-fruited berries at Concepci6n, 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 cultivated and appreciated throughout the world today.

Profitable strawberry production is challenged by several factors: diseases, pests, market

competition, and, arguably most importantly, by the phase-out of methyl bromide. This fumigant

is considered essential for the production of many crops, including strawberry (Rosskopf et al.,

2005), but because methyl bromide has great stratospheric ozone depletion ability, the Montreal

Protocol mandates that its use be reduced (Anonymous, 1998). Although traditional plant









breeding has been used to remedy several of the above-mentioned challenges, the knowledge of

the Fragaria genome structure may streamline the variety improvement process, potentially

permit discovery of gene function, and ultimately 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 strawberry's commercial value of 1.4 billion dollars as a

fruit crop (Folta et al., 2005), 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-Kohler et

al., 2002), clone characterization in germplasm banks (Harrison et al., 1997), (James et al.,

2003), identification of cultivar proprietary (Arulsekar et al., 1981), (Bringhurst et al., 1981),

(Gidoni et al., 1994), (Nehra et al., 1991), (Bell and Simpson, 1994), (Hancock et al., 1994),

(Levi et al., 1994), (Parent and Page, 1995), (Landry et al., 1997), (Degani et al., 1998),

population genetics studies (Degani 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-Kohler et al., 2003), (Sargent et al., 2003), (Sargent et al., 2004).

Pioneer molecular markers were based on polymorphisms observed on punctual loci or the

whole genome: isozymes and intron length polymorphism; Randomly Amplified Polymorphic

DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), and Amplified Fragment

Length Polymorphism (AFLP). More recently, Simple Sequence Repeats (SSRs) have been









employed to address the challenge of marker transferability (Monfort et al., 2005), (Nourse et al.,

2002), (Ashley et al., 2003). The present work discusses 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 noncoding DNA segments, in the chromosomes of an

organism." (The American Heritage, 2006)

The complexity of plant genomes began to be investigated in the mid- to late-1970's using

quantitative DNA reassociation kinetics (i.e. Cot curves) (Goldberg, 2001). It was determined

that plant genomes had families of repetitive sequences and that these repeats varied in copy

number and arrangement in the genome (Flavell et al., 1974), (Goldberg, 1978).

By the end of the 1970's, with the ability to construct cDNA clones, there was the

surprising finding that the coding regions of eukaryotic genes were interrupted by introns

(Gilbert, 1978 ), what led to investigation of posttranscriptional splicing mechanisms (Jeffreys

and Flavell, 1977).

The first plant gene was cloned in 1979 (Bedbrook et al., 1980), demonstrating that plant

DNA was not different from the DNA of other organisms and therefore could be manipulated

using the same enzymes, cells, and vector systems. 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 expressing bean phaseolin seed storage

protein gene (Murai and Sutton DW, 1983). In addition to being a vector to foreign genes, T-









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.









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









achieved by removal of membrane lipids 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; however, because plants have a

solid cell wall in addition to the cellular membrane, solvents alone are not enough to expose

organelles, and mechanical force must be applied. Samples can be sonicated but generally are

either treated with ethyl ether (Watson and 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 nucleic acids. The first membrane lysis is induced

by osmotic pressure generated by 0.35M sorbitol (Fulton et al., 1995; Hanania et al., 2004),

0.35M glucose (Chaudhry et al., 1999) or Triton 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 ethylenediaminetetraacetate

(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 dissociated by SDS (Kay and Dounce, 1953),

proteases, chaotropic agents, chloroform (Sevag et al., 1938), and phenol. Because phenol

solubilizes proteins (Cohn and Conant, 1926), it has been used to deproteinize preparations of









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).









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









polysaccharides during DNA isolation is to differentially precipitate the sugars by manipulating

the 2-butoxyethanol concentration (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, 1983). Reducing agents like B-mercaptoethanol,

dithiothreitol, ascorbic acid, sodium bisulfite, and diethylcarbamic acid can be added to the

extraction buffer to inhibit the oxidation process and protect DNA from quinones, disulfutes,

peroxidases, and polyphenoloxydases. Polyvinylpyrrolidone (PVP) and its insoluble, cross-

linked 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 and Shattuck, 1983; Leutwiler et al., 1984),

purification by cesium chloride gradient (Travaglini 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 strawberry research, it is imperative to devise

a standard protocol that is effective across cultivars and species of different ploidy levels.

Examination of the literature on strawberry (Fragaria spp.) indicates that the many published

DNA isolation methods are not universally transferable between cultivars or species. An optimal

protocol should use readily available plant material (such as mature leaves), be inexpensive,

rapid, reproducible, and have high yields of high molecular 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.









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









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









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









resuspended in 200l water and DNA quantities were estimated by a NanoDrop ND-1000

spectrophotometer.

Nucleic acids were extracted from 96 individuals that belong to a diploid Fragaria

mapping population. Minimal quantities 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 further study tissue-to-buffer ratios, under different

conditions from those tested above. This time, tissue was macerated in buffer after having been

ground in liquid nitrogen and incubated at 650C for Ihour. The absorbance values at 230, 260,

and 280nm were determined by a NanoDrop to make inferences about nucleic acid purity.

Absorbance ratios A260/A230 and A260/A280 are measures of contamination by polyphenols or

carbohydrates (Craigie and McLachlan, 1964; Logemann et al., 1987), cited by (Manning, 1991)

and protein, respectively. The ultimate usefulness 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 GAT 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 appreciable amounts of DNA

from mature plant leaf tissue, or yielded plenty of material that was not amenable to further

manipulations, such as restriction digestion or PCR (data not shown). However, a variable

previously considered minor had an unexpectedly great impact in the retrieval of nucleic acids:

further maceration of tissue in extraction buffer. Most of these preparations do not separate DNA









from RNA, so quantification is generally a combination of nucleic acids. This is important for

two reasons. First, the RNA isolation protocols for strawberry are principally revisions of DNA

extraction methods. Those that yield high amounts of RNA also contain proportionate amounts

of DNA, and RNA is removed with selective LiCl precipitation. In these preparations RNA and

DNA recovery is generally parallel and so quantification of both as nucleicc acids" provides a

general measure of DNA recovery. Also, in an attempt to identify an efficacious method, the step

of removing RNA, and verifying its removal would limit the number of protocols and

experimental conditions that could be tested.

Table 2-1 lists yields from the different DNA isolation protocols 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 comparison between treatments, values for yield shown

in the table are averages of replications, standardized using 1 g of plant tissue as the

denominator. Table 2-2 ranks the four methods that 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 30-tissue 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 extraction 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-borate or guanidine thiocyanate. Only after

purifying the guanidine thiocyanate prep utilizing the DNeasy Plant Mini kit, did the DNA









become PCR-amplifiable. It is interesting to note that the difference in spectrophotometer

readings before and after the purification was minor (treatments 8 versus 10), suggesting that the

kit may be a viable alternative to oher methods used to purify PCR-recalcitrant DNA.

The 4th highest ranked protocol type in table 2-2 is in fact the only one of the four listed

that resulted in tractable DNA. Of the many CTAB protocols that were investigated, the ones

that required maceration of plant tissue in buffer cluster together at the top in terms of tg 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 superiority were made. The objective was to discover the

variable responsible and incorporate it into a protocol that would yield DNA amenable to

enzymatic reactions. The factors tested were: i, nucleic acid precipitation by 2-butoxyethanol; ii,

boric acid (rather than HC1) used to adjust the pH of Tris for the extraction buffer; iii, second

round of extraction from plant tissue after chloroform treatment; iv, dilution of upper phase with

Na+ solution before DNA precipitation.

Treatments T30-T37 (comparing precipitations by isopropanol against 2-butoxyethanol)

verified that the latter has a detrimental effect on DNA precipitation. Considering all 4

experimental variables, 65 to 200% more nucleic acids were recovered by isopropanol rather

than by 2-butoxyethanol precipitation.

The absolute importance of boric acid to nucleic acid isolation has not been tested, though

borate appears to contribute to higher in yields when in combination with other factors. In the

extractions using guanidine thiocyanate, borate-containing buffer (T36) had on average 10x

higher yield than HCl-containing buffer (T8, T9). However, this increase may be attributed to the

different tissue-to-buffer ratios among treatments. A second comparison, this time between









CTAB buffers, strengthens the argument for the contribution of borate: T30 (Tris-borate) versus

T82 (Tris-HC1), where T30 had a tissue-to-buffer ratio = 16mg/ml and T82 had the ratio that was

determined to be optimum for DNA extraction (illustrated in figure 2-2). Perhaps borate was at

least partially responsible for T30's 25x greater yield than with T82. When used in substitution

to Tris, though, boric acid alone was not able to increase the retrieval of nucleic acids. T38 (1M

boric acid, no Tris) was a similar treatment to T30, but the yield was 60x lower.

A second round of extraction from plant tissue increased approximately 50% the DNA

recovery relative to a single incubation in extraction buffer. T34 and 35 yielded 60 and 45% of

single-extraction treatments T32 and T33, respectively. 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 the recovery of nucleic

acids. Observing the results for treatments T24, T27: no dilution; T25, T28: dilution by 2.5

volumes ofNa+ solution (detailed in Appendix A); T26, 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 the highest yield of tractable

DNA. Therefore, an optimum protocol was devised to further investigate the following factors:

leaf tissue state, incubation temperature and duration, tissue-to-buffer ratio, leaf tissue

maceration.

Leaf tissue state

DNA was extracted from the same mass of fresh and lyophilized tissues. As expected,

yield per gram of sample was generally higher from lyophilized samples. However, this likely is









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









water. Figure 2-3 illustrates the result of the optimization of the 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 determined in the experiment above (40 mg/ml),

the procedure of extracting DNA with incubation at 40C for 5 min was tested on ten strawberry

cultivars, 2 replicates each. 'Strawberry 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. x ananssa 'Diamante' had negligible yields.

Perhaps solely grinding leaves in liquid nitrogen is not sufficient to break down the cells and

expose contents to the extraction 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 deviation of 2 mg. Tissue was macerated in liquid

nitrogen and, subsequently, in extraction buffer for approximately 30 s. Grinding in buffer was

conducted until the material was the consistency of paste. Incubation was performed at 650C for

1 hour. No correlation between amount of tissue processed and DNA recovered was apparent

(figure 2-4).

PCR was performed using 1 1l of the extracted DNA at variable nucleic acid concentrations

(40ng/Cl to 4.5igg/[l) and the primer 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-amplify 50% of the diploid mapping population samples, an effort was

made to monitor for correlations between PCR outcomes and i, nucleic acid concentration in the

sample (figure 2-4); ii, tissue-to-buffer ratio during DNA extraction (figure 2-4); and iii,

A260/A230 ratios (figure 2-5) that could be indicative of carbohydrate contamination. The









absorbance ratios at 260nm and 230nm wavelengths were grouped into seven categories, and the

number of samples in each category is indicated in figure 2-5.

No conclusive correlation between 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. Surprisingly, there was no pattern

suggesting a relationship between template concentration and PCR amplification. This outcome

indicates that other factors are contributing to inhibition of the process. In an attempt to dilute a

possible polymerase inhibitor, lower tissue-to-buffer 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 indicates the purest nucleic acid sample. 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 observed 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 sample's potential to be used

successfully in downstream applications.

Tissue maceration method

The processes of breaking leaf tissue down solely in liquid nitrogen versus preliminary

pulverization in liquid nitrogen with subsequent grinding in buffer 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 prominent absorbance peak at 260nm was observed for samples









that were processed at 600C and ground in buffer (figure 2-6). Samples macerated this manner

and incubated at 40C appear to contain many polysaccharide contaminants, as a peak is seen at

230nm. The desired A260:A230 and A260:A280 ratios are equal to 1.80. Samples that were

ground in liquid nitrogen only and incubated at 40C absorbed more at 230nm than 260nm (ratio =

0.61, table 2-4), indicating that they probably had low content of nucleic acids.

Due to the extraordinary increase in DNA content 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 investigated. Incubations post-homogenization were

carried out at 650C for Ihour. The more aggressive the treatment, the higher the amount of DNA

obtained (figure 2-7). None of the samples appeared 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 isolation. 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 the isolated nuclei were not 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 denaurant by breaking intramolecular hydrogen bonds

(Kauzmann, 1954) and, therefore, it causes inhibition of enzyme activity. We hypothesized that

the lack of amplification by PCR and digestion by restriction enzymes occurred due to the

presence of this chaotropic salt in the DNA preparation. To test this hypothesis, two approaches









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.







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.










Table 2-1. Nucleic acid yields from isolation protocols. 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-to-buffer ratio (mg of tissue per ml of buffer). n/a: not
aplicable; Yield: [g of nucleic acids obtained if 1 g of tissue had been used for DNA
isolation
P T Status T/B Yield Brief Description
mgtissue Ugnuclac
/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
2.5vol dilution
(not amenable to restriction digestion, even after treatment with
28 F 30 18800 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/Tris-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











Table 2-1. continued
P T Status T/B
mgtlssue
/mnlbuffer
40 F 100
41 F 333
15 42 F 635
16 43 F 125
17 44 F 2.5
45 F 25
18 46 F n/a
47 F n/a
19 48 F n/a
49 F n/a
20 50 F n/a
51 F n/a
21 52 F n/a
22 53 F n/a
23 54 F 14
55 F 70
56 L 14
57 L 70
24 58 F 66
59 L 66
25 60 L 1.6
61 L 8
62 L 16
26 63 F 250
64 F 250
27 65 F 100
66 F 100
28 67 F 40
68 F 40
69 F 40
70 F 40
71 F 40
72 F 40
73 F 40
74 F 40
75 F 40
76 F 40
77 F 40
78 F 40
79 F 40
80 F 40
81 F 40
82 F 40
29 83 F 75
84 F 75


Yield
[gnuol ac
tissue
15
5
0
8.5
150
40
18
5
12
14
3
1
0
0
0
25
0
1300
45
50
1250
60
100
0
1
0
0
16
80
98
69
34
28
95
142
28
41
34
57
41
25
76
211
387
28


Brief Description



Epicentre kit
Epicentre kit
Mo Bio kit
Qiagen DNeasy kit
Silica
Silica
Isolated nuclei, Qiagen DNeasy kit
Isolated nuclei, Qiagen DNeasy kit
Isolated nuclei, CTAB
Isolated nuclei, CTAB
Isolated nuclei, SDS
Isolated nuclei, SDS
Isolated nuclei, guanidine thiocyanate
Isolated nuclei, SDS, TIPS
Murray and Thompson + solid CTAB, ppt by low ionic strength
Murray and Thompson + solid CTAB, ppt by low ionic strength
Murray and Thompson + solid CTAB, ppt by low ionic strength
Murray and Thompson + solid CTAB, ppt by low ionic strength
Murray and Thompson, 6% CTAB, ppt by low ionic strength
Murray and Thompson, 6% CTAB, ppt by low ionic strength
Murray and Thompson, precipitation by ethanol
Murray and Thompson, precipitation by ethanol
Murray and Thompson, precipitation by ethanol
Murray and Thompson, 5% CTAB, ppt by isopropanol
Murray and Thompson, 5% CTAB, ppt by isopropanol
CTAB + SDS
CTAB + SDS
40C, Omin
40C, 5min
40C, 30min
40C, 60min
20C, Omin
200C, 5min
20C, 30min
200C, 60min
420C, Omin
420C, 5min
420C, 30min
420C, 60min
650C, Omin
650C, 5min
650C, 30min
650C, 60min
Unexpanded leaf
Expanded leaf










Table 2-1. continued
P T Status T/B
mgtlssue
/mlbuffer
30 85 F 100
86 F 100
31 87 F 50
88 F 50
89 F 50
90 F 50
32 91 F 40
92 F 40
93 F 40
33 94 F 40
95 F 40
96 F 40
97 F 40
98 F 40
99 F 40
100 F 40
101 F 40
102 F 40
103 F 40


Yield
[gnuol ac
tissue
92
400
2476
300
3048
700
1400
1450
665
660
1000
940
1155
1605
955
975
1335
1455
2245


Brief Description


Powder
Slurry
Slurry, 40C
Powder, 40C
Slurry, 600C
Powder, 600C
2% CTAB
6% CTAB
20% CTAB
No polytron
/2 speed, 5sec
/2 speed, 15sec
/2 speed, 30sec
/2 speed, 60sec
Full speed, 5sec
Full speed, 15sec
Full speed, 30sec
Full speed, 60sec
Full speed, 120sec


Table 2-2. Ranking of 4 best nucleic acid extraction protocols
Average Treatments included in Protocol #
[g nucleic acid/g tissue average calculation
11,750 T24, T25, T27, T28 9
4,415 T30,T31 10
3,450 T36, T37 12


T83, T84, T86, T87,
T89, T91-T103


Protocol type

Strawberry
CTAB with tris/borate
Guanidine thiocyanate

CTAB with slurry


1,232


29-33




















S0 min
S5 min
S30 min
S60 min


200


100


0


20 42 65
Temperature (oC)


Figure 2-2. Effect of incubation temperature and time on DNA yields. The standard plant DNA
extraction procedure of carrying out incubation at 650C for 1 hour displayed, as
expected, superior yields to other incubation time lengths and temperatures.


0 50 100 150 200
Tissue-to-buffer ratio (mg tissue/mi
buffer)


Figure 2-3. Effect of tissue-to-buffer ratios on DNA yields. The optimum ratio for DNA isolation
was 40 mg of leaf tissue per ml of extraction buffer. The yield declined rapidly as
more tissue was processed by the same volume of buffer.









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











1200


1000 -* amplification
E no amplification


800



600



400 -
B 13

se 0 *3
D **
200 0 o [ 0


0 0 *

0 2 4 6 8 10 12 14 16
Tissue-to-buffer ratio (mg tissue per ml buffer)

Figure 2-4. Relationships between DNA yield, tissue-to-buffer ratios, and sample amenability to
amplification by PCR. DNA was extracted utilizing lyophilized tissue from 94 F2
individuals from a Fragaria diploid mapping population. The 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 lml, the tissue-to-buffer ratios also
represent the amount of tissue (in mg) processed per sample. Correlations between
amount of tissue processed, tissue-to-buffer ratio, DNA yield, and PCR outcomes
were not apparent.














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.












800
750
700
650
600
550
o 500
- d450
S400
E 350
S300
250
200
1 50
1 00
050
000
-0 50
-087
22


-- 600C

C--

600C

-40C


slurry


no slurry


230 240 250 260 270 280 290 300 310 320 330 340
Wavelength nm


Figure 2-6. Effect of interactions between maceration method and incubation temperature in the
absorbance at 220-340nm. The most desirable product from a DNA isolation
procedure has a peak at 260nm. A peak at 230nm indicates contamination by
polysaccharides. The more aggressive maceration method, combined with higher
temperatures, appears to be the best combination of treatments.


3o

200

1 00

00

1 00
220 230 240 250 260 270 280 290 300 310 320 330 340
Wavelenoth nm


- Full speed, 2min

12 speed, 60sec

- Full speed, 60sec

- Full speed, 30sec

- /2 speed, 30sec

speed, 5sec

- Full speed, 15sec

- Full speed, 5sec

- /2 speed, 15sec

- No Polytron


Figure 2-7. The effect of Polytron homogenization 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.









CHAPTER 3
PRIMARY ANALYSES OF Fragaria GENE DISTRIBUTION

Introduction

Although the cultivated strawberry genome is complex and polyploid, its monoploid

genome is particularly small and tractable (approximately 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 extended to other rosaceous 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 features of the genome of strawberry.

Genome annotation has been defined as "the 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." (Stein, 2001) The first challenge to annotate any genomic sequence

information is to discriminate between two types of sequences: coding (DNA sequences

encoding a protein) and non-coding (DNA is not transcribed into RNA or it is transcribed but not

translated into a protein). Regulatory sequences such as promoters and enhancers are examples

of non-coding DNA sequences. Other non-coding DNAs are transfer RNA, ribosomal RNA,









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









(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









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









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









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 contained in the large query nucleotide sequence and

one of them has very high similarity to more than 100 hits, this condition may mask the

similarity results to the second gene, appearing as if the second gene was non-coding sequence.

Similarity searches with BLASTX were performed using sequence segments for which

BLASTP detected amino acid matches. The translated nucleotide query was delimited to

sequence fragments of 8kb whereas the non-redundant (nr) amino acid database against which

the F. vesca sequences were compared was confined to green plants (green algae and

embroyphytes) "Viridiplantae". BLASTX was carried out to determine coding sequence

orientation, to assign tentative gene identification 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 used to assess microcolinearity between Arabidopsis and F.

vesca.

The BLASTN algorithm was utilized in separate searches against the EST and the "nr"

nucleotide collection databases. The query sequences originated from fragments for which a

gene had been predicted by FGENESH. EST databases 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 repetitive elements and non-translated sequences









features such as rRNA, tRNA, and was also useful 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 duplication 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 carried out. This time, the query sequences were 8-

12kb fragments of genomic sequence (regardless of whether or not genes were predicted in that

segment), compared against Rosaceae ESTs. The objective was to determine ifMedicago

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 available online at the Gramene website: http://

www.gramene.org/ db/searches/ssrtool.

Results

The average fosmid insert fragment size 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 numbered predicted genes and their corresponding

BLASTX results is available in Appendix B. The software specificity was 55%, since out of the

235 genes predicted, 129 had hits in the amino acid database having as threshold E < 1015.

Enzymes related to mobile elements like transposase, integrase, polyprotein,

retrotransposon polyprotein, transcriptase, and reverse transcriptase were putatively present

ubiquitously: 14 out of 26 fosmids contained at least one of those types of enzymes. In some

cases, several of these enzymes were present in tandem, as depicted for fosmid 18A19 in figure









3-2. The second fosmid diagrammed in figure 3-2, fosmid 34D20, contained putative protein-

encoding sequences, including inverted repeats of a gene next to a transposase.

Expressed Sequence Tags (ESTs)

ESTs facilitate genome annotation (The Arabidopsis Genome Initiative, 2000) because

they are strong evidence that a sequence is transcribed. In the case of Fragaria, only a small

percentage of protein hits was supported by EST hits (32 of 129), exacerbating the need for more

rosaceous ESTs. Three classes of ESTs were identified (figure 3-3): i, those that displayed

identity to predicted, putative protein-encoding genomic sequence; ii, those that were

FGENESH-predicted genes, but for which there was no protein hit; and, more interestingly, iii,

those that were identified 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 identified in all fosmid insert

sequences, except three: 11D02, 15B13, and 32L07. It is interesting to note that these fosmids

putatively contain plastid and RNA genes and belong to the 50% class 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% occurred in regions that were predicted to be

intergenic. The great majority (92%) of the repeats were dinucleotides (table 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.









(Bennetzen et al., 2005) proposed that retrotransposon removal as well as small deletions caused

by unequal homologous recombination and illegitimate recombination, lead to genome

shrinkage. Grasses like rice, maize, sorghum (Bennetzen 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 appeared to be one such

gene-empty space, since the only similarity detected between its 32kb sequence and the protein

database was to polyprotein, which comprised only a small portion of the fosmid sequence.

The pattern of gene distribution 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 gene per 3-6.6kb, whereas rice has one gene per 6.46 kb (Yu et al.,

2002) and barley has one gene per 15-20 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 FGENESH or BLASTX similarity-based

approach at E<10-15, respectively. In either case, strawberry ranks among the more gene-dense

species. Since a portion of the fosmid sequences analyzed arose from non-random, "gene of

interest" selections, it is possible that the sample was biased toward genic regions, and that the

number of kb containing one gene 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 following 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 enough that a sequence encoding a protein in Medicago









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.
















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.










A)34D20


Protein binding


Cystelne peptidase w



Transposase [

Anthocyanin 5-aromatic


NAC/NAM ]


5 kb


I Cytohrome P450


Integrase
Integrase



Integrase





Integrase


I Transferase

Figure 3-2. Diagram of two fosmid inserts of variable 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 transposon-related enzymes, integrase and
transferase. SSRs were identified both in genic and inergenic spaces.


B) 18A19










ESJs : .:I Lz- y tv F'.. ESH
32



Putative protein-encodinr sequences ESTs NOT predicted by FGENESH
identified by BLAS

Figure 3-3. EST classes identified by homology searches between large genomic F. vesca
sequence and Rosaceae ESTs. BLASTX similarity searches were carried out between
genomic sequence and the Viridiplantae protein database. A fraction (25%) of the
protein matches was validated by BLASTN-detected 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 dinucleotide 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









CHAPTER 4
GENE-PAIR HAPLOTYPES: NOVEL MOLECULAR MARKERS FOR INVESTIGATION
OF THE Fragaria x ananassa OCTOPLOID GENOME

Introduction

The cultivated strawberry, Fragaria x aananssa, contains 8 copies of a set of 7

chromosomes (2n=8x=56). The amount of DNA contained in a single complete strawberry

chromosome set is approximately 200 million bases (Nehra et al., 1991; Akiyama et al., 2001;

Folta and Davis, 2006), a very small genome size relatively to other angiosperms. There is some

controversy as to which angiosperm contains the lowest C-value (or "Cx-value", terminology

proposed by Greilhuber (Greilhuber et al., 2005) to specify the monoploid genome of

polyploids), due to different size standards used among various flow cytometry studies.

However, likely candidates to the smallest genome are Arabidopsis thaliana with 157Mb

(Bennett et al., 2003), and perhaps the green strawberry Fragaria viridis with 0.108pg (Antonius

and Ahokas, 1996). If the formula proposed by Dole2el (Dole2el et al., 2003) (where Ipg = 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 1000-

fold between species (Bennett and Leitch, 2005), the difference in monoploid genome sizes

between F. x aananssa and A. thaliana is negligible.

Although strawberry's small basic genome size makes Fragaria species attractive

organisms for genomic studies, the process of sorting 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 polyploid where reassortment amongst all homologous


chromosomes occurs, the number of possible genotypes for a single locus would be 1 + ,
2\ i)









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.









GPH markers were also used to investigate polymorphisms in diploid Fragaria species, in

an attempt to identify genome contributors 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 published elsewhere (Hancock et al.,

2004) (Darrow, 1966) (Staudt, 1973) (Hummer et al., 2005) (Staudt, 2003) (Staudt, 2005). F. x

ananassa is not included in the figure, as cultivated 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 long 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 1300's, 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 Gardener 's Dictionary (Darrow, 1966) and it received the name of. x aananssa

due to its resemblance to pineapple in odor, taste, 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 spontaneous hybridization occurred in Europe. F. virginiana

with its rather small fruits was transported overseas in the 1600's. Because of its relatively large

fruits, F. chiloensis was collected by the Frenchman Amedee Francois Frezier, during a

reconnaissance mission to the Spanish West Indies ordered in 1714 by the king Louis XIV.

Disappointingly, no fruits were observed during the first years, probably because, in an attempt









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









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 website (http://signal.salk.edu/

cgi-bin/tdnaexpress). The next 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 sensitive 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 the micro-colinearity detection level. Two-

hundred and fifty F. x aanassa EST sequences were randomly selected for similarity searches

against Arabidopsis utilizing FASTA and all (rather than only the best match) of the Arabidopsis

sequences that had a similarity E-value < 10-4 were considered for further analysis. The loci tags

were recorded on two separate tables, one table keeping the correspondence between F. x

aananssa and Arabidopsis similar sequences, and the other table had the Arabidopsis loci tag

sorted in crescent order. When the difference between two consecutive Arabidopsis loci tag

numbers was equal to or lower than 10, a putative gene pair was detected and the F. x aananssa

EST sequences were retrieved from the non-sorted table.









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









laboratory using and the rubidium chloride method (Hanahan, 1985). The putative recombinant

plasmids and competent cells were gently mixed, iced for 30min, heat-shocked at 420C for 2min,

and immediately iced again for at least 5min. XL1-Blue cells with no vector were included in

each transformation round as a negative control. When large fragments were cloned, a separate

treatment with a smaller fragment for which transformation had been successful before was

included as a positive control. Two-hundred pl ofLuria-Bertani (LB) broth (10g tryptone, 5g

yeast extract, 10g NaC1, per litter of deionized water) were added to the transformed cell and

were incubated in a shaker for 1 hour at 370C, with agitation of 220rpm, after which 100[l of

cells were spread onto LB-agar plates containing 50g ampicillin/ml medium. The TOPO vector

has the 1-galactosidase reporter gene. Therefore, when this vector was used, an overlay of 50l

of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-1-D-galactoside (X-gal) at 20mg/ml

dissolved in N-N'-dimethyl-formamide and 10ld 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-Gal were not added to the LB plates when the pJET1 vector was

used. This vector contains a gene for a restriction endonuclease in the cloning site. If disrupted

by an insert, the lethal endonuclease is not expressed and the transformants are able to propagate.

After the cells were plated, they were incubated at 370C overnight and single colonies were

selected for screening for transformants-white colonies for TOPO and, supposedly, any colony

for pJET1. The screening procedure was carried out by setting up individual PCR reactions for

each colony using annealing temperature of 55C and primers specific for the vector (pJETIF: 5'

GCC TGA ACA CCA TAT CCA TCC 3', pJET1R: 5' GCA GCT GAG AAT ATT GTA GGA

GAT C 3'; TOPO, M13F: 5' GTA AAA CGA CGG CCA GTG AAT TGT A 3'; M13R: 5' CAG

GAA ACA GCT ATG ACC ATG ATT AC 3'). Approximately 10 colonies were initially









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









detected in the pool of 1,500 was approximately 10% as calculated by


1,500 x +258- Therefore, the amplification of 2 loci out of the 10 investigated (table
(25,999 25,998)

4-2) may be regarded as fairly successful. A third set of primers (GPH4) permitted amplification,

though after fragment cloning and sequencing, amplification was determined to be unspecific.

There was no similarity between sequences obtained and the 770bp from the template sequences

for primer design.

The gene prediction from F. vesca genomic sequence enabled detection of 18 potential

gene pairs. Of those, primers designed for 11 loci rendered amplification of at least the positive

control DNA template ofF. vesca.

Table 4-2 summarizes the results of PCR amplification 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 most cases the PCR reaction number, followed by the

colony that was determined to be a transformant by PCR and/or restriction enzyme digestion.

The hypothesis that a fingerprint for each allele belonging to the octoploid 'Strawberry

Festival' would correspond to alleles from different diploids could be tested by GPHs 5, 23, 10,

27F10, 34D20, and 72E18. The full alignments for every 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









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









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









evidence of relatedness. Table 4-3 represents the length and position of indels and SSRs

identified in the sequenced clones. In this table, 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, presenting 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 present in the same region of a given locus, as the corresponding

genotype deviates from a consensus sequence compiled 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 consensus. The sequence of the clones listed in

the table is conserved with the sequence as they appear in the cladograms 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. x ananssa, since F. virginiana is the pollinating parent to F. x ananassa. Another study

published a year later showed that the crosses between F. vesca x F. chiloensis and F. vesca x F.

virginiana produced sterile hybrids (Mangelsdorf and East, 1927). The occurrence of natural

hybrids between F. chiloensis and F. vesca (Bringhurst and Gill, 1970), the geographical

predominance ofF. vesca, and a recent study on chloroplast DNA showing that the F. vesca is

closely related toF. x annanssa (Potter et al., 2000), support the hypothesis that F. vesca is a

contributor to the genome of octoploid strawberries. In this study large intergenic regions were

sequenced from a series of octoploid and diploid alleles to assess the relatedness between the

cultivated strawberry and potential subgenome donors. Two central methods were used to detect









relatedness, both based on multiple sequence alignments. The first used cladograms to display

consolidation of single nucleotide polymorphisms (Table 4-2). The second method was as

assessment of multinucleotide polymorphisms, 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 describe 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. x ananssa (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. This surprising finding contrasts directly with cytological evidence

and suggests that F. vesca may not be a contributor to at least 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, 27F 10, and 32L07, respectively. None of the deletions, however, corresponded to

any of the F. x 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', 'Diamante', '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 (three 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. x ananssa,

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









octoploid alleles. Phylogenetic analysis of the 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 octoploids (Davis and DiMeglio, 2004).

The data identified here provide further evidence to support the hypothesis that F. iinumae

is a subgenome donor to the modem octoploid. In all comparisons herein where octoploid

sequence was obtained, the octoploid related more closely to the F. iinumae haplotype. Thus,

one conclusion that can be made is that F. x 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 discrepancy, although all of them are speculative. The one

important caveat is that 'Strawberry Festival' is only one octoploid accession and was used

almost exclusively as the octoploid 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' Strawberry 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 variants were derived from the A genome donor.

One alternative explanation is that perhaps the A genome underwent extensive modification,

such as expansion, therefore preventing amplification of octoploid sequences by PCR.

Alternatively, these regions could have been deleted from the octoploid genome, as the octoploid

genome is smaller than four diploid genomes, indicating a loss of genetic material (Folta and

Davis, 2006). A final explanation is that not all diploid species, including many F. vesca

accessions, were tested, so the A genome may be represented by a genotype not tested in this









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-Kohler et al., 2003). If polysomic inheritance led to

a trait of interest early on, it may have been selected as beneficial in breeding populations and

fixed in subsequent lines.

Another unlikely explanation is that changes in F. iinumae paralled those in F. x ananassa

in two separate and unrelated instances. Probability suggests that this cannot be the case, yet it

remains a formal possibility, especially if the changes induced result in regulatory alterations that

affect gene expression, biological function and possibly selection. It is also possible that

cultivation and selection have important consequences 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 have 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.









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













-' E3


Srandom selection of EST


FASTA
b-


C-iI


Fragaria EST
Arabidopsis gene
- intergenic region of unknown size
- intergenic region of known size
> PCR primer


retrieval of Arabidopsisadjacent gene sequences



TBLASTX ITBLASTX

x


no Fragaria sequence


identification potential micro-colinearity


II II


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 strawberry EST and the intergenic region is
amplified if the adjacency is true, the gene space is smaller than 4kb, and the gene
orientations are conserved.


-14-"
-til


I I I


I ']i Ml











Table 4-1. PCR primers designed for amplification of micro-colinearity-inferred putative
intergenic fragments
Primer Fragaria EST Arabidopsis Intergenic Primer sequence


gene


FA SEa0007-G07
FA SEa0015-B08
FA SEaij""i2C''r
FA SEaOO18E1Or


At2g20120
At2g20140
At3g07320
At3g07330


fragment size in
Arabidopsis (bp)

3,415


2,196


acgagggcttggaagaaagg
gcccaacaacagaaagacc
caatgccatggtctccggtc
tqccqttqcacacaccttcc


GPH20 FASEa0012D10r At5gl3440 1,043 gagggtaacgctcatggtt
GPH20 FASEa0012E08r At5g13450 gtctccttcaattctttctcctc
GPH21a AY679587 At5g06750 tgacatcccataagccatca
GPH21b DQ011163 At5g06760 1,509 gggaggactacggcacataac
GPH21c DQ011163 At5g06760 atcagatgtcggcactgc
GPH22 gi48249442 At5g11250 tttcagctcagcaagcaagg
FaSCH6rgene 1,632
GPH22 FASEa0004E09r At5g11260 gctcccaggaccaaacca
FaHy5
GPH23F FASEa0013H07r Atlg23750 982 cttgagggccatcagcac
GPH23R V01014C10 558132 At1g23740 tacacccacgccttcatctc
GPH27F FASEa0014E11r At1g74260 2,300 tgccgctgccatttctct
GPH27R FA SEa0011H07 At1g74280 ccatgctcttgataggccaaat


FA SEa0014B05r
FA SEa0016G12r
CX662192
AY961594
AB211167
AJ414709
AJ414709


At2g30100
At2g30110
At3g176600
At3g17670
At4g38970
At4g38990
At4g38990


724

1,867


3,017


aatggagctgatggtttcgat
aaggatgatgacacgaactatca
ggacacatggctcccaga
caagacagcgggagcagt
ccagggacgatgttttgctc
ggtggattacattttgggtgaca
ttcaagctttggacaactaacg


name


GPH4a
GPH4b
GPH5a#2
GPH5b#2


GPH31F
GPH31R
GPH51F
GPH51R
GPH56F
GPH56R
GPH56R2











Table 4-2. PCR primers that allowed amplicon generation. A "minus" sign in the "amplification"
and "clone #" columns signify, respectively, no amplification and no transformants
were observed.


Primer
name


Template


GPH4 'Strawberry
Festival'
GPH5 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica

'Strawberry
Festival'
GPH23 F. vesca
F. viridis


F. iinumae


F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'


GPH 10


'Strawberry
Festival'


10PPR
1/10A
B22*


F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica


'Strawberry
Festival'


11D02


F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'


PCR
product
size (kb)


2.0, 1.0,
0.5
2.8
2.8
2.8
2.8
2.8
2.8

2.8


2.0



2.0
2.0

4.4
4.4
4.4
4.4
4.4
0.728
0.726
0.266
0.722
0.652
0.724
528
644
584
584
643
1.6
1.6
1.6
1.6
1.6
1.6
1.6


Clone # Vector


13
15
21
5
5
7
19
1
2
6
7


2
5


3
3
4
2
7
18
19
20







2
7
18
19
20
library
2031-1
2032-1
2033-1


TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO


TOPO
TOPO


TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO
TOPO







pJET1
pJET1
pJET1


E. coli
strain


TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10


TOP10
TOP10


TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10
TOP10







XL1-Blue
XL1-Blue
XL1-Blue


Sequence
obtained
from
forward
end (bp)
1,109
638
1,268
1,249
1,256
1,261
1,262
Fu
1,257
749
Fu


Sequence
obtained
from reverse
end (bp)


ill clone


ill clone


1,299
1,287
1,298
1,263
1,295

1,306
755


Full clone
Full clone


Full clone (2,024)
Full clone (2,081)
Full clone (2,111)
Full clone
Full clone
Full clone
Full clone
Full clone
Full clone
Full clone
Full clone
Full clone
Full clone
Full clone
Subset of GPH10 sequence
Subset of GPH10 sequence
Subset of GPH10 sequence
Subset of GPH10 sequence
Subset of GPH10 sequence


1,403
Full clone
1,299


10PPR1/10AB22 is a locus within GPH10











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











Table 4-2.
Primer
name


continued
Template


PCR
product
size (kb)


63F17 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'
72E18 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'
73122 F. vesca
F. viridis
F. iinumae
F. nubicola
F. nilgerrensis
F. mandshurica
'Strawberry
Festival'


Clone # Vector


library
2
3


3
1

library
1096-4
1097-1

1099-1
1100-6
1101-10


pJET1
pJET1


pJET1
pJET1

TOPO
TOPO
TOPO

TOPO
TOPO
TOPO


1834-16 pJET1


1836-1

1838-5


pJET1

pJETI


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


XL1-Blue
XL1-Blue


XL1-Blue
XL1-Blue

TOP10
TOP10
TOP10

TOP10
TOP10
TOP10


XL 1-Blue

XL 1-Blue

XL1-Blue


Sequence
obtained
from reverse
end (bp)


Full clone



Full clone


1,052 1,515
1,000 973

1,958
Full clone
1,170 1,245


No seq

No seq

No seq










GPH5 ananassa clone7 CAGAAGGTAATATGCATGATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAATA 582
GPH5 viridis TAGAAGGTAATATGCATGATATAAATATCAAGTTAATTGTACAGTGATAT---TTGTAACC 57 6
GPH5 iinumae TAGAAGGTAATAT-------------ATCAAGTTAATTGTACAATAATAT---TTGTAATC 566
GPH5 nilgerrensis CAGAAGGTAATATGCATGATATAAATACCAAGTTAATTGTACAATGATAT---TTGTAATC 579
GPH5 mandshurica TAGAAGGTAATACGCATGATATAAATATCAAGTTAATTGTACAATGATAT---TTGTAATC 578
GPH5 nubicola TAGAAGGTAATACGCATGATATAAATATCAAGTTAATTGTACAATGATAT---TTATAATC 583
GPH5 vesca TAGAAGGTAATATGCATGATATAAATATCTAGTTAATTGTACAATGATAT---TTGTAACC 579


GPH5 ananassa clone2 GGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAGAAAAGAGCT 240
GPH5 ananassa clone7 GGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAGAAAAGAGCT 240
GPH5 viridis GGAACAAGAAGTAGCACCTCCAAGAAGAAGAGAAAAAATGGGATCTACAGAAAAGAGCT 240
GPH5 iinumae GGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAGAAAAGAGCT 240
GPH5 nilgerrensis GGAACAAGAAGTAGCACCTCCCAAGAAGAAGAGAAAAAATGGGATCTACAGAAAAGAACT 240
GPH5 mandshurica GGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAGAAAAGAGCT 240
GPH5 nubicoa GGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAATGGGATCTACAGAAAAGAACT 240
GPH5 vesca GGAACAAGAAGTAGCACCT CCCAAGAAGAAGAAGAAAAATGGGATCTACAGAAAAGAGCT 240

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



170bp 124bp
ananassa4 4 I




-nandshu 11
'"3 ".-l"" I ^114 4 A1H4ft-l- ,,I,, ,, L



uinumae 2 V1
- Mmh IIIlllItII-+HHJti-4

^ i IIIII-II-I-1H I1-- i N

Iinumae I I I Iij 1194


SNPs indels
A G C T deletion insertion coding
I II A V non-coding

Figure 4-5. Diagrammatic representation of alignment of full GPH23 clones, depicting all
polymorphisms identified, such as Single Nucleotide Polymorphisms, insertions, and
deletions. Numbers in triangles indicate the length of indels.





























Figure 4-6. EcoRI Restriction patterns observed for GPH10 clones from the octoploid
'Strawberry Festival', indicating four different allele classes. M: molecular weight
marker; V: empty vector; 2-20: polymorphic clones



2,121 2,166 3,716
GPH10a 10PPR1F #3 Col32Rev2 #7 #22 #16 #20 #11 -#121 #GPH10b #19 #14 #6 #13. #5 GPH10c

4280bp 19 121 207 114 66 347 228 90 685 311 64 655 382 184 267 342
#10

85 2,206 2,251 3,799
GPHlOa 10PPR1F #3 -Gol32Rev2 #7 #22 1 #15 #20 #11 -#12 #GPH10b #19 #14 #6 #1 #5 GPH1O
18 _6 P -- --- ---- --- --1- 4- 4-a 4- 4i- *- *4-- 4- 4-
4363bp 19( Z'1 263 114 35 22 'J 686 311 4 Il Is 26 ,4
24

2,204 2249 3,799
GPH10a 10PPR1F #3 Col32Rev2 #7 #22 #15 #20 #11 -#12 #GPH10b #19 #14 #6 #1. #5 GPH10c
19- --- ---W --- N ----- -0. ---? 4- -4 4- 4 4-- 4- 4, *- ,
4Siup 121 i F'I 114 FIt ', 7', 'I r4 I. i'! q 1,l4 hl7 '4.


#1 2 2,265 2,310 3,61
GPH10a 10PPR1F #4 #3 Col 2 #7 -#2 #16 #20 #11 #GPH10b #19 #14 #6 #1" #5 GPH10c

4422bp 1~. 1U 12 4 144 t 114 66 362 226 102 997 64 655 383 184 264 342


-- F primer R primer [ Most polymorphic loco + EcoRI restriction site


Figure 4-7. GPH10 clones, 4 alleles from the octoploid Fragaria x ananssa, detected by
distinct EcoRI (green, vertical arrows) restriction patterns. The primers designed to
amplify and sequence all 4.4kb clones are represented by black and blue arrows. The
numbers between primers are the distances (in bp) between primers. The boxed
region contained most of the polymorphism observed for GPH10, and it is
comprehended between primers 10PPR1 and 10AB#22.


#AO 60


2.3kb
2.0kb












72E18 esca GAAAA-AAGAGAGAGA--AAATTACAGATTTAAAGCGACAACAA-TGAAAAGGAATGA 6(
72E18 mandshurica GAAAA-AAAGAGAGAGA-AAATTACAGATCTAGCGACGCAG-TAGAAGGATGA 5
72E18 nilgerrensis NNAAA-AAAAGAGAGAGA---TTACAGATCTAN-GACAACAA-TAGAAGGAATGA 5'
7E18 viridis AGAAATAA AGAGAGAGA--AAATTACAATCTAAGTGACAACAA-TGAGAATGA 6(
72E1 iinumae GAAAAAAAGAAGAGAGA--AAATTACAGATCTAAAGCACGAACAAATGAGAAGGAATGA 6;
72E18 ananassa GAAAAAAAAAAGAGAGAAAATCTAAAGCACGAA -TGGAAAGGATGA 6'


72E18 vesca GAGGCAAAGAGAAGAGAT GAGGAAGTTACCTTTGTGAAT GAAGTGAGT GGAGAGA 6
E18 mandshurica GAGGCAGAGAGAGAATGAGGAGTTGACCTTTGTGTAGAGTGAGTGAGGGAGAGA 6
72E18 nilgerrensis AGGAGAGAGAAGAGATGAGGAAGTTGACCTTTGTAATAGAGTAGT----- GA 6
72E18 viridis GAGGCAGAGAGAA GAGATGAGGAGTTGACCTTTGTGTAGAGTAGTAGG--GAGA 6
72E1 iinumae GAGACAGAGAGAAGAGATGAGGAAGTTACCTTTGTAATAGAGT----- GAGAGA 7
72E18 ananassa GAGGCAGAGAGAAGAGATGAGGAAGTTACCTTTGT ------GTAGGAGA 6
*** ** ******************************* **** *** ***

72E18 vesca GAGAGAGAGATCGACAC AGAGCGAAAGACAGTGTGGTGTTTGTAGTTAG 7;
72E18 mandshurica GAGAGAGAGATCGACGACGAAGCAGAGCGAAAGAGACGAGTGGTGTTTGTGAGTTGAG 7
72E18 nilgerrensis GAGAGAGAGATCGAAGACGAAGCAGAGCGAAAGAGACGAGTGTGGTGTTTGTGAGTTGAG 7(
72E18 viridis GAGAGAGAGATCGAAGACGAAGCAGAGCGAAAGAGACAGTGTGGTGTTTGTGAGTTAG 7
72E18 iinumae GAGAGAGAGATCGAAGACGAGGCAGAGCAAAGAGACAGTGTGGTGTTTGTGAGTTAG 7
72E18 ananassa GAGAGAGAGATCGAAGACGAAGCTAGCGAAAGAGACAGTGTGGTGTTTGTGAGTTAG 7


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










10PPR1AB22_vesca
10PPR1AB22_nubicola
10PPR1AB22_mandshurica
10PPRlAB22_nilgerrensis
10PPRlAB22_iinumae
10PPR1AB22_ananassacione2
10PPR1AB22_ananassaclone18
10PPR1AB22_ananassalone20
10PPRlAB22_ananassa_clonel9
10PPRlAB22_viridis

GPH5_vesca
GPH5_viridis
GPH5_iinumae
GPH5_ananassa_clone2
GPH5_ananassa_lone7
GPH5_nilgerrensis
GPH5_nubicola
GPH5_mandshurica

GPH23_ananassadone3
GPH23_ananassa_lone4
GPH23_iinumaeclone2
GPH23_iinumae_clone5
GPH23_mandshuricadone3
--_------------------------- 34D20_mans huia
34D20_vesca
34D20 mandshurica
34D20_nilgerrensis
34D20_iinumae
34D20_ananassa
34D20_viridis
34D20_nubicola

27F10_vesca
27F10_viridis
27F10_iinumae
27F10_ananassa
27F10_nubicola
27F10_mandshurica

72E18_vesca
72E18_mandshurica
72E18_nilgerrensis
72E18_viridis
72E18_iinumae
I 72E18_ananassa


Figure 4-9. Cladograms ofF. x 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 most related diploid
toF. x ananassa clones in every locus analyzed. F. vesca clones, on the other hand,
were the furthest from the octoploid, contrary to prediction based on data of other
author's previous studies.











Table 4-3. Overview of insertions and deletions detected through alignment of all sequenced
clones. Each column represents an aligned 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 clones 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 orplus signs, respectively) in relationship to the consensus
observed. White boxes represent accordance to the consensus sequence for the region
in focus.


Clone
10PPR1AB22
nubicola
mandshurica


I II
-5
-5


Indels and SSRs in Polymorphic Loci
III IV V VI VII VIII IX X XI
6 TA
5 TA


XII XIII XIV


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
viridis
nubicola
vesca
iinumae


II Ill
+8


V VI


17022
vesca
mandshurica
viridis
nubicola
iinumae

27F10
vesca
mandshurica
nubicola
iinumae
ananassa
viridis


I II III
-36

+5


-7 +6
-14 +6


+12 -5
+12 -26


II III IV V VI VII VIII
-2
-2 -2
-9 -2


IX X



-3
-3
+3 -3


+505


29G10 I II III IV V
vesca
mandshurica
nubicola -1 +2 -13 +7
nilgerrensis -1











Table 4-3.
Clone
32L07
vesca
viridis


continued


I II III IV
-9


Indels and SSRs in Polymorphic Loci
V VI

-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









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-









fruited, runnerless) and ssp. americana wild accession WC6 (Davis and Yu, 1997). The map was

populated with 3 isozymes and 75 RAPD markers, of which 11 were codominant. This was

possible due to a novel approach to Polymerase Chain Reactions (PCR), using mixed DNA

templates for formation of heteroduplex bands (Davis et al., 1995). The locations of six genes

involved in the anthocyanin pathway were assigned into this map later (Deng and Davis, 2001).

The second strawberry linkage map was developed for F. x ananssa, to increase the

knowledge of the octoploid genome and to address questions on inheritance patterns in

strawberry (if disomic or polysomic) (Lerceteau-Kohler et al., 2003). Amplified Fragment

Length Polymorphism (AFLP) markers were used to generate separate maps for the male and the

female parents, with 235 markers in 30 linkage groups, and 280 markers in 28 linkage groups,

respectively. Though the study generated very detailed maps, with a total of 789 markers, AFLP

markers are not easily transferable between species or even populations. The density of markers

did add evidence of polysomic inheritance, since 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

pistillatee parent) and F. nubicola FDP601 (pollinating parent). These diploids have been shown

to be the most closely related diploid relatives to the cultivated octoploid species (Potter et al.,

2000). The creation of a reference map using a diploid 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









(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.









Materials and Methods

The diploid mapping population 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 lml of extraction buffer (2% CTAB, 1.4M NaC1,

100mM Tris-HCl pH 8.0, 20mM EDTA 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-tl

tubes, samples incubated at 650C for lh, and 1 vol of 24:1 chloroform:octanol was added to each

tube. After mixing the organic solvents with the 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 volume of isopropanol, centrifuged, the supernatant

discarded, the pellet air-dried, and resuspended in 50tl TE pH 8.0. The DNA concentration

varied from 40ng/pl to 4,543ng/.l.

Target regions for marker development were derived from the F. vesca 'Pawtuckaway'

sequence annotation described in Chapter 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 for 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.









ii, Tandem and non-tandem duplications were avoided as potential targets for PCR primer

design. Target sequences should be unique to yield locus-specific amplification, since the

assessment of more than one locus at a time would complicate data scoring. Tandem duplications

were detected when adjacent F. vesca query sequences that had the same BLASTX hit, which

appeared to indicate gene family clusters (e.g.: putative genes in fosmids 05N03, 13103, 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

genome's 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 evidence of potential duplication in F. vesca.

iii, Some fosmid clones did not appear to contain gene pairs when similarity to database-

deposited 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 displaying similarities, one to

proteins and the other to ESTs.

Once apparent single copy, PCR-amplifiable putative gene pairs were identified, the

software Primer3 (Rozen and Skaletsky, 2000) was used to facilitate design of PCR primers. For

each primer pair, the forward primer was designed 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 primer pair was designed to generate a single

band product, polymorphic between the parents-before or after restriction digest. In those









cases, a primer with the fosmid clone name and orientation (F or R) had a suffix added to

indicate another set. Figure 5-1 illustrates the case of primers designed for fosmid 40M11.

The PCR amplification 50pl-reaction components and conditions for the parental DNA and

for the 94 F2 samples were: lx buffer (at 10x concentration, composition was 35mM MgCl2,

37.5ig/ml BSA, 160mM KC1, 400mM Tricine-KOH pH 8.0), 0.2mM each dNTP, 0.2iM each

primer, 0.05unit Taq polymerase, 1 .l DNA template, at variable concentrations (40ng/pl to

4.5ig/pl). Initial denaturation: 94C, 2 min, followed by 35 cycles of: denaturation at 940C for

15 sec, annealing for 45 sec, and extension at 720C. A last extension of 5min at 720C after the 35

cycles was executed. Table 5-1 contains functional information of the gene pair amplified, as

well as annealing temperatures and the extension 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). Positive control primers FvLFYintron2F/ FvLeafy3' are

anchored to the exons that flank Leafy gene's 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 extension time were variable, since the primer

pair under investigation was their determinant.

Following successful PCR amplification, 10pl of each single-band amplicons were

digested with lunit of different restriction enzymes (table 5-2) in a total volume of 201p.

Amplicon polymorphisms were resolved in 2% agarose gel, lx TAE buffer, 0.5[ig/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.









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 linkage groups utilizing the software

JoinMap 3.0 (Van Ooijen and Voorrips, 2001) with 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 pairs, 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 were 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 are detected, one of the expected size, the other with higher

molecular weight. Amplification by the other primer pairs displayed multiple bands, similar to

non-specific amplification. There were primer pairs 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 amplifiable. 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 observed for 34D20 were expected, according to the









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









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.









vesca ssp. bracteata DN1C x F. vesca ssp. vesca 'Yellow Wonder' F2 population (Deng and

Davis, 2001). This may be evidence of multiple 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 poplar, are mapped to different 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 with different gene functions. In common morning

glory (Ipomoeapurpurea, 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 validate 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 subgenomes, indicate that these may 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 preparation from recalcitrant

tissue, to computational analyses, development and proof-of-concept assessment of a novel

molecular marker, these trials present new facets 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 barriers that guard the DNA. The results of over 103









systematic tests of various experimental conditions indicate that the most important

consideration is complete disruption of the tissue via maceration, and that this process may be

greatly enhanced by co-application of chemical lysis to disrupt tissue. 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 ofF. vesca. The data indicate that

the small genome ofF. vesca maintains a character and composition similar to other model plant

species, suggesting that this species will have utility in answering questions within the Rosaceae

family.

Annotation of fosmid inserts leads to the understanding of gene content and distribution,

and permits marker generation for linkage mapping. More importantly, this initial survey of the

strawberry genome is the first opportunity to compare strawberry to sequences to those of other

organisms. Here relationships between the general properties of the genome have been

deciphered. Strawberry is a gene-dense 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 searching for micro-colinearity between F. x ananassa and

Arabidopsis is a clever approach, but it needs to be automated to increase the chances of finding

adjacent genes. This approach has the advantage that it is not based on F. vesca sequence.

Therefore, amplification of haplotypes is not biased towards F. vesca-like alleles. In addition,









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.

















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










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
















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.










LGVI


EMFvi072
BFACTO42
EMFn049
EMFnO4EOB
UDF002
EMFn136
TSA3
17022
F3H
UFFxa2 F02
UFFxal16H07
EMFn1B2
ARSFL013
ARSFL092
CAD-3
EMFn002
EMFn12B
EMFv143
EM Fvl BSAB
EMFv1 64
EMFn152
CFVCT005 B
EMFn115
ARSF 10 0
EMFv025
EMFv027
CFVCT004
PC21
AC8


0.0
4.4
10.5
10.7
11.1
19.4
20.4
24.4
26.4
27.1
27.8
29.5
29.9
36.6
38.4
39.0
39.6
41.0
422
43.0
433
434


Ii


0.0
6.3
6.4
8.3
13.9
19.1
34.5
38.3 I
41.3 .
41.9
43.3
44.7
46.1
47.4 .
48.5
49.7

502

50.4 ;
51.0
51.3

51.7

51.8
54.3
57.7


LGVII


IDH-2
34D20
UFFxa01 E03
AG106
EMFv1 60BC
FACu012a
PC12
ACO
EMFv1 60AD
EMFn153
EMFvO10
FACO05
CCB
CFVCT017
EMFvi133
ARSFLO22
EMFn119
AG56
AG1 02
AG104
CFVCTO36
EMFn01 7
MC225
EMFv1 04
AG49
Fvi020
CFVCT030
BFACT047
EMFn225
CFVCT010
CFVCT002
EMFvDO6
UDF019
XLRR-B
EMFn117
EMFn123
GOT-i
EMFn1BS
FAC004d
PGLM
CC2
PGL1
S1AS 1INV-B
10K105
EMFn22B
BFACT010
CC116
Fvi6b
EMFvio25
ARSFL007


0.0 63F17
7.1 AC31
7.5 73122
13.0 72E8J
13.2 AG35
16.7 EMFv190
18.2 CFVCT009
18.7 EMFn201
19.0 EMFn14D23
ARSFL011
19.1 CFVCTO31A
19.7 EMFv021
SARSFLu024
19.9 BFACTu029
FACuOO1
CFVCT031C
20.4 BFACTLOO4
20.5 UFFxa20G06
UFFxal9B10
21.0 CFVCT026
CHS
25.4 i 1L18
28.1 | CHI
28.4 I EMFn213
30.8 CFVCT019
31.4 BFACTuI1B
32.1 XLRR-C
37.0 EMFviOO8
38.5 ARSFLu099
43.6 01K06
50.3 B FACT31
51.3 MC45
53.8 CFVCT023
53.9 EMFvi109
56.1 BFACT044
63.0 EMFv023
73.2 CoMET


Figure 5-3. Gene-Pair Haplotypes assigned to linkage groups of the reference Fragaria map.


LGI


438
44.4
44.6

45.0

45.1
452
45.4
45.5
48.4
51.6
523
54 .
55.4
57.7
592
59.7
605
66.0
713
732
74.1
76.6
77.7
78.6
82.0









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-mercaptoethanol, sodium bisulfite or sulfite, these
reducing agents were added just prior to use of buffers. Most procedures included at least one
25:24:1 phenol:chloroform:isoamyl alcohol deproteination step followed by one 24:1
chloroform:octanol extraction. When RNAse-treated, the enzymatic reaction was carried out at
50g/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 thiocyanate, which were kept at room temperature, plant material in
buffer was incubated 30-60 minutes at 650C, unless otherwise stated. When product was
obtained, 5-10 t-g of DNA were digested with 2-4 restriction enzymes. Below is a brief
description of each protocol.

DNA Extraction from Leaves

1. Tomato [Fulton, 1995]: utilizes a combination of a DNA extraction buffer (0.35M
sorbitol, 0.1M Tris-base, 5mM ethylenediaminetetraacetic acid, EDTA, pH 7.5) and a nuclei
lysis buffer (0.2M Tris, 0.05M EDTA, 2M NaC1, 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), Ig (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% 2-
mercaptoethanol, 10% polyetheleneglycol, PEG), the supernatant 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 NaC1, 0.1% CTAB). Used 0.lg of two cultivars ofF. vesca ssp. vesca f
semperflorens: Yellow Wonder (T4, T6) and Alexandria (T5, T7); fresh expanded leaf tissue,
extracted by lml (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 650C. Buffer composition: 4M guanidine
thiocyanate, 100mM Tris-HC1, 10mM EDTA, 0.5M NaC1, 1% sarkosyl, 1% sodium sulfite.
Newly expanded (T8) and unexpanded (T9) leaves of 'Sweet Charlie' were used for extraction
from 100mg tissue in 1001l buffer. Further treatments to aliquots of the product of this prep were
performed, aiming removal of contaminants: adsorption to a column from the DNeasy Plant Mini
kit (T10) or dialyses into TE pH 7.0 at 40C (T11). Dialyses was performed overnight, TE buffer
replaced by fresh buffer, and dialyzed again for another day. Sample was 50[tg/ml RNAse- and
150[g/ml proteinase K-treated. DNA isolation was continued with phenol extraction and
standard downstream steps.
4. Guanidine thiocyanate and CTAB utilized 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









DNA prep of polysaccharides. The incubations were carried out at room temperature and 65C
with guanidine thiocyanate and CTAB, respectively.
5. Guanidine thiocyanate and CTAB used simultaneously: extraction buffer kept at room
temperature, 15 minutes: 4M guanidine thiocyanate, 100mM Tris-HC1, 10mM EDTA pH 8.0,
0.5M NaC1, 1% sodium sulfite, 1% sarkosyl, 2% CTAB, 1% PVP, 2% 2-mercaptoethanol.
Treatments included extraction from 10mg (T13, 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 extraction from 100mg (T17, T19) and 500mg (T18, T20)
oflyophilized (T17, T18) or fresh (T19, T20) tissues.
7. Pine tree [Chang, 1993]: this protocol was originally designed for RNA extraction and
was adapted here to DNA extraction by omitting the lithium chloride step. Buffer: 2% CTAB,
2% polyvinyl pyrrolidone (PVP), 100mM Tris-HC1, 25mM EDTA, 2M NaC1, 0.5g/L
spermidine, 2% 2-mercaptoethanol. After the addition 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 step was done together with incubation with
extraction buffer, at room temperature for 20 minutes in 8M urea, 0.4M NaC1, 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 supplementation with 1% sodium sulfite and 1% PVP to
prevent oxidation of phenols (T23).
9. Strawberry (Manning, 1991): Buffer: 0.2M Tris, 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 temperature, 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 (called "Tube A" here). "Tube B" contained inter-
and lower phases from this first round of chloroform 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 Tube 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 other 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) and water. The relative volumes of water and 1M Na acetate/acetic
acid buffer were calculated to raise the Na concentration 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, 664 1l of the 1M Na acetate/acetic acid 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 volume as 3.3ml, the final volume was 13.2ml. The sample received 10ml of
(water+ sodium buffer), of which 9.2ml were water and 800.l were the 1M Na acetate/acetic
acid buffer. After dilutions were made, T25, T26, T28, and T29 were precipitated as before: 2-
butoxyethanol, were iced, and centrifuged. The goal of the centrifugation here is to precipitate
polysaccharides, not nucleic acids. The six supernatants were transferred to new tubes and equal
volumes of 2-butoxyethanol were added to precipitate nucleic acids. After icing for 30 minutes,
the tubes were centrifuged for 10 min at 3,500rpm, the supernatant discarded, and the pellet









washed with a 1:1 solution of 0.2M boric acid/Tris, 10mMNa2EDTA (pH 7.6) : 2-
butoxyethanol. Pellets were washed with 70% ethanol, 0.1Kacetate/acetic acid (pH 6.0), then
with absolute ethanol. After dry, pellets were resuspended in lml water, and 10g DNA digested
with restriction enzymes. An aliquot of one of the treatments (T28) was EcoRI-digested before
and after treatment with 150Ig/ml Proteinase K and with phenol:chloroform. A second attempt
to isolate digestible DNA using the strawberry protocol was made, adding 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 possibilities raised were: i, the SDS, rather than
CTAB, nature of the protocol. Treatment numbers T16, T18, and T24 used SDS, therefore
testing this variable; ii, the boric acid, instead of HC1, used to adjust the pH of Tris; iii, the re-
extraction of interphase formed after chloroform treatment; iv, the dilution that raised Na
concentration to 80mM prior to DNA precipitation; v, precipitation by 2-butoxyethanol in place
of isopropanol or ethanol. The isolated roles of boric acid and 2-butoxyethanol 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 HC1 (buffer: 200mM Tris/borate,
200mM EDTA, 2.2M NaC1, 2% CTAB, 2% 2-mercaptoethanol, 2% PVP), and precipitating one
treatment with isopropanol (T30) and the other with 2-butoxyethanol (T31).
11. The strawberry protocol suggests two different dilutions (2.5 volumes or 4 volumes) to
elevate the Na+ concentration to 80mM. The chosen dilution here was the 2.5vol. An experiment
was set up to test the merits of the combinations of two factors: i, re-extraction 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 and 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-butoxyethanol; T34=second extraction
round/isopropanol; T35=second extraction round/2-butoxyethanol.
12. Finally, 2-butoxyethanol was used in the guanidine thiocyanate protocol (number 3).
The treatments were essentially the same as described for T8 in protocol number 3, except that
2% 2-mercaptoethanol was added to the extraction buffer and the Tris was adjusted by boric
acid, not HC1. 100mg of tissue processed by 6ml buffer. Nucleic acids precipitations were done
by isopropanol (control, T36), and 2-butoxyethanol (T37).
13. According to an article that proposes a method to isolate 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 NaC1, 4% CTAB, 0.2% 2-mercaptoethanol.
14. Epicentre kit. Used 10mg (T39), 30mg (T40), 100mg (T41) of 'Strawberry Festival'
leaf tissue with 300.l buffer.
15. PowerPlant DNA Isolation kit from MO BIO (T42). A leaflet (350mg) of fresh
FRA520 (F. nubicola) was ground with liquid nitrogen in microfuge tube. The remaining steps
were carried out according to manufacturer's directions.
16. Qiagen DNeasy Plant Mini kit (T43). Followed company's directions for fresh tissue.
17. Silica-based DNA extraction. Nucleic acids tend 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 at concentrated solutions and pH<7.5 (GeneClean Manual). Silica









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









followed by phenol:chloroform and chloroform extractions. DNA was precipitated by
isopropanol.
22. Use of triisopropylnaphthalenesulfonic acid (TIPS) as a hydrotrope in a surfactant
system (Bies and Folta, 2004). Hydrotropes stabilize surfactants (e.g. SDS) to allow them to
remain soluble. Nuclei (150.l) were incubated with 1200.l of extraction buffer 1 (10mM
EDTA, 10mM Tris, 1%SDS) at 65C for 20min (T53). The sample was treated with Proteinase K
for Ih at 37C. After a phenol:chloroform extraction and centrifugation, the interphase was re-
extracted with 5 volumes of extraction buffer 2 (50mM Tris-HCl pH 8.0, 5% SDS, 1%TIPS, 2%
2-mercaptoethanol, 4% PAS-p-aminosalicylic acid). The supernatants of both extractions were
combined and nucleic acids precipitated by isopropanol.

Modifications of Murray and Thompson DNA Isolation Protocol

A series of modifications of the protocol proposed by Murray and Thompson were tested.
Though the original protocol included cesium chloride gradient, this step was suppressed for all
variations tested.
23. Extraction buffer: 200mM Tris, 2M NaC1, 50mM EDTA, 2% CTAB, 2% PVP, 2% 2-
mercaptoethanol. After initial 45min incubation at 650C, solid CTAB was added to extraction
buffer, raising CTAB concentration to 6%. Further 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 deproteination 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 concentration of CTAB here is maintained, but, since not
salt was added, the ionic strength of the solution decreases from 2M NaCl to 0.5M. In low ionic
strength, CTAB precipitates nucleic acids during a 30-minute incubation. The pellet formed after
the incubation and successive centrifugation, the supernatant is discarded and the pellet dissolved
in 0.5 volume of 1M NaC1. Prep was treated with RNAse and downstream stages of DNA
precipitation by alcohol followed as the standard procedure cited above.
24. Increase in CTAB concentration to 6% as above, with the difference that here CTAB
was not added as powder, instead as equal volume of 10% CTAB, 2 M NaC1. For DNA
precipitation, 3 volumes of 6% CTAB, 100mM Tris-Hcl, 25mM EDTA were used, decreasing
concentration of NaCl to 0.5M. Both fresh (T58) and lyophilized (T59) leaves were used.
25. Pea: extraction buffer: 0.7M NaC1, 1% CTAB, 50mM Tris-HCl pH 8.0, 10mM EDTA
pH 8.0, 1% 2-mercaptoethanol, 0.01% sodium bisulfite. Departs from Murray and Thompson
protocol in that DNA precipitation 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-HC1, 50mM EDTA, 2.2M NaC1, 2% CTAB,
0.06% sodium sulfite, pH 8.0; after homogenization of the tissue and buffer, 0.5 volume of each
5% sarkosyl, 10% PVP, and 20% CTAB were added, elevating the CTAB concentration from
2% to 5% and decreasing NaCl concentration to 0.8M. Plant tissue: 'Strawberry Festival', fresh,
mature, leaves of 'Strawberry Festival' (T63) or 'Sweet Charlie' (T64), 3.5g, 4ml buffer/g tissue.
27. Cacti (de la Cruz et al., 1997). Combination of CTAB and SDS extraction buffers.
CTAB buffer: 100mM Tris-HCl pH 8.0, 20mM EDTA pH 8.0, 4% CTAB, 1.7M NaC1, 4% PVP,









5mM ascorbic acid, 10mM 2-mercaptoethanol. STE buffer: 100mM Tris-HCl pH 8.0, 50mM
EDTA pH 8.0, 100mMNaC1, 10mM 2-mercaptoethanol. Fresh 100mg of 'Strawberry Festival'
leaf tissue were ground in liquid nitrogen and subsequently incubated for 10 min at 650C with
lml 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 650C was carried out; 1.25ml of
cold 5M KOAc was added and incubated in ice for 40 min, centrifuged at 3,500rpm for 10 min
T65). The upper phase was transferred to a new tube and the nucleic acids precipitated by
isopropanol. An alternative method (T66) substituted the addition of KOAc and ice incubation
by addition of equal volume of 24:1 choloform:octanol, keeping steps after centrifugation the
same.
28. Extraction buffer/plant material Incubation temperatures and durations were tested: 4C
(T67-T70), 20C (T71-T74), 42C (T75-T78), 65C (T79-T82), and Omin (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 NaC1, 100mM Tris-HCl pH 8.0, 20mM EDTA pH 8.0, 1% 2-
mercaptoethanol) 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 incubation time "zero", an aliquot was taken right
after buffer and ground tissue were mixed and chloroform was added. Samples were centrifuged
at 4,000rpm for 10min. The upper phase was transferred 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 treated with RNAse. The precipitation steps were repeated to
obtain virtually RNA-free DNA. DNA was quantified 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 NaC1, 100mM Tris-HCl 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 transferred 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 above) to the tissue and further grind until slurry
is formed (T86). The 100mg per treatment of FRA520 plant material was mixed before nucleic
acid isolation to eliminate the leaf age factor. A NanoDrop was used to quantify the nucleic acid
content.
31. A factorial experiment tested interactions 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 grinding method) in one of the two fashions tested, the material
was split to be incubated for 1 hour in the two different temperatures. The downstream steps
were followed as described above, including quantification of nucleic acids and absorbance at
230nm and 280nm by a NanoDrop ND-1000 spectrophotometer.









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









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










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










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










Predicted
Number of Genes
Fosmid
ab Similari
initio ty


6

7

17022
1
2AA
3A
4

5
6



9A
18A19
1
2
3
4
5
6
7
22H18
1
2
3
4A
5
6 k
7A
8
A
22L05
1
2
Not
predicted
3

4A
5
6

7


Protein Hit


EST
Hits
(gb no.)

CX6616
57.1


9 6


7 6


8 4


8 3


Fosmid
Insert Size
(bp)


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


34,090 3.8


homeodomain
X
X

oligopeptidase

hypothetical

X
unknown
lectin protein kinase
hypothetical

cytochrome P450
X
integrase
integrase
integrase
integrase
transferase


+ BQ1046
55.1
DY675
330.1


+


40,908 5.8


37,851 4.7


polyprotein
X
hypothetical
X
unknown
pre-mRNA
processing factor 38

X
X
X

oxidoreductase
oxidoreductase
sulfate transporter
X


+

+
+

35,112 4.4 11.7



DY674519.1 EST starts upstream of predicted
gene 3, and spans oxidoreductase
+ DY671
565.1
+

C03800
67.1










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










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










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










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










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










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










APPENDIX C
PCR PRIMERS USED TO AMPLIFY AND SEQUENCE GENE-PAIR HAPLOTYPES


GPH name
GPH4

GPH5





GPH10


































GPH23


11D02


Primer name
GPH4a
GPH4b
GPH5#2a
GPH5#2b
GPH5A2
GPH5B#2
GPH5A3
GPH10a
GPH 1Ob
GPH10c
10ABCol7Rev2
10ABCol32Rev2
10AB#3
10AB#4
10AB#5
10AB#6
10AB#7
10AB#8
10AB#9
10AB#10
10AB#11
10AB#12
10AB#13
10AB#14
10AB#15
10AB#16
10AB#17
10AB#18
10AB#19
10AB#20
10AB#21
10AB#22
10AB#23
10PPR1F
PPR1R1
10PPR1R2
GPH23F
GPH23R
GPH23F2
11D02F
11D02R


Primer sequence
ACGAGGGCTTGGAAGAAAGG
GCCCAACAACAGAAAGACC
CAATGCCATGGTCTCCGGTC
TGCCGTTGCACACACCTTCC
GCTCTTTGGTGTTCAAAGTTGGAT
ATCCAGCCAAACTGAAGGTG
CAGCCATGAAGTCAAGGTCA
GGCTTCTTCTTGTCCGGCAGC
GAACTCCAGGTCAGATCTTCG
CTCGCTGCAAATCAGCTACC
GAGTTTGTCGAGCTGATC
ATAGAGGCGATGTTGTAG
GGCCCTGATCACTCGACA
GGTTTGGTTGGTTAAGGTG
GACAGTACCTGAAAATTTGG
AAGTATCATTAACAGGC
ATCATATATGCGGGTGTG
TAACGAGCAGTGGCGG
ATCACCTCTACTCCCACGC
CACCGTAACAGCTGAGCAAG
ACACAAATGCCTCATCCACA
ACTAAAGCCCAGCAACCCTC
TTCTCTGTCAACCCTGCCTT
GGGGCAAAGTTTACATAGCA
AACTCGCCGGAAGACACTTA
GCCGGAAGACACATATCGAT
GCATCCCCTTTACATCCAAA
GTTAGAGACGACGACGGGAG
TGCCTGGCAAAGTAAACCTC
GGCGTGTCAATTTGTGAATG
TCATCTTCCTCTGTATGCGACT
GGTTTTGTTTTTGGTGGGAA
GTCGAGTGATCAGGCCGTA
AACGGAGAAGAAGACTGTCG
GATCGAACGGCTGATATTAAA
TGTAGCTCATACTTTTGTTCTC
CTTGAGGGCCATCAGCAC
TACACCCACGCCTTCATCTC
GAACTGCGAAGATCTATCTGA
GAGCTGCTGTGTGAACCAAA
GTTCAACTCCAGATGAAGTGAGG










GPH name
17022

27F10

29G10

32L07

34D20


40M11

63F17

72E18


Primer name
17022F
17022Rb
27F10F
27F10R
29G10F
29G10R
32L07F
32L07Rb
34D20Fb
34D20Rc
34D20Fb2
40M11Fd
40M11Rd
63F17F
63F17R
72E18Fb
72E18Rb
72E18Fb2


Primer sequence
AAAATGGGTTGCACGAGTTC
GGGTTTCCTCACAAACTTCG
CCTGCAGGGTTTTTCATCAT
TGGAAATGTATTCTGGTTCTCC
TGGCCTTGTTTCCTAAACTCTT
AGAAGAAGGCAGCACCCAAT
GAGTTGAAAAACGGGTCGAA
AGGAAAATGCGGGAGAAAGT
GCAGAAAGAAACTGATGTGCTT
CGCAGTCGTAAAAATTCGTCT
TGGGTGTGGATGAACTATACG
CAACATTTTGGTGGCCTTCT
CGGCCTATGAAACCACAGTT
GCAGAAAGAAACTGATGTGCTT
CGCAGTCGTAAAAATTCGTCT
GCAGAAAGAAACTGATGTGCTT
CGCAGTCGTAAAAATTCGTCT
GCAGCAATCAAAT CATTCCA












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












TGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGAGTACCTTGTCTCGCATGGGCTTAAGAATGCTTCATATA
CTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGAT
TTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTGCATATGATATTA
AATCTTTTGTTTGGTTCCTCATTGTATAATTTGGTTCTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTA
ATTCAGGGTTCAAAATTGCACCTGATCAGCTTACACGAAAGAGAATGCCGGATGTGATAAATTCAGGTGTCAATGAC
CCTCCACAAAAGAGATCACTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTATGATAGAAGATCTTCTAT
TTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATTTTTCAGCAGCCAGCGTCTTCTATGGTTAATTCAGGCTT
CAAAATTGCACCTCATCAGCTTACGCGAATGAGATTGCCGGATGTGGTAAATTCAGGTGTCAATGACCCTCCACAGA
GGACATTGCCGGATGTAAGTATCTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACC
TTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAATGATGTTTTTTGGCTT
CCAGTTTTTCTTCACATAAGCATTTTAAAGCTGATCATTGTAATCGAACTCGAATTATTCTACTACTGGTGTAAGTT
GCCTTGTGTCACCACCACTAAGATCACAATTTCGTATTTTATGATCAACACCGAAGACCTATGTCTAGTGTCGTGAT
TATGGTCATGTGAAGTGGATTTCTTAATATGCCTCGTCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTG
ATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTTCGTGCTGAAGATGGGAGTGTTGTATGT
GGAGTTCATGAAAACATTGTGAATTTTTTTGATATATATTTTTGTTTTTGTAAAAATGGATCTCTTCATAACATTGG
GGTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAACGGCA


>GPH5 viridis clone5
CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAACCGGACCGGAAGATCAT
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












CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA CCGGACCGGAAGATCCT
CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT
GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG
AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT
ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT
CAAACAAAGCATCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTT
TGGTGTTCAAAGTTGGATTCAATTACATGTAGACATAGTTACCATTTTCCCATTTGAAATAGAAGGTAATATATCAA
GTTAATTGTACAATAATATTTGTAATCAGTGAAAATAATGACAATCTTTATAACAAAATTTCAGTTATCTTTTCACT
GCTGTATGAACTGTCACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACC
AATATAAAACAGAATTGGATTTCCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGACAAAGAAACCTTTTGT
CATAGGGATTGAACCTGAATTATCTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCATGTTACATGTCTGCAT
TTGGTGACAAAAGCTAAATCTTAAGAATTTAGACGTATTAGACCATTGGGCTTAATCATCGTCCGAGCCCAAATCTG
CACTAGCCCGTAATATGCTTTTTATAGAAAACAGACTCTCTGTGATCTTCGCCATTGAGGAGTCAGGTTACTCAGCT
CTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTCTTCAAAGTTTGAAACTTTAAGCT
TCAATGGAGGAAGATAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATGTGGTTGGCATATATAAAAGCTTGAAGGA
TTGCCAAAACCAAGCTGGTTCCTCGGTAAAGTTTTGATCTTTTAAGCCCTTTATAATTTGATTACTCTCATTGTTTT
ATCAATTTTTGATTTCCCATTTGATnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnACTA
GTTTTTCTAAGAACTTGATGTCATTTACTTGATGAGTTGATGGAGGATTACACATGTGGTTTGGTTTTGTTTTTAGG
TATGCAATCCTTCTGTAAGTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCGAGGAGTACCTTGTCTCACATGGG
CTTAAGAATGCTTCATATACTATCAGCGCCAGTGATGTGAAAGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTA
CCAGGTTTGAATTGATTTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAGCTA
GTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAATCTTTCAGCAGCCA
GCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTAATCAGCTTACACGAAAGAGAATGCCGGATGTGGTAAA
GTCAGGCGTCAATGACCCTCCACAAAAGAGATCATTGGATGTAAGTATCATATGCTACATGGAACTTTTGTAGTTTG
ATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTATAATCTTTCAGCAGCCAGCGTCTTCTA
TGGTTAATTCAGGCTTCAAAATTGCACCTTATCAGCTTACACGAATGAGATTGCCGGATGTGGTAAATTCAGGTGTC
AATGACCCTCCACAGAGGACATTGCCGGATGTAAGTATCTTATGCTACATGGAAATTTTGTTCTGGCCAGATTGGCA
TGAAAATCCGGATACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTGCAAA
TGATGTTTTTCGGCTTCCAGTTTTCTGCACATAAGCATTTTAAAGCTGATAATTGTAATCGAACTCGAGTAATTCT
GCTACTGGTGTAAGTTGCCTTGTGTCACCACCACTAAGATCACAATTTCGTATTTTATGATCAACACTGAATACCTA
TGTCTAGTGTTGTGATTATGGTCATGTGAAGTGGATTTCTTAATATATGCCTCATCTATGTCTTCATCCAGCAATCC
TGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGTGCAGGAGCTGTACTCCGTGCTGAAGA
TGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATCTTTTAGGATATATATTTTTGTTTTTGTAAAAGTGGA
TCTCTTTATAACATTGGGTTTACTATAGTTGCACCGGCTGCGGGAAGGTGTGTGCAACGGCA


>GPH5 nubicola clone7
CAATGCCATGGTCTCCGGTCTATTTCAACCGGGAAGTTCTTATGAGTGGGTGGTGACAAA CCGGACCGGAAGATCAT
CAGAATCAGATTTGTTTGCTCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT
GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG
AAAAGAACTTGCTTGCGCTTTCTCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT
ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT
CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAATGAACTGTAAGAGACATATTTCAAGC
TCTTTGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAATAGAAGGTAATACG
CATGATATAAATATCAAGTTAATTGTACAATGATATTTATAATCAGTGAAAATAATGACAATCTTTATAACAAAATT
TCAGTGATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGACCAACAACACCAAACAAACAGAA
CCAGACCAAATCACACCAATATAAACAGAACTGGATTTTCATGAAAGGGCGCAAGGCACAATCAATGAAGGAGAAGA
CAAAGAATCCTTTCGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCGTATGCAGGCA
TGTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGC
TTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATTCTTTTTATAGAAAACAGAGATTCTCTGTGATCTT
CACCATTGAGGAGTCAAGTTACTCGGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTG
GGTTCTTCAAAGTTTGAAGCTTTAAGCGTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGTTCGAAAGGGAGATG
TGGTTGGCATATATAAAAGCTTGAAGGATTGCCAAAACCAAGCTGGTTCATCGGTAAAGTTTCGATCTTTTAAGCCT
TTTATAATTTGATCACTCTCATTGTTTTAnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn












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












ATATATTTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTACTATAGTTGCACCGGCTGAGGGAAGGTG
TGTGCAACGGCA


>GPH5 mandshurica colonel
CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAAGAAGACCGGAAGATCAT
CAGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGACAAGATCCTAAGGAGGAACTCCGAGTCT
GGTTTAGAATTGTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG
AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT
ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGGAGCAGGTTAGCTAAAAGCTTC
AAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGTTGAACTGTAAGAGACATATTTCAAGCTCTTT
GGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTCCCATTTGAAATAGAAGGTAATACGCATGA
TATAAATATCAAGTTAATTGTACAATGATATTTGTAATCAGTGAAAATAATGACAATCTTTATAACAAAATTTCAGT
GATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGA
CCAAATCACACCAATATAAACAGAATTGGATTTTCATGAAAGGCAGCAAGGCACAATCAATGAAGGAGAAGACAAAG
AATCCTTTCGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCATGTTA
CATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAAT
CATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATTCTTTTTATAGAAAACAGAGGTTCTCTGTGATCTTCACCA
TTGAGGAGTCAAGTTACTCGGCCCTGAAGTAAAAGTCCAGTCAAGTAGTGCAGTTGAGTTCAACTTGTTCTGGGTTC
TTCAAAGTTTGAAACTTTAAGCGTCAATGGAGGAAGAGAAGGATGCCTTTTATGTTGCTCCAACGGGAGATGTGGTT
GGCATATATACAAGCTTGAAGGATTGCCAAAACCAAGCTGGTTCATCGGTAAAGTTTTGATCTTTTAAGCCTTTTGT
AATTTGATCACTCCCATTGTTTTATCAATTTTAGATTTCCCATTTGATTACATTACTGGCTCTTGTTTATTTTGTTG
AACTAACTATGCCCTTTCGTTCTAACATGCAACTGAAAATAACTGCTAGATTGTATAGCTGAGCCTTTATGGTGTTC
ATTATGTAAAAAGAATTCTGGTGTGTTCTGGTGGTGGGTATAAAGCACCTCCCTGAGATTATATGAGATACTATGCTTCTGG
AAAATGTTATAAGATGAAAACAACTTTTTCTAACAACTTGATGTTATTTACTTGATGAGTTGATGGAAGATTACGTA
TGTGGTTTGGTTTTGTTTTTAGGTATTCAATCCTTCTGAAATTGTGTTTAAAGGGTATGGTTTGCCTAAGGAGGCCG
AGGAGTACCTTGTCTCGCATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAAAGATGGTCTGTTT
GGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCATGTGTTCTAGTTTCTGTTTGGGTATCTGTTA
TTTTCATGGCATGTCGCGTGGAACTAGTTGCATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGT
TTTTTATTATAATCTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTGATCAGCTTACA
CGAAAGAGAATGCCGGATGTGATAAATCCAGGTGTCAATGACCCTCCACAAAAGAGATCACTGGATGTAAGTATCAT
ATGCTACATGGAACTTTTGTAGTATGATAGAAGATCTTCTATTTGGTTACTCATTGTATGATTCGGGTTTTTATTAT
AATTTTTCAGCAGCCAGGGTCTTCTATGGTTAATTCAGGCTTCAAAATTGCACCTCATCAGCTTACGCGAATGAGAT
TGCCGGATGTGGTAAATCCAGGTGTCAATGACCCTCCACAGAGGACATTGCCGGATGTAAGTATCTTATGCTACATG
GAAATTTTGTTCTGGCCAGATTGGCATGAAAATCCAGATACCTTCAGTTTGGCTGGATTATGGAGTTGCGTTGATCA
CTTGTTTATTGTATTTATTCTGCAAATGATGTTTTTTGGCTTCCAGTTTTTCTTCACATAAGCATTTTAAAGCTGAT
CATTGTAATCAAACTCGAATAATT CTACTGGTGTAAGTTGCCTTGTGTCCCCACCACTAAGATCACAATTTTGT
ATTTTATGATCAACACCGAATACCTATGTCTAGTGTCGTGATTGTGGTCATGTGAAGTGGATTTCTTAATATATGCC
TCATCTATGTCTTCATCCAGCAATCCTGCATACTTGAGTTTGATGGAGCTTCAAAAGGAAATCCTGGATTATCTGGT
GCAGGAGCTGTACTCCGTGCTGAAGATGGGAGTGTTGTATGTGGAGTTCATGAAAACATTGTGAATTTTTTTGATAT
ATATTTTTGTTTTTGTAAAAATGGATCTCTTTATAACATTGGGGTTGCTATAGTTGCACCGGCTGCGGGAAGGTGTG
TGCAACGGCA


>GPH5 ananassa clone2
CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA CCGGACCGGAAGATCAT
CGGAATCAGATTTGTTTGCGCTTGCAGAAAGAGAATCCTCGAGTGAAGAGAAGATCCTAAGGAGGAACTCCGAGTCT
GGTTTAGAATCCTTGAGCAAACTCAAGGAACAAGAAGTAGCACCTCCCAAGAAGAAGAAGAAAAATGGGATCTACAG
AAAAGAGCTTGCTCTTGCTTTCCTCCTACTCACAGCATCAGCAAGAAGTTTCCTATCAGCTCATGGAGTTCACTTCT
ATTTCTTGCTTTTCCAAGGCTTGTCCTTTCTTGTTGTAGGCTTGGACTTAATAGGTGAGCAGGTTAGCTAAAAGCTT
CAAACAAAGCGTCAATTGCCCACAGTTATTCTTTGATAGATATATGCTGAACTGTAAGAGACATATTTCAAGCTCTT
TGGTGTTCAAAGTTGGATTCAATTACATGTAGACACAGTTACCATTTTTCCATTTGAAACAGAAGGTAATATGCATG
ATATAAATATCAAGTTAATTGTACAATGATATTATTTGTAATAAGTGAGAATAATGACAATCTTCATAACAAAATTT
CAGTTATCTTTCCATTGCTGTATGAACTGTTACCATTAGCCTCTCACACAAGAACAACTACACCAAACAAACAGAAC
CAGACCAAATCACACCAATATAAAACAGAATTGGATTTTCATGAAAGGCGGCAAGGCACAATCAATGAAGGAGAAGA
CAAAGAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTCATATCTCATATGCAGGCA
TGTTACATGTCTGCATTTGGTGACAAAAGCTAAATCTTAACATGACCTAAGAATTAAGACATATTGGACCATTGGGC
TTAATCATAGTCTAAGCCCAAATCTGTACTAGCCCATAATATGCTTTTTATAGAAAATACTGTGATCTTCACCATTG













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
CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAACCGGACCGGAAGATCAT
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












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












GTAGAAATAATTGAACATTAGAAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAA
AGCGGCTTCATATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATT
CCCAATTTTCATTTCCCACCAAAAACAAAACCCACACGACGCCGTTTTGCTCCAATCCCCCTTTCTTCTTCAACCCC
ATAGTCGCCTCAGCTCAGTTCCATTTGTCTCAGATGCGATGGCCTCCGGCGACCCAATCTCCGACTACACCCAAACA
CATCGCATTGTCCTTCTAATCGACCTCAACCCACTCCTCCATCTCCAAGATCCAACCCAATTCCTCACCTCTGTCCT
CTCCTCAATCAAAACCCTAACCTCCTTCCCTTCTCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCTCCTCT
CCTCTCCGCCTCCAAGCTCCCGTCTTCGTCTCTAACCCAA CGATCCCCTAT
CTCAAACCCTGGCGTCTCTCTCGTTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAACGCTTGTTGCGGCT
GCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAGCAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGATACGTT
TTCGAATTGCTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAATTTGTGAATGAGTTCTTGA
ATTGTGAGGGTTTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAGGCATAT
GTGTATAGAGATATTCAATTGAGTCGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGTTGGATT
GAAATGTGGTGTTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGCTTGGTT
CGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTGGGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATAT
AAGAAGGTTAGAGCGCATTTGAGTCTTGAGATATCGGATGTAAAGGGGATGCCTTTGGAGTGCAAGTTTTGTGATCT
TGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATCGCTTGTTTTCGGTGGAAGGCATGAGCTCGC
AGACAAGAGGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAATGGAGTGTCGAAGATTCAGGTTAAGGCT
TTGCAGAAGGATAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCTGATTCTGGTCTATGAAGTTTCAGGAAA
AGATGGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCTATCAGTGGAATTGGGTGAGTTTG
TACCGAGGAAATTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTAGTTTCT
ATTTCAAATGATAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATTTTTGTTAT
GGATGAAGGAATTCACCCTCATAAAAAAGGGCATGGCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAAAGATGA
AGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCATCTAATAAG
CATTCTGCTGAGATTGATGGAAAGAAAAAAAGTAGCAAAAGAAGTTCACATTCACTCAAAGATCTCACCTGGAGTTC
TTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGTTTACTTTGCCAGGCAACGTAGCAGCTCAA
AAAAGTTGAAATTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAATCTAAGGTG
CACCAGGAAAAACAAAAGGAGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATGTCGTCATC
TGGTTCAGCTGGAGAAATTTCTTTCCCTGTGGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAGATTACAAA
CCTCAGAAGATTTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTGAATCTGAAGTAGTAGACTTGGGGGCA
TTCGCACATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAACATAGCACAACAACCCCTTCAGAAGATCA
AACTCCTGTAAAATATGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGG
ATATGGTTGCCAGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCCTGGATGTGAAGGCTTTACTTCAGAA
ATAATAGTTCGAGAGTATCCTTTCATTTCTCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCTAC
TTTAATGCTATGTAAACTTTGCCCCTTGTTAGTGTTACACTTTTCCTTCACTAGCACAAAGATATGAATTACAGATA
CTTTTCCGGATGGAGATTTTACAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTGAAACA
TATTTGCACGCTTTTGGAGACCATTCGTGCTCGGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAA
ATTATGCTGGAAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTATTTTCT
AATGAAATTGTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAATATATGTTTATGGT
TTGCCTGTTAATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATTTGTATG
CCCTCCCAAAAGGCCTTTGGGGGTAGTATGAAGAAGGGAGACATTGACAGTCAAAAATATTATCTCCTTATTTTACG
TACAAAATTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTCCTTTGC
ATAATTTCATATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGTATGCAT
ACAACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACTGCCCAAA
TTTATGTGAAAACACTGCATTTATGTTTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCAAATTTT
CAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATGATGAGG
AAGAACTCCCTAATAATTTATTCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGATGAGGTG
GGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAACATTACAATGGAAG
GCCAAGTGCTCAAGTACTTAAACAAGAAGAGCATGCTCGCAAGTTGATGAAAGCTCAAGAGAGTAGAGAGAGGGCTT
GGAGAATTGCTTCTTTCACAAGTCGGGTAGCTGATTTGCAGCGAG


>GPH10_ananassaclone7_(samerestrictionatternas clone20)
GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT
CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT
AGCGAACTGAGTAGTTTGGATTTGAGAAGAGGATGTAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTGGAGAA
AGCTTTCGGCTTTGAAGTGGAGTGTAGGGTAATAACAAACTCGTGATTAAAAGACAGGATTAATGTCAGTGAGGTTT
GGTTGGT GTTAATTTAAGGTCATAGGTTCAAATTT TCT TTC CCTCACGACATATGTAGGGGTGTATGAATTAT












TAATAAAAGACAAATTTAATATCAGCCGTTAGATCATATTACGGCCTGATCACTCGACATATGTTGATATACGCCCA
ACTCAAATTCGATATATATTTTCGATATACGTATATTTTATTTTTTTAAAATAATTAAATAACTATTTACGTTGTTT
AACAAAAGAAACAATTGAAGTTAAATTAAGAGCACCGTAACAGCTGAGCAAGAGTACGAGAACAAAAGTATGAGCTA
CATCATTTGTTCATATAGAGAAAATATAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTAA
AAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCATATCATCCGCTTGATCATATATGCGGGT
GTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCAAAAATAAAACCCACACG
ACGCCGTTTTGCTCCAATCCCCCTTTCTTCAACCCCATAGTCGCCTCAGCTCAGTTCCATTTGTCTCAGATGCG
ATGGCCTCCGGCGACCCAATCTCCGACTACACCCAAACACATCGCATTGTCCTTCTAATCGACCTCAACCCACTCCT
CCATCTCCAAGATCCAACCCAATTCCTCACCTCTGTCCTCTCCTCAATCAAAACCCTAACCTCCTTCCCTTCTCTCT
CTTCCTCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTCTCCTCTCCTCTCCTCTCCGCCTCCAAGCTCCCGT
CTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACATATCGATCCCTATCTCAAACCCTGGCGTCTCTCTCG
TTTGACCGGAAGTTGACCGGGTCCGATTCGCCGCGGGGAACGCTTGTTGCGGCTGCGATGCGGCAGCTGGTACATGA
TTACGCTTGGGAGCAGGTGATCTGCGACGCCGTGGCGGCGGAGACAGGTACGTTTTCGAATTGCTGTGGTTTGAGGT
CTAATTTGGCTGTTGTGTTTTTACCGGCGTGTCAATTTGTGAATGAGTTCCTGAATTGTGAGTTGAATTGTGAGGGT
TTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAGGCATATGTGTATAGAGA
TATTCAATTGAGTTGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGTTGGATTGAAATGTGGTG
TTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGCTTGGTTCGGCTCTTGTT
CCATTTGGTTTGATTTATCCAGAGATTGGGTGTCATCTAGGATTTTCGGGTGTAATGATCGATATAAGAAGGTTAG
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 clonel8












GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT
CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT
AGCGAACTGAGTGGTTTGGATTTGAGAAGAGGATGAAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTAGAGAA
AGCTTTCAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTTATCTAAAAGACAGGTTTAATATCGGCCGTTAG
ATCACATTACGGCCCTGATCACTCGACATATGTTGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACA
TTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTTTTTTTAAAGTAACTAAATGAC
TATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAAGAGCACCATGACAGAGTACGAGAA AGTATG
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
CCCAAGGATATGGTTGCCAGGCACAAAAGCTAAGCTCTGATCCTGGATGTGATGGCTTTAC
TTCAGAAATAATAGTTCGAGAGTATCCTTTCATTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCA
ATTCTACTTTAATGCTATGTAAACTTTGCCCC CTGTTACACTTCCTTCACTAGCACAAAGATATGAATTAC
AGATACTTTTCCGGATGGAGATTTTACAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTG
AAACATATTTGCACGCTTTTGGAGACCATTCGTGCTCAGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCT
AGAAAATTATGCTGGAAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTAT
TTTCTAATGAAATTTGTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAGTATATGTT
TATGTTTTGCCTGTTAATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATT
TGTATGCCCTCCCAAATGGCCTTTGGGGGTAGTAAGAAGAAGGGAGACATTGACAGTCAAAAATATTATCTCCTTAT
TTTACGTACAAAATTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTC
CTTTGCATAATTTCATATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGT
ATTCATACAACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACGG
CCCAAATTTATGTGAAAACACTGCATTTATGTTTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCA
AATTTTCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATG
ATGAGGAAGAACTCCCTAATAATGTATTCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGAT
GAGGTGGGTGAAAATAGTAGAATGAAGAAATTGGTATCAGCAGAAGATGAATCCCCTGATCCACAGAAACATTACAA
TGGAAGGCCAAGTGCTCAAGTAGTTAAACAAGAAGAGCATGCTCGCAAGTTGATGGAAGCTCAAGAGAGTAGAGAGA
GAGCTAGGAGAATTGCTTCTTTTACAAGTCGGGTAGCTGATTTGCAGCGAG














>GPH10 ananassa clonel9
GGCTTCTTCTTGTCCGGCAGCCTCTTCAGCCACTCGTCCTCCGGCGCCGCCGATACCTCCTCCGCCTCCGACGACTT
CGAACACAGCGGAATCGCTAGCCTCCTTATCGGAGACCGAACGAGCCGAAACGGCGTCGCTTTAGGCGAGAGTGAAT
AGCGAACTGAGTGGTTTGGATTTGAGAAGAGGATGAAATTGGTAACGGAGAAGAAGACTGTCGACATTTTTAGAGAA
AGCTTTTAGCTTTGAAGTGGAGTGTAGGATAATAACAAACTCGTTATCTAAAAGACAGGTTTAATATCAGCCGTTAG
ATCATATTACGGCCCTGATCACTCGACATATGTTGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACA
TTTTTTTTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTTTTTTTAAAGTAACTAAATGAC
TATTTACGTCGGTTAATAAAAGAAACAATTGAAGTTAAATTAAGAGCACCATGACAGAGTACGAGAA AGTATG
AGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATATAGAGGCGATGTTGTAGAAATAATAGAACATTAG
AAAATTAAATTACCTAAAAGCCGATGAGTAAAATAATAACAAACTCGTAACCTAAAAGCGGCTTCATATCATCCACT
GGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAACCCGCCAAAGCCTAATTCCCAATTTTCATTTCCCACCA
AAAACAAAACCCACACGACGCCGTTTTGCTCCAATCCCCCCCCCTTTCTTCTTCAACCCCATAGTCGCCTCCTCAGC
TCAGTTCCATTTGTCTCATGCGATGGCTTCCGACTCAAATTCCGGCGACCCAATCTCCTCCTACACCCAAACCCATC
GCATCGTCCTTCTAATCGACCTCAACCCACTCCTCAATCTCCAAGATCCAACCCAATTCCTCACCCCTGTCCTCTCC
TCAATCAAAACCCTAACCTCCTTCCCTTCTCTCTCTTCATCTCTCTTCGCCGTCAGGCCCTTCTTCTCGTCTCTCTC
TCCTCTCCTCTCCGCCTCCAAGCTCCCGTCTTCGTCTCTAACGATCTCTTTCAACTCGCCGGAAGACACTTATCGAT
CCCTATCTCAAACCCTGGCGTCTCTCCTGGTTTGACCGCAAGTTGGCCGGGTCCGATTCGCCGCGGGGAACGCTTGTT
GCGGCGGCGATGCGGCAGCTGGTGCATGATTACGCTTGGGAGCCGGTGATCTGCGACGCCGCGGCGGCGGAGACCGG
TACGTTATCGAATTGCTGTGGTTTGAGGTCTAATTTGGCTGTTGTGTTTTCACCGGCGTGTCAATTTGTGAATGAGT
TCTTGAATTGTGAGGGTTTGGAGGATTTCAATGTGTTTTGTGAGAGGTTTCGAGGGTTTTTCGAGAATGTGGATGAG
GCATTTGTGTGTAGAGATATTCAATTGAGTTGGGTTGATGTGAGGTATGGATTCGATAGCGGTGAGGATGAGGTAGT
TGGATTGAAATGTGGTGTTTTCGAGAGGGGGGTTAGGAGTTTAGGGTGGGGGTTTTGCTCATCTGATTCGATTGTGC
TTGGTTCGGCTCTTGTTCCATTTGGTTTGATTTATCCAGAGATTGGGGTGTCATCTAGGATTTTCGGGTGTAATGAT
CGATATAAGAAGTTTAGAGCGCATTTGAGTCTTGAGATATCGGATGGAAAGGGGATGCCTTTGGAGTGCAAGTTTTG
TGATCTTGAGTTGGCTGATTTGAAAATGTTGTGTAGGAGTAGAGGTGATGATGGCTTGTTTTCGGTGGAAGGCATGA
ACTCGCAGACAAGAGGTCATGAGGTGAAGAGGCTGTTTTGGGGAAGTGTTGGCAACGGAGTGTTGAAGATTCAGGTT
AAGGCTTTGCAGAAGGATAGTGAGTTTGGGAAATTTAAGGGGGAATTGTCGGATCCGATTCTGGTCTATGAAGTTTC
AGGAAAAGATGGAAAAGAAGTTTCTGGTGGTTTGTTTGTAGATAAGGTTCTTGAAATGCTATCAGTGGAATTGGGTG
AGTTTGTACCAAGGAAATTGCCACCTGTTTGGCAGATTCTCTTGAGTTTTATATACAGGGAGGGTTGCTGGGCATTA
GTGTCTATTTCAAATGATAGTGGTGTATCACATACTGGAATCCTTAAGCCTTTTACAGTTTCTTCAGCTCTTATTTT
TGTTATGGATGAAGGAATTCACCCTCATAAAAAAGGGCATGTCATTGGTGCAGTGAATAAGGGTCAGTCTCGTCCAA
AGATGAAGAATGAGATGTGCAAACCTGATGCTGATTTGAACGACTTTTGTGGGTCGCAAACTGGGCCTTCACCATCT
AATAAGCATTCTGCTGAGATTGATGGAAAGAAAAAAAGTAGCAAAGAAGTTCACATTCACTCAAAGATCTCACCTG
GAGTTCTTTCTGTAAGGCAGCATTCGAATTTTCAGACTTACATTTGGAAGAGGTTTACTTTGCCAGGCAACGTAGCA
GCTCAAAAAAGTTGAAATTTCTAAAATGCTGGATGAAACAGATTAAAAAACTGAAGTATCCAATAACGGAGGAGTCT
AAGGTGCACCAGGAAAAACAAAAGGGGATGAGCAATAGGTTGGATTTGTTGCACCAAGAGAGCGAACAGCCAATGTC
ATCATCTGGTTCAGCTGGAGAAATTTCTTTCTCTGCGGCCTTTGGAGTACAGGATGAAGCTGCTCAGGAACATAGAT
TACAAACCTCAGAAGATTTTTTCTGTAATTTCTCTGATAAGATCCAACAAGGGCTAGAATCTGAAGTAGTAGACTTG
GGGGCATTCGCACATCGGCTTTTGAGTCAATCAATATATTTTTTGACTCAAAAGCATAGCTCAACAGCCCCTTCAGA
AGATCAAACTCCTGTAAAATCTGACAATCTTGATGATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATC
CCAAGGATATGGTTGCCAGGCACAAAAGCTAAGCATCTGATCCTGGATGTGATGGCTTTACT
TCAGAAATAATAGTTCGAGAGTATCCTTTCATTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAA
TTCTACTTTAATGCTATGTAAACTTTGCCCC CTGTTACACTTCCTTCACTAGCACAAAGATATGAATTACA
GATACTTTTCCGGATGGAGATTTTACAATCAGAAGTTGGGGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGTGA
AACATATTTGCACGCTTTTGGAGACCATTCGGTGCTCAGTGTCATCTGGAGGGAGGCTTCTTTGGTGACTGGACCCT
AGAAAATTATGCTGGAAAGATTATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAACTATTAT
TTTCTAATGAAATTTGTATTCATGAACACTGAAATGGTAGATACTCAGTTATTTACAATGAAACTCCAATATATGTT
TATGTTTTGCCTGTTAATGATACTTTTATCAGTACTTCGATGAAACATATAGTGTTGAAACAATTATGTGATTGATT
TGTATGCCCTCCCAAATGGCCTTTGGGGGTAGTAAGAAGAAGGGAGACATTGACAGTCAAAAATATTATCTCCTTAT
TTTACGTACAAAATTGATGACTCCTCATCAGGCTGTTGAAGGCAGGGTTGACAGAGAACAGAAAAGCTAAATACCTC
CTTTGCATAATTTCATATGACTTAAGTGACTTTCCTTATTAATCTAGATTTGCAACCTTGTTTTTCTGACACTATGT
ATTCATACAACTTTTGCAATTGTATTCTGTATGTTGCAATAGTTCATTCCTTTGTTTTCCAGACCCAAAAAAAACGG
CCCAAATTTATGTGAAAACACTGCATTTATGTTTGAAGAAGTAGGATTAGGCAGGTAGACTGATGATTCAATTCCCA
AATTTTCAGGTACTGTCAGACTCTTGAAGACGTGGTTCATAAAATCTACACAAAAATGGATTTGTTACTGTTTGATG
ATGAGGAAGAACTCCCTAATAATGTATTCAACAGCGAGGATAGCAGTCATTCATACAAAGAAAAACCAGGGAAAGAT












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











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












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












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












GACCTGCCAGAATCCACGCCACCAAACTCTTTAGCACTAATCCAATCCATAACAACTTCATAAAACACACATAGCAT
CAACATGCAATAATGTGGGTCCATAAGAACCATGAGTATGACATAGAGTCTTCAAGCTTCGATTTCCTTATTTGCTT
CGAAAGAAGCAAGTTCAGAGTCCACACAACCAGAATATAGATCTCAAATTTAATAAACATATTCCTAAGAACCTAA
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
GAAAGAAGCAAGTTCAGAGTCACAAACCAGCTGTCTCAAAAATTTAATGAACAT AGAACCTA
AAGAAATATAAAATCGTAACTGAACTTATTCTGAAATTGTCGTATAAATTGTAAACCGATCAACACAAACTTCAAGT
TCAGATTCACAGAGCAGATCAGAGATAGCATACCAGTGACCTAAGAAACAAAACAACCTTCTAACAAGATCGCAAAC
ATTGGAGATTAAATACGATGAGCTATGAGACAACTTTTCCATGCAAATCTAACAAAAGAGAATAAAGGGATCTGGAG
AATTAGGGGTTAGAGGTCACCTTAAGAGTTTGGGTGAAACACAACCTGGGGAGACAGAGACAGAGGAGGAACTGCCA
AGATCTATCTGACCACCAAACCAAGTaAAAAGGgTtaaGCTATCAgTAACgAgCtCCtAACCGTCCaTCTCCAATCt
TGTCagGgGtGAtCCTACGCGTCCACTTGTCCTCTCCCAGTTCTAACTGTGTAGACGCTAGCTGCGGATTGTTATTA
TTTTTTGGATAGAATACCTTTGCAAAATAGGAAGCTCCTCCTTGTTTTTCGGCAAGAGAAAGGCCAAAATATCTGAC
CATTCCGACGCCGGAGCTTCCTCAGAAAGCCAGTACTATCCGCCACATCGATGCCAGGCCTTGCGAGGTTTGCCTCC
GCTTCTTTGGATTGTGTTTTTCGTGGTTTAGGAGATTGCTGAACAAAAAAGAAAAACATAACATATGATGAATGAAT
TATCAAATTAATTAATCAAGGTGACAATACAAGATGAGAACACCAAAGATTCAATAGTGTGTACTCTCAAGCCTAAT
ACTAACACAACAAAGAAAGATTCTATCGTTCCATTCCCAAATCAAAAACCATTACAATAGACCGCCGGCAAAGTGCT
GCTAGGAGGTAGTGTCATCGTGACACTCTATGAAGGTGATGCTTAAGAGGGACACGTCAATAGCAAGTATGAGGGAA
AAAAAGGATGTTTACTGGCATGTCGAATGATAATTTCGTAATTAGTTAAGTGAAACTTATATTTCAAGGTACTTTGA
CCTAGTATTTGGAAAAACCGTAACATCTAAATACACTTGACGACATATTTTTATGAGATTTCTATGAATTAGTTATG
AGAGATGGTTTTCTTTAGACTATTTTGATTTTGGATGTTTCCTTAACACACATTATATTTCTCCATTTTCTTTTTAT
GTATAAGTAATTTTCTGTATCACTTAAAACATTTCTTACTCTTTCCAGAAGCATCTCTATCCCCCTAAACCAAATA
CACTAACATGTCAATGTCCACCCAAAAGAAATAAATAAGCACCTTCATTTTTAAGCGCCATAAAAAATAGAGAAGAA
TATAAGGTTTGAAGTGAACAAGGGGATAAGCAGTTTAAGGTCGACTTGTTCGGAAACAATGCTGACCACCACCACTG
CCACTCTCAGTCTCAGCTCCTCCTCCTCTACTTCCCAACTCCCACCACTCTTCCACTCTCTAACACCAAACCCAATC
TCCCTCAGATTCTCCTCCACATTACAGCTAACTAAAACCAGAACCAGACCAACCCTTAAAACTCTCACTCGCCAAAA
ATGCCAGCTCCCTGCTCTGAGAGTGTCTGCTAAATACGAAGCTTCCCCTGCCACAGCTGAGGCCTCCACGGTGCCGT
CGGAGATGAAGGCGTGGGTGTA












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












GAGCTGCTGTGTGACCAAATGGGTACAGAAGAAGCCNGTTTGCCAAACCTACCCATGATCCAATCAATGCATAAACT
TTAAGAACTCAAATACCAAACGATCAAACATAATGACTGAAATGAACAAAAATCAAATGAGCAAAGACTAAATGAGA
AAACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCTGCCAT
TCCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAAACACAATTTGATACAGAAGT
CTTCACACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATAGTAATAGTATAAGGAGAAGAT
GAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCCTAACTTGGTT
TCCTTTCAGTGTCTTCGACCTTCTTCTAAAACGACGGAGTCGGTGAAACTGTGCAAGTCTTCTTGTGAATTTTCTTT
TCTAGGTGATTTTTTTTTTCTTTATAATTAATTTGGTTTTATTTTTCCAAATAATACCTGAAAGACTTTTTTTTTT
TTTTTTTGATAGAAATACCTAAAAGACTTCATAAAAGCTGTTAAGGCTTCATTTAGGATTGCAGTAATTTTTTTTGG
ACAGTATTACGGGACACTGTGACAGCTTGAGTTTGAATCTTAGGTGGGATGATTTAAGTATCTTAGTTGAATGGATG
TTATGACATATTGGTGATTAGTATTAGAGTTATGAGAAAATAAAATGAAAATACAGTACTGGCAATAAACACAATAC
GGTGGAGCAATCAACAAAGCAATAGATTGACAAGAAATGAAGACCTAAAAAAAACCATTGCATTAATGCAATAGTGT
TGATTTTCCAATCTCTCCTGAATAGTATTACAACTCTCCTGGACAAGTCATAACTGTGGGGGGTAATGGTGTAAGCA
AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACATAGCACCCCATAAATCATATCAGATGG
GGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAAACCTAATTTTGTCTAAAGAATGCTAAAATCGAACTCCC
AAGCAACCAAATCTTCTGTTCCCCTGCTTTAGTATGTTGTGATTATGCCTCTGCTTCCCCAGCAGCATGAATCCGCT
CGTCTGGAGTTACAGCATGAAGCAGTTCGTCTGTCTCTTGTTGGCAGTTTGAGGT
AGCTCGTCTGGCATTGCAGCATGAAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGG
ATCTAGGTCCCAACACTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNN


>110D02 iinumae
GAGCTGCTGTGTGAACCAAATGGTACAGAGAAGCCGTTTGCCAAACCTACCCATGATCCAATCAAATGCATAAACTT
TAAGAACTCAAATACCAAACGATCAAACATAATGACTGCAATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAA
AACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCTGCCATT
CCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAATTTGATACAGA
AGTCTTCACACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATACTATAAGAAGGA
GATGAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCATAGCATAGGATTCATTGCTGTATTCTTCCCTTAACTTG
GTTTCCGACGGAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCAATTTTCTTTTCTAGGTGTTTTTTTTTCTTTT
ATAATTAATTTGGTTTTATTTTTCCAAATAATACCTAAAACTTTTTTTTTTTCATAAAATACCTGTTAAGACT
TAAGACTTCATTTAGTATTGCAGTAATTTTTTTGGACAGTATTACGGGACACTGACAGCTTTAGAGTTTGAATCTTA
GGTTGGATGATTTAAGTATCTTAGTTGAACGGATGTTATGACATATTGGTACTTAGTATTAGAGTTATGAGAAAAGA
AAAAATGAAAATACAGTACTGGCAATAAACACAATTCGGAGGAGCAATCAACAATGCAATAGATTGGCAAGAAATGA
AGACCTAAAAAAACCATTGCATTAATGCAATAGTGTCGATTTTCCAATCTCTCCTGAATAGTATTACAACTCTCCTG
GACAAGTCATACCTGTGGGGGGTAATGGTGTAAACAAACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCT
GGGCAGACGTAGCACCCCCTAAATCATATCAGATGGGGTTAATACTACCCAGGTGTGACATATTTGTACAGTTAAAC
CTAATTTTGTCTAAAGAATGCTAAAATCGAACTCCCAAGCAACCGAATCTTCTGTTCCTCTGCTTTAGTATGTTGTG
GTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGGAGTTACAGCATGAAGCAGCTCGTCTCTAGTTGCA
GCATGAGGTAGCTCGTCTCTTGTTGCAGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCGTCTGGCAT
TGCAAGATGAAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAGGTCCCAAC
ACTTACCAGGTAGGTTAGTCTCTTCTGCGTCGAGTAACCATGCGGGTACCTGGTGGGAAAAGCGTAACATCTCTCTT
CTCGGAATCCATAGAATGGCGCTTCTGTCCGTATCAGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGG
TGAGACACAACACCAATCTCTTCAGAGTAATCATCAAGAACCTCCACCATTTGATATGGTGCCTCACTTCATCTGGA
GTNNNNN


>11D02 nubicola
GAGCTGCTGTGTGACCAAATGGTACAGAGAAGCCNGTTTGCCAAACCTACCCATGATCCAATCAAATGCATAAACTT
TAAGAACTCAAATACCAAACGATCAAACATAATGACTGAAATGAACAAAAATCAAATGGGCAAAGACTAAATGAGAA
AACAGACCTCTTTGTAAACTGGGTTTTGGGGTTTAAAGCCATGGGCACCATATGAGCCCAACCCAAGAGCAGCCATT
CCTGTTTATCACAAAATCACAAATTGGGTCCTCTCAGATTGATGCAAAATCACCAGACAAACACAATTTCATACAGA
AGTCTTCCCACAGAGAATATGACATTTGTAATTAAACACAGAATAAAAATGATAACTTTTCAATAGTATAAGAAGGA
GATGAGGACAGTACCAGAGACTGCAGCTACTTTGTGCCACAGCATAGGATTCATTGCTGTATTCTTCCCTTTGCTTG
GTTTCCTTTCAGCCTCTTCGACTTTCTTCTAAAACGACGTAGTCGGTGCAACTGTGCAAGTCTTCTTGTGATGCAAT
TTTCTTTTCTAGGTGATTTTTTTTCTTTTATAATTAATTTGGTTTTATTTTTCCAAATAATACCTGAAAGACTTTTT











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













>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












TGCGAAAACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAG
AACAAAAGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCT
CAGATTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTGCTACAAAAGCCCAT
AACTTGTAAACATCATCGAAGTTTGTGAGGAAACCC


>17022 iinumae
AAAATGGGTTGACGAGTTCGTGAACAACACTTTACGACCCAAAGCGTCCAATACTTCTTAATTTGACAACGGACATA
GTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCTTTTCTTATAAAAGCAATGGAATCGTA
GAGACTGCATATCTTACTCAGCACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTATAACTTCC
CGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATCCCCTTATCAC
TGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTTTAGTTTCTTATGCTGA
AGCAAAATATGTAATCACCTAGGCTACAGAAGAGTGTTACTTATTACCGGACATGTTCACAATCTTTGAAGATGAAG
AACGGTACCAGTTACCCAACATAATCATAGTTATTTCGGAATATTGATATTTTGATTAATATGTAATTGATCGCTAC
TTGACTGATGTATATTATGAATGTCACTATTTAATATTTTGGGCTGCTACCTACTCTTCAACAAACTTTAATTAATT
AACCAAACATCAGTGTCACAAGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAACATACCT
CCCAAAAATAGTTACTTTAAAGCTGGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTT
GTTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTCGGTCATCAGTGTCAAACAAGAA
GAACCGTGAATTGCGACTATACCAGGATGCCTTTGGCCACTTGCCAACCTCAAGAAAAGGACCCCTCAGAATGTCAA
GATGAGATCACTGTGATTCCTTTTAAAATTTGACAGTGATTCTTCTACAAAAGATGGACCAAATTCCACCTTGTAC
TGTACAAAAAACGAGTTTGAGCAGTGGGAATCGTTCCAATATATTTCTGCTCTGTTTACCAATTGCCAGGATGATAC
AAACATCTAAACTCTACAGGAACCCTTTTCTAGCAAAAAAAATGAGAAGAAAGAACTCTACAAGAATCCAAAGCGCG
AAAACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGAACA
AAAGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTCGGAACAATATCATTGATTTCAGATTTTGTCTCAGA
TTCTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTGCTACAAAAGCCCATAACT
TGTAAACATCATCGAAGTTTGTGAGGAAACCC


>17022 nubicola
AAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATTTGACAACGGACAT
AGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCGT
AGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTC
CCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACTCTACATTCATTCGATCCCCTTATCA
CTGTAGGGCTCCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTTTAGTTTCTTATGCTG
AAGCAAAATATGTAATCACCTAGGCTACAGAATAGTGTTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATGA
AGAACGGTACCAGTTACCCAACATAATCATAGTTATTTTGGCCTAATTTTGATTAATATGTAATTGATCGCTACTTG
AATGATGTATATTATGAATGGCACTATTTAATATTTTGGGCTGCTACCTACTCTACAACAAACTCTAATTAATTAAC
CAAACATCAGTGTCACAAGTCACACCAACCTAATTAAACTTTCCATTACAAGTAGCTTTCCCAATAACCTACCTCCC
AAAAATAGTTACTTTAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTGCCTATAATTCTGGATTTGTT
ACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCATCAGTGTCAAACTAGAAGAA
CCGTGAATTGCGACTATACCAGGATGCCTTTGGTCACTTACCAACCTCAAGAATGTCAAAATGAGATCTCTGTAATT
CCTTTTAAAATTTTAACAGTGATTCTTCTACAAAAGATGGACTAAATTCCACCTTGTACTGTACAAAAAACGAGTTT
GAGTAGTGGGAATCGTTCCAATATTATTTCTGCTCTGTTTACCAATTGCCAGGATGATTCAAACATCTAAACTCTAC
AGGAACCCTTTTCTAGCAAAAAAATGAGAAGAAGGAACTCTACAAGAATCCAAAGCGCGAAAACAAAATCAGAACTA
AGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGATGAGCTTCTCCAGCAAGTACAAAAGAATCAAACCAGATA
AAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAGATTCTTCGTCAACAGTAGA
TAGTCCGCCTTCTCTGATGAAGGAAGGATTCAGAAAATTTGCTACAAAAGCCCATAACTTGTAAACATCATCGAAGT
TTGTGAGGAAACCC


>17022 mandshurica
AAATGGGTTGCACGAGTTCGTGAACGTACAATTTACGACCCAAAGCGTCCAATACTGCTTAATTTGACAACAGACAT
AGTAGAGGAAAACAGGTACCTCCAATGCAAGGAATCGGCACTAGAGACTGCATTTCTTATAAAGGCAATGGAATCGT
AGAGACTGCATTTCTTACTCAGTACTGAATCTGTTGGTCAGCAACACAGAAACTAGCTGTGGGCAATGTTTAACTTC
CCGAAATTCAACAGCCATCAGAGTTCATCTGCCAATCAGGGCAAATATGACCACATTCATTCGATCCCCTTATCACT
GTAGGGCTTCATTGGAAACGCTTTGGTCAGCGCAAGACTGATGTTGATTGTAGCCTAGTTTAGTTTCTTATGCTGAA
GCAAAATATGTAATCACCTAGGCTACAGAATAGTGTTACTTGTTACCGGACATGTTCACAATCTTTGAAGATGAAGA
ACGGTACCAGTTACCCAACATAATCATAGTTATTTTGGCCTATTGATATTTTGATTAATGTGTAATTGATCGCTACT
TGAATGATGTATATTATGAATGGCACTATTTAATATTTTGTGCTGCTACCTACTCTTCAACAAACTCTAATTAATTA












ACCAAACATCAGTGTCACAAGTCACACCAACCTAGTTAAACTTTCCATTATAAGTAGCTTTCCCAATAACCTACCTC
CCAAAAATAGTTACTTTAAAGCTAGTTCTTGTCAAATAGTGAACCACCATCAACTCTTCCCTATAATTCTGGATTTG
TTACTCGCTAGTATGTGTTGAACTTTGTTTCTTTTACAAAGACAAAAGGACTTTGGTCGTCAGTGTCAAACTAGAAG
AACCGTGAATTGCGACTATACCAGGATGCCTTTGGTCACTTACCAACCTCAAGAAAAGGACCCCTCAGAATGTCACA
ATGAGATCACTGTGATTCCTTTTAAAATTTAACAGTGATTCTTCTACAAAAGATAAGACTAAATTCCACCTTGTAC
TGTACAAAAAACGAGTTTGAGTAGTGGGAATCGTTCCAATATATTTCTGCTCTGTTTACCAAGTGCCAGGATAC
AAACATCTAAACTCTACAGGAACCATCTTCTAGCAAAAAAATGAGAAGAAAGAACTCTACAAGAATCAAAGCGCGAA
AACAAAATCAGAACTAAGACTAGACATGAACAAATTTGCTGCAGCCTCCACTGAGGAGCATCTCCAGCAAGAACAAA
AGAATCAAACCAGATAAAATGGAAAATCTCCTCTCACGTTGGAACAATATCATTGATTTCAGATTTTGTCTCAGATT
CTTCGTCAACAGTAGATAGTCCGCCTTCTCTGATGAAGGATGGATTCAGAAAATTTGCTACAAAAGCCCATAACTTG
TAAGGCATCATCGAAGTATTGTGAGGAAACCC




>27F10 vesca
CCTGCAGGGTTTTTCATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATTATATTTATATATCATCACAACAGCTGAA
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


>27F 10 iinumae
CCTGCAGGTTTTTCATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAAG
CAGCTCATGATTCCTTTAAACACACAAAAAAAACCCACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGAAC
GCACAAAATTCAGTAAAAAAGAAAAAAGGGATCCGCTTCAATACAATCCCATCAAACTTGCAGACCTTTGGAGACAA
ATTTCGTTGCTTAATGTAATAAGCAACAAAAATCCAGCTCAGCTGGATCAAAGCCCAGATGAAAAAGATTAAAACTT












TACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGATGGGTAGATCGGGAGAGATAAAATTACCAGAATCTGAAGT
GGGGGAAGTGAATCAGTGAAGGACTGAGTTGCTGGAGTCTTGGGAGATCTGAGCTCTAAAGCCGGCGAAGGATGCGC
GGCGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATACGCTAGTGATTTTGA
TTATGAATTCTATAAATTCTACAAAA ATTTATTTCTTAATTCTTACTCTGTTTCGGTGTTGGCCAGATTTGA
CTCTTCTGTGCTTCAGTCATGACTTTGACCATTTACTTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGT
GGTGAATTCTGGTTCGTGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCGGGT
AACAGTAACTTTATCGTTTTACCGCTAGCATGTCTCTGTCTGTCGACATATATAACGCTTCTTATTTGTTTTGGGTC
TCTACGCTTTTGGGTCTTATTTGTCAAGTTTCAATCACTTGAAACTATTGAAATAGCTGAAATACACACTTACTATA
TGCAAACACAAGGGAGAGAGGAGAACCAGATCATTCTA


>27F 10 nubicola
CCTGCGGGTTTTTATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAAGC
AGCTCATGATTCCTTTAAACACACACAAAAAAAACCCACAATCAAAATGAGAAAATGAACAATACCCAAGTCATGAA
CACACAAAATTCAGTAAAAAAGAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGACAA
ATTTCGTTGCTTAATGTAATAAGCAACAAAAAATTCAGCTCAGCTGGATCAAAGCCCAGATGAAAAAGATTAAAACT
TCAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGTAGATCGGGAGAGATAAAATTACCTGAATCTGAAG
TGGGGGAAGTGAGTCAGTGAAGGACTGAGTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCGAA
GGATGCGCGGCGCAGGATAGGAGGGAAAAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATACGCTAGT
GATTTTGATTATGAATTCTATAAATTCTACAAAAAATTTATTTCATTTCTTAATTCTTACTCTGTTTCGGTGTTGGC
CAGATTTGACTCTTCTGTGCTTCAGTTTTGACCATTTACATTTATAACCCCGGGAAGGGTTCAAGCGCGGCCTGCCA
CGTGGTGAATTCAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTAATCGTCTCCCCTCCGGTAACAG TAAC
TTTATCGTTTTACCGCTAGTATGTCTCTGTCTGTCGACATGACGCTTCTTATTTCTTTTGGGTCTCTACGCTTTTGG
GTCTTATTTGTCAAGTTTCAATCACTAGCAGGAAGACTTGCGTATAATTGAAATAGCCATTATCTATACTCTATATG
CAAACACAAGAGAGAGAAGGAGAACCAGAATCATCCA


>27F10 mandshurica
CCTGCAGGGCTTTTTATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCTGAA
GCAGCTCATGATTCCTTTAAACACACACAAAAAAACCCACAATCAAAATGAGGAAATGAACAATACCCAAGTCATGA
ACACACAAAATTCAGTAAAAAAGTAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCAGACCTTTGGAGAC
AAATTTCGTTGCTTAATGTAATAAGCAACAAAAAAATCAGCTCGGCTGGATCAAAGCCCAGATGAAAAAGATTAAAA
CTTTAAACAAGAAAATAAAGATCAGAGGAAGAAAATATGATGGGTAGATCGGGAGAGATAAAATTACCTGAATCTGA
AGTGGGGGAAGTGAGTCAGTGAAGGACTGAGTTGGTGGAGTCTTGGGAGATCTGAGATATGAGCTCTAAAGCCGGCG
AAGGATGCGCGGCGCAGGATAGGAGGGAACAGGGTGCGTAGGATAACCCAATCAATGAACCAAATGAGAATACGCTA
GTGATTTTGATTATGAATTCTATAAATTCTATAAAAATTTATTTCATTTCTTAATTCTTACTCTGTTTCGGTGTTGG
CCAGATTTGACTCTTCTGTGCTTCAGTTTTGACCATTTATTTTTATATCCTCAGGAAGGGTTCAAGCGCGGCCTGCC
ACGTGGTGAATTCAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCCCGGCAG TAAC
TTTATCGTTTTACCGCTAGTATGTCTCTGTCAGTACTCTTCTGTCGACATAACGCTTCTTATTTGTTTTGGGTCTCTACG
CTTTTGGGTCTTATTTGTCAAGTTTCAATCACTAGCAGGTAGACTTGCGTATAATTGAAATAGCCACTATCTATACT
CTATATGCAAACACAAGAGAGAGAAGGAGAACCAGAATACATTTCCA


>27F10 ananassa 2
CCTGCAGGTTTTTATCATGTAAGGACCTCCATTGTCAGTAGCTTTATGCATATCATCTTCATCACAACAGCGGAAGC
AGCTCATGGACTCCTTTAAACACACAAAAAAAACCCACGATCAAAATGAGGAAATGAACAATACCTAAGTCATGAAC
ACACAAAATTCAGTAAAAAAGAAAAAAGGGATCCGCTTCAATCCAATCCCATCAAACTTGCACCCCTTTGGAGACAA
ATTTCGTTGCTTAATGTAATAAGCAACAAATTCAGCTCAGCTGGATCAAAGCCCAGATGAAAAAGATTAAAACTT
TACCCAAGAAAATAAAGGTCAGAGGAAGAAAATATGGTGGGTAGATCGGGAGAGATAAAATTACCAGAATCTGAAGT
GGGGGAAGTGAATCAGTGAAGGACTAACA GTTGCTGGAGTCGTGGAAGATCTGAGCCCGGCGAAGGATGCGCGGCGCAG
GATCGGAGGGAAAAGGGTGCGTAGGATAACCCAACCAATGAACCAAATGAGAACACGCTAGTGATTTTGATTATGAA
TTCTATAAATTCTACAAAA ATTTATTTCTTAATTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACACTTCT
GTGCCTCAGTCATGACTTTGGCCATTTACTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAAT
TCTGGTTCGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCCTCCCGGCAACAGTA
ACTTTATCGTTTTACCGCTAGTATGTCTCTGTCTTATTTGTTTTGGGTCCCTACGCTTTTGGGTCTTATTTGTCAAG
TTTCAATCACTTGAAACATAGCAGGAAGACTTGCATATAATTGAAATAGCTGCAATACACACTTGCTATATGCAAAC
ACAACAAGAGAGAGGAGGAGAACCAGAATCATCCA













>29G10 vesca
TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGAGCTTTGGAGAGGTAGGAAGAGTTTATTTCTAGAGGGAGGCTACC
CATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAGGAAGATGGGGAGGATAAATAAAAAGGATAGAAACTGCTC
AAGTGCTTAACAATGGTTGTAGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGA
TGAAGACGCCGTATCTTAAATTTTGATTTGGTTCTTCTCACACACCAGAGATTGAGTTCGGATCATCGGATCCGAAA
AATCAAGTCCTTGTGTATAAAAGCACGTTACGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATTATTGTCA
CGGTGGACCACGTCAGTCTGGCATATCGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCATATCAGAC
CCAAGCCATTACTTGCTTCTATGAACGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATTTCTTATTC
ACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGACAGTTCATTGAATTTCAGGAATCCACAATTGGGTG
CTGCCTTCTTCT


>29GI0 nubicola
TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGGGCTTTGGAGAGGTAGGAAGAGTTGATTTCTAGAGGGAGGCTACC
CATTTGAAGTAGAGATTGACTAAAAACAACTTGAAAGAGGAAGATGGGGAGGATAGAAACTGCTCAAGTGCTTAACA
ATGGTTGTAGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGATGAAGACACCGT
ATCTTAAATTTTGATTTGGTTCTTCTACACACACCAGAGATTGAGTTCGGATCATCGGATTCGAAAAATCAAGTCCTT
GTGTATAAAAGCACGTTACGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATTATTGTCACGGTGGACCATG
TCAGTCTGGCATATCGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCATATCAGACCGAAGCCATTAC
TTGCTTCTATGAATGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATTTCTTATTCACAACGACGATT
GGCTTCTTGGTGTGTTGCGCTTTGTTAGGACAGTTCATTGAATTTCAGGAATCCACAATTGGGTGCTGCCTCT


>29G10_nilgerrensis
TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGGGCTTTGGAGAGTTAGGAAGAGTTGATTTCTAGTGGGAGGCTACC
CATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAGGAAGATGGGGAGGATAAATAAAAAGGATAGAAACTGCTC
AAGTGCTTAACAATGGTTGTGGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATTATATAGAGGTGCATGTAGGA
TGAAGACACCGTATCTTAAATTTTGATTTGGTTCTTCTCACACACCAGAGATTGAGTTCGGATAATCGGATTCGAAA
AATCAAGTCCTTGTGTGTAAAAGCACGTTGCGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATTATTGTCA
CGGTGGACCACGTCAGTCTGGCATATCTATCATCACTCTCAATCTTGTCGATCATCAATTTGGCTACATATCAGACC
GAAGCCATTACTTGCTTCTATGAATGTATTTATATCATTACTAATCACCCAGAATTATGGATAATATTTCTTATTCA
CAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGACCAGTTCATTGAATTTCAGGAATCCACAATTGGGTG
CTGCCTTCTTCT


>29G10 mandshurica
TGGCCTTGTTTCCTAAACTCTTCAGGGTCTAGAGCTTTGGAGAGGTAGGAAGAGTTTATTTCTAGAGGGAGGCTACC
CATTTGAAGTAGAGATTGGACTAAAAACAACTTGAAAGGAAGATGGGGAGGATAAATAA ATAAAA GACTGCTC
AAGTGCTTAACAATGGTTGTAGACGAGTTGTGTCTTGCTGCATATATTGAAGAGATCATATAGAGGTGCATGAAGGA
TGAAGACACCGTATCTTAAATTTTGATTTGGTTCTTCTCAGTTCTCACACACCAGAGATTGAGTTCGGATCATCGGA
TCCGAAAAATCAAGTCCTTGTGTATAAAAGCACGTTACGGAGTGATCCCACTCATCAATAAGTTATCGGACTTAATT
ATTGTCACGGTGGACCACGTCAGTCTGGCATATCGATCATCACTCCCAATCTTGTCGATCATCAATTTGGCATGCAT
ATCAGACCGAAGCCATTACTTGCTTCTATGAACGTATTTATATCATTTCTAATCACCCAGAATTATGGATAATATTT
CTTATTCACAACGACGATTGGCTTCTTGGTGTGTTGCGCTTTGTTAGGACAGTTCATTGAATTTCAGGAATCCACAA
TTGGGTGCTGCCTTCTTCT




>32L07 vesca
GAGTTGAAAAACGGGTCGAATCCCGGCACCACCGTCCGCGTCGCGTAGGACTTGAATCCTTCCAAGGTCACCTCCTT
GATGTACATAGCTGCCCTCGCCGGAGAGGTGCGGACGCTAATCGGAAGCCGATTTTGGAGAGATTTAGTGTCGGTGA
TAGATCGGAACCCTAGAAATCTGAGCTTCTGGTTTTTGCTTTCGGAAGTTGAGAGTCTGAAATGACATGGTTCGAAT
TTCTTTTTGTTGTTTTCCGCTTTTTTGGTGGGTTCGAATTTTTAGACCAAGGCGGGAGATATTTGGGCCAGTGATTT
ATATCTTGGGCTCACTCTGGGACTCATGTCTTTGGGCCTCGTCGACCTCGAGGTGCTCATGAAGTCCGGCCGTCCTC
AGGGTCGGAAACACCGCGGTACTACTGACTACTGTGTCATCGCTTTAGAATTTCATTAATTGGCTTTGCGAGCTATA
AATAATTGTGATTTGGTTTTAGTATTAGTATTTATCAACGGGAATTGCGGAGATGAGAAAA
GTTGAGGTTGATTTGGGGGAGTGTGGTGTTGTTAGTTAGTTGAATTATTAGAAACGAAAAAATAACAGAAGAATATA












AATGTGGATGGATTATTGGATTAAGATTTGATTCAACGGAAGAAGGAGGCGTGGTGTGTGTTTTGATAGTCTAATTT
GAACTGTTTTGCTTCTGACAGCTAAAATCTATCCGGTGGTGAAAAATCAGCATCGGCTACTATGTACACTTTTAATC
GGCAACGCATTAGCGATGGAGGTGACTTGTCTAATTTACTAAGTTTATTTAGGTTGTTACTGGTACATTTTATGTGT
TTATTGCCGTGGATGTAGTTTGTATGGGCCAGTTGACCAGCAGTTTCAAATGGCAGGCCAATAGGGCCAACCTAGAT
TGTAGTTGAATTTTGGGAAGGAAAAAAAAAAGCAAACCAAAAGACATCACCACGAGCCACTTTGGCCTATCTATATA
TATTACTTCCTTGCTTAATGTGTTGCTCAATTGCTAAACAATATCATCAATGTCTAAAATAACGCGCCTCAAGGCTA
AGGCAAGGGAAGGCGTGCCTTAGGACGACCTCTGAAAGACATTTGATATCAAAGGTGTGATTGAGGCGCGCGATCAA
GACGACGAGGTCAAGGTGCCTAATACAACATCAGGTTATAGGTTTGAATCTCACTTTGAGAAATGTGATGGTTTGAA
CGGTTAAATCTATTGTCTTTTTATATTGTATGGGCGGTAAAATTAAATGTTAAACTTCGGTAAATTGTCAAATGTTT
AATAGTATAAGAATCTACATATAGTAGGTGTAAAATAGATACCGAAATGATAATATTTTGTGAATAACGTACGTCAT
ATGATTTAATATTAAGACTTTGTACGATTTAACGTTACACATTAAAATTGTAGATAAAAAGTTTATATCATCATCAA
CATCGATGTTCGAATAAATTTATAACGTTCAATGCGGTACAATCTCCCAATGACTATTATCGAGTACAACGTCCA
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
CCCAACAATTCAAGGACCACACATGAGTGCACT TCACTGAATCGTCACCTTACAACCACACTACCTATCC
AACCCAGACACAGATGAAAATTCACAGCAGCTAAGAATATAGTACCAATTTTGCTCTATCTTTCTTCTTTACCAAAA
CAAAAAAGATCCTGTAGTAACTAATATAACAGCTAACAGCTTTTCCCATCCTGCCCATAACAGCTTTTCCCCTGCAG
TATGGGAAACCCTGATCTAAATCCCCCGATTTATAGTAACAAAAAAATAAATAAAATAATTTGCTTTCCTCATTTAC
CATTTTACCCTCATCTTCTCCTTCATTGCCACTTGAACCCCCACTCTCCATGCTCCTTGAACCTTCTCAACACCCTT
TCTAGGGCAATGTCAAAAGCGTCTTTTACCGTCTCCAACCCCTCCTGCGGTTTCGCGTACAGAAAATTCGGTATGTA
ATCGATAACTTTCTCCCGCATTTTCCT












>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














>34D20 viridis
NNNGAGAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGTGCAGTTCAGGAGATAAAGGAAGTA
TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC
GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT
TATGTGAATC CATTGGGCATATATAATCATTCAGAACTACAAGGAAAGATTATCGGC
GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG
ACTGCAAGTTACGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA
ATGGATTTAGTACTTCCATTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTTCATGCACAGGAGAACTGC
AGGTGAACCATATGTCTATATAGATATGTCGAATGTTAGATAGGATACATAGTATGAGGGTGTGGATGAACTATACG
TAGAGCACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGCATGAATCTCCCTCCCGGCCACAGTAGAC
CACACTTTTGAACTGGCGGATTCCATCCGGCCTAGATTTTGTGCCGACTATCACAATAGTGTAAGTTGGTCCTCCTA
GCTATAGTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGATGAATATGGAA
TGAACAAAAGCTGCTTTGTCCATCTTTTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATTCTCTTT
ATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGTATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGT
ATAGTTGTATTGATCTGATATAACTCCCATTAATGAATCTAATATAAATTTTCCTAGTTAACAGAGGATAGGTCT
CCGGCTGACCTTATCCAACAAGGAAACAGAAACATACAATTAACGCATTATACACAAGACTGGTCTATATAAGGCAT
CAAATTCTCTTTATCTGTTTCATTGATCATATTGTCTAGTACTGGAAGAGCTATATTCATCTGATAACAGAAAGTGC
TTACTTGCTGGTTCATACTCAATATGGATCCGAAGGTCCTTAGTTACAATGGTGTTGACCTGAGCATGAGCGACTTG
GATCTTCTTAGAGGCCCTTATTACTTAACCGATAGCATCATTCGATTCTATTTCACTAATCTTAGTTCCCATTATGA
TGATGATATCCTTCTGGTTTCCCCTAATATCTCTGATCTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAG
CCTTTGCGGAGTCTGACCAACTTGGTAAAAGGAAAGTTGTGATATTTGCAGTGAATGATAACGAAGATCCGAGTCGA
AGCGACGGCGGAAACCATTGGAGCTTGCTGGTGTATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGG
GGGTAACAATAGTTTGGAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCAC
CAATAACTCCAGCTGGGACTAGTAGTTTGGTTACCAACAACAGTTCTACAATGAGACACGAGTGCCACTCTACGCAG
TCGCGGCGATTTATAGACTATACCAAGACAATGCTTGGGGTTTGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTA
CTTGCGTCTGTTTGGAAATTATCATTATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAAATCACGAATGGTAATC
ATGGAGGTGATGATGATGTTAATGAACCTTGGTATAGAGAAGAGACTCTTATGCCTCAGCAGACGAATTTACGACGC
G


>34D20 mandshurica
NNNNNGAAGAACTGATGTGCTTTCCGGAGGGACTGACAGTGGAAAAGGACAGAGCAGTTCAGGGATAAAGGAAGTA
TTAATGTTAGGCATCCAAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGC
GAATTTGATTTCTACAAGGTGAGCAACATGCCTGCAAACTAGATATATTTTGTTTTTCTTACTATTACAGTGTGTGT
TATGTGAATCATCTGCAAATTATCTATATCTAACTCTATGGTATAATCATTCAGAACTACAAGGAAAGATTATCGGC
GAGAAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATG
ACTGCAAGTTGCGGGCAATAGGGGCTTCGGGTCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAA
ATGGATTTAGTACTTCCATTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGAACTGC
AGGTGAACCGTATGTCTATATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGGATGAACTATACG
TAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGTATGAATCTCCCTCCCGGCCACACTAGAC
CACACTTTTGAACTGGCGGATTCCATCCGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCT
CCTAGCCATAGTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATATGATGAATTT
GGAATGAATACAAAGACTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCAAAAGATGGGTACATGTTTGCTTATT
CTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTCATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGT
ACTTGTATAGTTGTATTGATCTGATATAACATAAATTTAATGAATCTAATAGACATTTTTCCTAGTTAACAGAGGAT
AGGCCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACACAAGACTGGTCTATATA
AGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCTCTTTATCTGTTTCATACTTTCATTGATCATATT
GTCTAGTACTGGAAGAGCTATATTTATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACTCAATATGGATCCGA
AGGTCTTTAGTTACAATGGTGTTGACTTGAGCATGAGCGACTTGGATCTTCTTAGAGTCCCTTGTTACTTAACCGAT
AGCATCATTCGATTCTATTTCACTCATCTTACTTCCCATTATGATGATGATATCCTTCTGGTTTCCCCTAATATCTC
TGATCTTCTGGTAAATTCTCCGGATCCCGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAAGGA
AAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTGGTG
TATTTCAGAAAATCAAACGCATTCGTACATTACGACAGCTTGGGGGGTAACAATAGTTTGGAAGCTAGGAAAATGTA
TACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCACCAATAACTCCAGCTGGGACTAGTAGTTTGGCTA
CCAACAACAGTTCTACAATGAGACACGAGTGCCACTCTACGCAGTCGGGGCGATTTATAGACTATACCAAGACAATG
CTTGGGGTTTGGGGTTTTGTTGTCAACTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCATCC












TTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTAATCATGGAGATGATGATGATGTTAATGAACCTTGGT
ATAGAGAAGAGACTCTTATGCCTCAGCAGACGAATTTTACGAGTGCG


>34D20 nubicola
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNATGTTGGCATCCAGACGGCATCTGGTTTTGGAGTCCCTCTCCAAGAAATGGAGCAAGTCCTACTTCCTACGCGA
ATTTGATTTCTACAAGGTTAGCAACAGCCTGCAAACTAGATATATTTTGTTTTTCTAACTATTACAGTGTGTATTAT
GTGAATCATCTGCATATTATCTATATCTAACTCTATGGTATAATGATTCAGAACTACAAGGAAAGATTATCGGCGAG
AAGGTGTTTTGCATGCAAGCAGCAGAAAATGCTATGGGCCAATTTCCCTTGCAAACACTTGCTATGGTGTAATGACT
GCAAGTTGCGGGCAATAGGGGCTTCAGGCCTTTTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATG
GATTTAGTACTTCCATTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGTTTTCCATGCACAGGAGAACTGCAGG
TGAACTTTATGTCTATATATGTCGTATGTTAGATAGGATACATAGTATGTGGGTGTGGATGAACTATACGTAG
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
GGTGAACCATATGTCTATATAGATATGTCGTATGTTAGATAGGATACATAGTATGTG GTGATGAACTATACGT
AGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGTTGGGTATGAGTCTCCCTCCCGGCCACACTAGACC
ACACTTTTGAACTGGCGGATTCCATCCGGCCTAGATTTTGTGCCGACTATCACAATAGTGTAAGTTGGTCCTCCTAG
CTATAGTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGATGAATATGGAAC
AAAGCTGCTTTGTCCATCTATTAGCATTTTTGAAACCCAAAAGATGGGTACATGTTTGCTTATTCTCTTTATCTAG
TGCATCATGTGAGTTATCAAGTTCATGTCTATGCATTCTGCTGATTTAGGAATAAGGATTGCAGTACTTGTATAGTT
GTATTGATCTGATATAACATAAATTTAATGAATCTAATATAAATTTTTCCTAGTTAAGCCAAAAGACCTTATCCTAC
AAGGAAACAGAAACGTACAATTAACGCATTATACACAAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTT
TCATTGATCATATTGTCTAGTACTGGAAGAGTTATATTTATCAGATAACAGAAAGTGCTTACTTGCTGGTTCATACT
CAATATGGATCCGAAGGTCCTTAGTTACAATGGTGTTGACCTGAGCATGAGCGACTTGGATCTTCTTAGAGGCCCTT
GTTACTTAACCGATAGCATCATTCACTTATCTTACTTCCCATTACGATGATGATATCCTTCTGGTTTCCCCTAATAT
CTCTGATCTTCTGGTAAATTCTCTGGATCCTGAGGATGAGCTTAGAGCCTTTGCGGAGTCTGACCAACTTGGTAAAA
GGAAAGTTGTGATCTTCGCAGTGAATGATAACAAAGATCCGAGTCGAAGCGACGGCGGAAACCATTGGAGCTTGCTG
GTGTATTTCAGAAAATCAAACGCATTCGTACACTACGACAGCTTGGGGGGTAACAATAGTTTGGATGCTAGGAAAAT
GTATACAGTATTCAAGAAACTTGTGGCAGCTCCAGCAACACAAGCACCAACTCCATCTGGGACTAGTAGTTTGGTTA
CCAACAACAGTTCTACAATGCGACACGAGTGCTACTCTACGCGGTCGCGGCGATTTATAGACTATACCAAGACAATG
CTTGGGGTTTGGGGTTTTGTTGTCAATTACATTTTGTCAAAGTACTTGCGTCTGTTTGGAAATTATCATTATCCTTC












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












ATGTTGTTAGTTGGGACTAAAAATCTTTCAATTGATCTTATGGCAGGTGGGCAAAAATTGTAAGCATTGCCCCCCAA
TTTCGAAGAGATGCCTTGAACTAAAAACCAGGAGTAGATGCAAAACAAAGACAAGAGCAACAAATTGGTAATAAACT
AGAGTGCATCTGAAACGTGCCCTATGCCAGCCAGCTTTGTCCATCGTAAGGACGAGAGCGACAATAACTATGTGACC
ACATGTGATGATGTGAACGGTACTCTTGTCATTTCGACGATCTTTCTTCTGAGATTTTTTCTATTCCTGGGTCACAT
ATGCGAAAGAAAACAGAGACAAAAGCATAGAAAAGAATTTGATATGATTGATTCCATAAGTGAAAGATGTATGTACA
TGTTAGCCGACACTACAAATGCTGCTAGAAAGAAATATACTACCTCCTAATACAATTTCTGTACCCCCATTTCTACA
CAGAATGTTGTAGCAAACTAGCAATGCCACCTTTCTGGTTTAAATTTCAAACAACAGAAACATTCTCCTTCATGTGT
TTGCATGTTTCCGAAAAATGTACAGAGCATCATTCAAATGGGATCTTTCTGCAATTGAAGCACCAGTATAGAACCAA
AGAGTCAGCAGTGTGTTTGTCGGAAGAGAACTATCGAGCCATGACTGTTTTCGCATCACAGGTTGCAGCTCAGATAT
TACAGCGTACAAGCTTTGGCGACACCACCATTTGGATTCCTGGGTTTCATTTGGTAGCCATGCATGAGAAGAAATGA
CAACAGAGGAGAAAAGAAAACTACGCGCATTCATAACTAAGTTCTATGCTTTTGTCTTTTATTTCTTGTAAGAGAAG
TTTTACCTCTGACGAATGTAAGCTGGTGGATTAAGGGTATGATTTTTCCACCATCTACCCAAAAGCATCCAGCTGCT
TGCAAGAAATCAAAGGAACAACATCAGTCAAGATGATTGTCTTCTTGCACAAGCCGAAAGCCCCAGCCAGCCCTCCT
CCCCCTTATTGCCAAACTTCACAACTAACCAAAAGCTCATCTAGCACCAAGTTTGGCTCTTCTAACTCAAGGAGTCC
GGCATCTGTAGTCACCACAGGAAACCAGCACTCTAAGCTCTGTGGCAGAAACATTACACGGTAGGAAAACAAAGAAT
GCCACCGAGCTCCTATGATGCAGAAAGAGGATTCATAACACCCATCAATCACTAGTTAGATCTCTCTCTTTCCAACA
ACCCCAGCAGGACTATGCTTCCTTTGTATACCATATTGCCAAGCATCTCTAGACTTATCACCCGACTGATCAAAGTC
GTCCAAAGAATTCTCAAACTGTGGTTTCATAGGCCG


>40M1 1 mandshurica
CAACATTTTGGTGGCCTTCTTGACATTCCAGTTTCTGGCCCTCAGATGCCTTGCAATGGATGCATCAGAACAGTATG
TGGACAGCTTCTCGGGTACTGCCTTTAACAATTTTCTCACCTCATTAATCTGCAAACAATAAGATTTTTTAGGCAAA
GCGGAACTATGAGTTCCCCAAACTAATAGCTTTCAAACAAGTAGAGGAGCACATTTACTAAAGATACCTTTGCCTGC
TGCTCTTCACTTGTTAAAATACTCTCAGAGCCATTTGAGGAAGATTTTTTTATTCCCGCACTCATAGTTTTGAGGGG
AAACTCTGCAAATCAACAATGGAGATTTCAAAACTTATGTCCTAGTTTCACAGTTCCCTTCGGTCTCCCATCACCAT
CAAATACAATAAATTTCAATATATTTAACAAAAAAATTGCTCTTCATCCCACAAAACACAGAGTCCTCATCTTCATT
GTTCAATATATCATTTGAAATTAACAACTTTTATTCTTCTAGTCAACCACATTTTGCAGCTACTTGTTTAACTCATA
AACCCTTTCTTCCGATCCATAGCTATCAAATATCCAATCTAAACGAGACTACTACTTTGTTCACAACGAATCCAACA
CAAAAGGATCAAAAAAACCATCCAAAACTCATGCACAACATAATCAACCAAATATTTTAACCACAAAAACAAGCACA
ATTCTCCAAAGTACAAAAAGAAATGGGCTTTAGACACCAGGAAGGCATATCAAACCGGCCCACACACGTTAAAGGGA
TACAAAGATCTCACCTGGACCAAAGACAGAACTGGGTGGTTGCTGACTGAGCAAAGCCAATATCTCGGAGCTCCTCA
GATGTCGGAGAGACCCATCTGAACCCAAGTCAACTGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN




>63F17 vesca
CGCTCTATGGAAGGGACAAGAGACACTGAAATAGCAATGGGGTCCTACCAACCTCATCATACATGGGCAAGAAATCA
TTCTAGTCCTCTCGGACAGGTAATCACAGAATCCAGATTAGATGCAGGTTTTGAATTATTAGAGTCTATAAAGGGAC
ATAGTTACAACTGTTTGTATGCTTTTCCATTTTTTTTATTTTTTTATTTTTTGAGAATGTATGCTTTTTCACTTGTA
TGGCCTGAAGTTGCGAATGTTTTGGTTGATAGATATTTGGATATAGAATGTCACTATGGGCAGAGCACACAGGAACC
GTTGAGGACTGTTTTAGAGAACCAGAGAGTCTTGAATGTGTTAGGAGAGTTAGAGCAATGGGTGAGATGAACTGGAA
ACAATTTGCTGCTGAGGAGGTTACAGAGATGAGGGGTCATCTATTGAAGTATCCAGTTGAAATTGATCGAAAAGGCA
AAGTCACATCCCTTCCTGGATGTGAGAGTTTCCCGATGCAGGAGGAAATATAACCGGTTCTTTCCTTGGCATTCAA
GAAAATTTGACAATTTGATCACCAGTTCAGTTTTATAGAAGAACTCAGTTAGTACAGTTTTGAAACGTTTTTTTGTT
GTATTTAGCAAACCCATAGGAGGATAGGGTTTTCTTTTATTCAACAGGGATATAGGCGCTTTTAGGGTTTCTTTTCC
TATTCAATTTCGTTCTTTGGTAGACCAAGTCGCTTCTTTGGCATTCAAGGAAACCTGAGCATTTGATCTGCCTGTCA
TCACATCCAGAGTTGCAGATTGTTTAGAGAAGAATTCCAATAAATTCCTTTTGTACAGTTTGGTTAACTTTTGGTAT
TCAACAACGCATTGTACAACTCTGCCAATTTGGCACATTATAATGTTGATATGCAGGTAACATCTCTGACTATGCAT
CTTTGCTTTTTCTTCTTTTTTTGAGAACAAGGCATCTTGTTTATGTGTAGCCAACTTGAAGCACTGTATTTAAATAA
TGCTAAAACAGTGTTAATTTTGTTATAAAGTGTAGGCAACAATGAACTTGAACTTGAACTTGAACTTGATAAGAAA
ATAGATCCCAGAGATGGTCTATCTACTACCCTTGACTACACAAGTTACTCATTCTTTACATGTGAAATGGCTATCCA
GAGCAGTCGTATTTCATGAGATATTAACAAGCTTTGGACGTCCAGTTGCCAGGTTCATCTCTACTCGGAGGCCAAGT
CGAGCAAGGGCAGGCACATCAACAGACCCCTTAA


>63F17 viridis












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















>72E18 vesca
GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAATCCGGCCTAGCATTATATCAAATCAGTCCTTGAGAT
TCGACATGCATAAAAAAGACAATAAAGGGTACAAAAACAACCACTCAAACAATCACAACATAATATCATTCAATACC
TTGACCATTCCGGTTCCATTATCACACAAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAACA
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
CTTGACCATTCCGGTTCCATTATCACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAAC
ATACACCACAATCAATGACAACAATGCCTCATTCACACAACAAAGAAATAGACATTCAAAAACAAAACACAATACAC
ACTACTAATGTGGCACAGAAACCAAAGCATGATTCAAAACAAAACTAGAACATCTACATAGTTCTCTCACAATAGTA
AAGAAACGATCTTTGACAATCAAAAGGCATCGAAAGCTAGTAAAGAAACGATCTTTCAGATGGGAAATACCCAAATT
TGATTGCTACATGCATAAAACCCTCAAATTGATACGAAATCAAACAATGCAGCAATCAAATCATTCCACATAAAAAA
AAAATT CAAGAAATAAAGAGAGAGAAAATTACAGATCTAAAGTGACGAACAATGAGAAGGAATGAGAGGCAGAGAGA
AGAGATGAGGAAGTTGACCTTTGTGAATGAGAGTGAGTGAGGGAGAGAGAGAGAGATCGAAGACGAAGCAGAGCGAA
AGAGACGAGTGTGGTGTTTGTGAGTTGAGGCGAAGAATTGNACCNNNATANAGGAGTGNGATTGACNAGTTATCTCN
GCNGNTTGATTTATGGACCGCGTCCATTGTGCCNTTGTGGGGCCATNACNGCTCCTNCCNCTGTNCCNGCCATNTTT
ATCTCCACCTTCTNCACNTTTNTGCCNCNCNTGNCCNTCCCTTTTCTCCCNNTTCTNNCTCANATCTTCTNAANGCT
NATNGCNTTTNCCNNANNGCATTTGNATTTNNGNGCTTCNATCAATNCGNNNNNAANGCTNCGTNTTGTCNTNNCAN
GGNTNCAAGGANCCTNNGGGANGCNNGTTGATCGTCAANTNCGGCCTGGTNATANANAATATNNGACNTNAAATGGT
TGATTGNNTCGTTAAATTNGAAATAAnnnnnnnnnnnnnnnnnnnnnnAAAAGGNCNTAATTTANTNNGTANNTCAA












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












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












ATGATGAGGATCTACTCAAAATCCAGACTACCATGGTTGGCAAAATTAGATCCTCACTGTAACCAGCTAGGCATTGG
TAATGCATAATAGCTATAGCTAACTATAGGTGGGAGACTCATCATTGAGATCATAGAAAAACAGAGATGAAAGAAAG
AAATGATGAAACAAGCAACAGCTATTCGAAAGCAAGTACAGAAGGGATTGTTCATGAAGTGTTCACCAAGTCACAGC
TTAGGGCATTCTTAGAAGCAACAAGCTTACCAACTTCCATTTACTTGTTTCAAGTTCATGATGATATTAACCATCCA
ACGAAAACATCCAAAGGTACTGTGACAGAAAGCTCAAGGGGATATCTGTGTTTAAAGCCACAACATGACTAAATATA
ATTGCTTCCAATTTCTAAAGTTACATTCGTTTTGTGCAAATGACAAAACAGTTCAAATTGACTGCATAAATAGATAC
TCTTGTATAGATCAACAAGCAAATCTCCAAGTTCTTATTACAAAGTCTAAGCAGAATACTAACATCAATATTGAAAT
TGGATAAATATGCGATCTGAACTTCTTCACGTTGATGACCTATCGTAGGAAATGGAATTGAACACTTGACACCAAAG
AGAACAATGAAGGTAGCCTCGCCAATCACTTCTACAAGAATGGGGGTAGAATCACCCATCGACGTGGATACTTGGGT
CTTCCGTCCTTCCCATCAAATAGCTGGACATGGCAGGGTGTCACAAAAGATCAATATTGCATGTAAAGAGCTTCTAC
ATACAAACTCAAATGGATATGTTCTGGCGCTTGTAGAATATAATTATGTATACAAATATGCATGTACAGAGCTTCCA
CATACAAACTCATATGTATACTTGTAAATTTATGCAATTTAATTCCAATAAAGGTGAGTTTAAATAGACCAAGATGT
TAGCTAAAAAAAAACAGACAAAACATTTAAGCAAAAGAAGAGCAGTAGAAGGTATTAAGATACCAAACAACATATTT
GGGTTGGAGGACAAAGTAGTATAGAGGAGTGTTCCTTCTTTAAACGGCGGTGTTTTCCTAGGGCCCAATTGGTCATG
ATAGAAGCAGCAACTACAGCAAAAAGATAACCAGCAACCGTCTGTGTTGCAATATTAAAAGCAAAAAGATAACCCAACCACTGATAAAT
CTCAGTCGTGTAATTTGCACATGTCACAATATTGAATAGAAAACCCATGTCAACGTGGTATTTGATAGCCTCCACTTCCATCAA
GACTTCTCAGATTCCTCAGTAGAATATGGCAATAGAAGTTCGCAATTTGATTTATTATCCCAAAACCAAACCCA


>72E18 ananassa
GCTAGGGAAAACAGCTCGTGGAGCATCATCTCCAGCAAACCCGGCCTAAACATTAACATCAAAATCAGTCCTTGAGA
TTCAACATGCATAAAAAAGACAATAACGGGTACAAAAACAACCACTCAAACAATCACAACATAATATCATTCAATAC
CTTGACCATTCCGGTTCCATTATCACACGAGCGGCTGAATGTCCTCGGTTTCTGCCATCTTCTTCTACCTGCAAC
ATACATCACAATCAATGACAACAATGCCTCAAACAAATAGACATTCAAAAACAAAACACAATACACAATGCTAACAT
TTCCCTAATATCAACTAACTCCATGCCAGATACCCACAGAAACCAAAGCATGATTCAAAACAAAC
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











APPENDIX E
GENE-PAIR HAPLOTYPE INDIVIDUAL LOCI ALIGNMENTS


To avoid identification of untrue polymorphisms and therefore poor alignments, "N"s 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 clone CAATGCCATGGTCTCCGGTCTATTTCAACT3GGGAATTCTTATGAGTGGGGGTTGACAAA 60
GPH5 ananassa clone7 CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAGTTCTTATGAGTGGTGGTGACAAA 60
GPH5 viridis CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGGGTTGACAAA 60
GPH5 iinumae CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60
GPH5 nilgerrensis CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTGACAAA 60
GPH5 mandshurica CAATGCCATGGTCTCCGGTCTATTTCAACTGGGAAGTTCTTATGAGTGGGTGGTTGACAAA 60


:AAGAAGAAGAAGI-AA
:AAGAAGAAGAAGIAA
:AAGAAGAAGAAGIAA
:AAGAAGAAGAAGI-A,
:AAGAAGAAGAAGI-A,
:AAGAAGAAGAAGI-A,
:AAGAAGAAGAAGI-A,
IAAGAAGAAGAAGIkAA


iridi
















































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
















iinumae CAATCTTTATAACAAAATTTCAGTTATCTTTTCACTGCTGTATGAACTGTCACC7
nilgerrensis AAATCTTTATAACAAAATTTCAGTTATCCTTCCATTGCTGTGTGAACTGTTACC
mandshurica CAATCTTTATAACAAAATTTCAGTGATCTTTCCATTGCTGTATGAACTGTTACC7
nubicola CAATCTTTATAACAAAATTTCAGTGATCTTTCCATTGCTGTATGAACTGTTACC'
vesca CAATCTTTATAACAAAATTTCAGTTATCTTTCCATTGCTGTATGAACTGTTACC7




ananassa clone2 CTCTCACACAAGAACAACTACACCAAACAAACAGAACCAGACCAATCACACCAA
ananassa clone7 CTCACACACAAGAGCAACAACACCAAACAACAGAACCAGACCAAATCACACCAA
viridis CTCTCACACAAAACAGCAACACCAACAACAGACCAGACCATCACACCAA
ignumae CTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACC( A
nilgerrensis CTCTCACACAAGAACAACAACACCAACAAGCAGACCAGACCAAAT(CACACCA
mandshurica CTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACCA
nubicola CTCTCACACAAGACCAACAACACCACACAAGCAGA CCAGACCAAATCACACCA
vesca CTCTCACACAAGAACAACAACACCAAACAAACAGAACCAGACCAAATCACACCA




ananassa cl AACAGAATTGGATTTTCATGAAAGGCGCAAGGCACATCATGAAGGAGA--AG
ananassa clone7 AACAGAATTATTTCATGTAAAGCACAAGGCACAATCAATGAAGGAGA--AC
viridis AACATAATTGGATTTTCATGAAAlGCAGCGGACATGATCATGAAGGAGA--A
inumae AACAGAATTATTCCATGCAAAGCACAA-GGCACAATCAATGAAGGAGA--A
nilgerrensis AACAGAATTGGATTTTCAT GAAAGGCAGCAAGGCACAATCAATGAAGGAGAGAAl
mandshurica A-CAGAATTGGTTTTTCAT GAAAGGCAGCAAGGCACAATCAATGAAGGAGA--AG
nubicola A-CAGAACTGGATTTTCATG"AAAGGCAGCAAGGCACAATCAATGAAGGAGA--A
vesca AACAGAATTGGATTTTCAT GAAAGGCAGCCAAGGCACAATCAATGAAGGAGA--A




ananassa clone2 GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTC
ananassa clone7 GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTC
viridis GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTC
iinumae GAAACCTTTTGTCATAGGGATTGAACCTGAATTATCTGGAGTGTTTCTGGCTGTC
nilgerrensis GAATCCTTTTGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTC
mandshurica GAATCCTTTCGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTC
nubicola GAATCCTTTCGTCATATGGATTGAATCTGAATTATTTGGAGTGTTTCTGGCTGTC
vesca GAATCCTTTTGTCATATGGATTGAATCTGAATTAGTTGGAGTGTTTCTGGCTGTC




ananassa clone2 TCATATGCAGGCATGTTACATGTCTG ----------CATTTGGTGACAAAAGCTP
ananassa clone7 TCATATGCAGGCATGTTACATGTCTG ----------CATTTGGTGACAAAAGCTP
viridis TCATATGCAGGCATGTTACATGTCTG ----------CATTTGGTGACAAAAGCTP
iinumae TCATATGCAGGCATGTTACATGTCTG ----------CATTTGGTGACAAAAGCTP
nilgerrensis TCATATGCAGGCATGTTACATGTCTG ----------CATTTGGTGACAAAAGCTP
mandshurica TCATATGCAGGCATGTTACATGTCTG ----------CATTTGGTGACAAAAGCTP
nubicola TCGTATGCAGGCATGTTACATGTCTG ----------CATTTGGTGACAAAAGCTP
vesca TCATATGCAGGCATGTTACATGTCTCATGATGTCTTCATTTGGTGACAAAAGCT7




ananassa clone2 TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTA
ananassa clone7 TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTA
viridis TAA ----------GAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTG7
iinumae TAA---------- GAATTTAGACGTATTAGACCATTGGGCTTAATCATCGTCCG7
nilgerrensis TAACATGACCTAAGAATTAACACATATTGGACCATTGGGCTTAATCATAGTCTAI
mandshurica TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCTA
nubicola TAACATGACCTAAGAATTAAGACATATTGGACCATTGGGCTTAATCATAGTCT-A
vesca TAACCTGACCTAAGTATCAAGACATATTGGACAATTGGGCTTAATCATAGTCTA









155













9AC 996
9AC 996
GGC 984
CGC 976


ii ii ii


CATGAAGTCAAGGTCAAGCC
CATGAAGTCAAGGTCAAGCC


CGAAGC


TTAAG 1096


AACTTTAAGCGa
\GCTTTAAGCGq


GCTCCAAC(


CCGAA


CCCACC









156









































'GAATTC
'GAATTC
NNNNTTC
JNNNNNM
JNNNNNM
'GAATTC


TTA(


'AAGGC'


3AAAT'












nanassa clone2 GTACCTTGTCTCAATGGCTTAAGAATGCTTCATGTACTATCAGTGCCAGTGATGTGAA 1679
nanassa clone7 GTACCTTGTCTCACATGGTTA TGCTTCATGTACTATCAGTGCCAGTGATGTAA 1678
ridis GTACCTTGTCTCGATGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1666
numae GTACCTTGTCTCACATGGCTTAAAATGCTTCATATACTATCACGCCAGTATGTGAA 1658
ilgerrensis GTACCTTGTCTCACATGGCTTA TGCTTCATATACTATCAGTGCCAGTGATGTAA 1684
andshurica GTACCTTGTCTCGATGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1681
ubicola GTACCTTGTCTCGATGGCTTA TGCTTCATATACTATCAGTGCCAGTATGTGAA 1694
esca GTACCTTGTCTCGCATGGGCTTAAGAATGCTTCATATACTATCAGTGCCAGTGATGTGAA 1716




nanassa clone2 AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTAATTGAT---TTCAT 1736
nanassa clone7 AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGAT---TTCAT 1735
iridis AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1726
inumae AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGAT---TTCAT 1715
ilgerrensis AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGAT---TTCAT 1741
landshurica AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1741
ubicola AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1754
esca AGATGGTCTGTTTGGAAGCCTTGTTGCTTGTCCTTACCAGGTTTGAATTGATGATTTCAT 1776




anassa clone2 GTGTTCTAGTTTCTGTTTGGCATCTGTTATTTTCATGGCATGTGGCGTGAGCTAGTTG 1796
anassa clone7 GTGTTCTAGTTTCTGTTTGGATCTGTTATTTTCATGGCATGTGGCGTGAAGCTAGTTG 1795
iridis GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAGCTAGTTG 1786
numae GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGAGCTAGTTG 1775
ilgerrensis GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAGCTAGTTG 1801
landshurica GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTCGCGTGGAACTAGTTG 1801
ubicola GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTG 1814
esca GTGTTCTAGTTTCTGTTTGGGTATCTGTTATTTTCATGGCATGTGGCGTGGAACTAGTTG 1836




nanassa clone2 CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCAGTTTTTTACTATAAT 1856
nanassa clone7 CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCAGTTTTTTATTATAAT 1855
iridis CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAAGTCGGTTTTTTATTATAAT 1846
inumae CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1835
ilgerrensis CATATGGTATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1861
landshurica CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1861
ubicola CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTCGGTTTTTTATTATAAT 1874
esca CATATGATATTAAATCTTTTGTTTGGTTCCTCATTGTATAATTTGGTTCTTTATTATAAT 1896




nanassa clone2 CTTTCAGCAGCCAGCATCATCTATGGTTAATTCAGGGTTCAAAATTGCGCCTAATCAGCT 1916
nanassa clone7 CTTTCAGCAGCGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCGCCTAATCAGCT 1915
iridis CTTTCAGCAGCCAGCATCTTCCATGGTTAATTCAGGGTTCAAAATTGCACCTAATCAGCT 1906
inumae CTTTCAGCAGCCAGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTAATCAGCT 1895
ilgerrensis CTTTCAGCAGCGCATCTTCTATGGTTGATTCAGGGTTCAAAATTGCACCTAATCAGCT 1921
landshurica CTTTCAGCAGCGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTGATCAGCT 1921
ubicola CTTTCAGCAGCGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTGATCAGCT 1934
esca CTTTCAGCAGCGCATCTTCTATGGTTAATTCAGGGTTCAAAATTGCACCTGATCAGCT 1956




nanassa clone2 TACACCAAAGAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAG 1976
nanassa clone7 TACACCAAAGAGAATGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAG 1975
iridis TACACGAAAGAGAATGCCGGATGTGGTCAATTCAGGCGTCAATGACCCTCCACAAAAGAG 1966
inumae TACACGAAA3AGAATGCCGGATGTGGTAAAGTCAGGCGTCAATGACCCTCCACAAAAGAG 1955
ilgerrensis TACACGAAAGATTGCCGGATGTGGTAAATTCAGGCGTCAATGACCCTCCACAAAAGAG 1981
landshurica TACACGAAAGAGAATGCCGGATGTGATAAATCCAGGTGTCAATGACCCTCCACAAAAGAG 1981
ubicola TACACGAAAGAG ATGCCATGTGTGATAAATTCAGGTGTCAATCCCTCCACAAAAGAG 1994
esca TACACAAAGAGAATGCCGATGTGATAAATTCAGGTGTCAATGACCCTCCACAAAAGAG 2016








158




























CGGCAGTC


viridis TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCT(
iinumae TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCT(
nilgerrensis TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCC(
mandshurica TTTGGCTGGATTATGGAGTTGCGTTGATCACTTGTTTATTGTATTTATTCTC


viridi


iridi


iridi


iridi


























































































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















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


















































































CCTTAAGI
CCTTAAGI
CCTTAAGI
CCTTAAG?
















































886




937
936
929
928
936


987
986
979
978
984


1037
1036
1029











































'GCTTAAG
GCTTAAGG
'GCTTAAGG

GCTTAAG
'GCTTAAGJ









































































-GCTCCTCCT
-GCTCCTCCT
QGCTCCTCCT
-GCTCCTCCT
\GCTCCTCCT


CCTC
CCTC
CCTC
CCTC
CCTC















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














GPH10 ananassa clone --------------------------------------------------
GPH10_ananassa clone20 TGAGGTTTGGTTGGTTAAGGTGTTAACTGATAAATTTAAGGTCATAGGTT 350
GPH10 ananassa clonel8- - - - - - -
GPH10 ananassa clonel9 --------------------------------------------------


GPH10ananassa clone -----------------------------------TAAAAGGAGGT 291
GPH10ananassa clone20 CAAACCTCACGACATATGTAGGGTGTATGAATTATTAATAAAAGACAAAT 400
GPH10ananassa clonel8 -------------------------------------TAAAAGACAGGT 291
GPH10ananassaclonel9 ------------------------------------TAAAAGACAGGT 291


GPH10ananassa clone TTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGT 341
GPH10ananassa clone20 TTAATATCAGCCGTTAGATCATATTACGGCC-TGATCACTCGACATATGT 449
GPH10ananassa clonel8 TTAATATCGGCCGTTAGATCACATTACGGCCCTGATCACTCGACATATGT 341
GPH10_ananassa clonel9 TTAATATCAGCCGTTAGATCATATTACGGCCCTGATCACTCGACATATGT 341


GPH10ananassa clone TGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACAT----- 386
GPH10ananassa clone20 TGATATACGCCCAACTCAAATTCGATATATATTTTCGATATACGT----- 494
GPH10ananassa clonel8 TGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACATTTTTT 391
GPH10_ananassa clonel9 TGATATACGCCTAACTCAAATTCGATATATATTTTCGATATACATTTTTT 391


GPH10ananassa clone ----------------------------------------ATATTTTATT 396
GPH10ananassa clone20 ----------------------------------------ATATTTTATT 504
GPH10ananassa colonel 8 TTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTT 441
GPH10ananassa colonel 9 TTTTAAGTAACTAAATGACTATTCGATATATATTTTCGATATACATTTTT 441


GPH10ananassa clone TTTTTAAAGTAACTAAATGACTATGTACATCGTTTAACAAAAGAAACAAT 446
GPH10ananassa clone20 TTTTTAAAATAATTAAATAACTATTTACGTTGTTTAACAAAAGAAACAAT 554
GPH10ananassa clonel8 TTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAAT 491
GPH10_ananassa clonel9 TTTTTAAAGTAACTAAATGACTATTTACGTCGGTTAATAAAAGAAACAAT 491


GPH10ananassa clone TGAAGTTAAATTAAGAGCACCATAACAGCTAGAAAGAGTACGAACAA 496
GPH10ananassa clone20 TGAAGTTAAATTAAGAGCACCGTAACAGCTGAGCAAGAGTACGAGAACAA 604
GPH10ananassa clonel8 TGAAGTTAAATTAAGAGCACCATGACAG---------AGTACGAACAA 532
GPH10_ananassa clonel9 TGAAGTTAAATTAAGAGCACCATGACAG---------AGTACGAGAACAA 532


GPH10ananassa clone AAGTATGAGCTAAACAA--------------------ATAGAAAAATA 526
GPH10ananassa clone20 AAGTATGAGCTACATCATTTG------------TTCATATAGAGAAAATA 642
GPH10ananassa clonel8 AAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATG 582
GPH10_ananassa clonel9 AAGTATGAGCTACATTGTTTGCTCGTCGGTTTGTTCATATGGAGAAAATA 582


GPH10ananassa clone TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTA 576
GPH10ananassa clone20 TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTA 692
GPH10ananassa clonel8 TAGAGGCGATGTTGTAGAAATAATTGAACATTAGAAAATTAAATTACCTA 632
GPH10_ananassa clonel9 TAGAGGCGATGTTGTAGAAATAATAGAACATTAGAAAATTAAATTACCTA 632


GPH10ananassa clone AAAGCCGATGAGTAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCA 626
GPH10ananassa clone20 AAAGCCGATGAGTAAAATAATAACGAACTCGTAACCTAAAAGCGGCTTCA 742
GPH10ananassa clonel8 AAAGCCGATGAGTAAAATAATAACAAACTCGTAACCTAAAAGCGGCTTCA 682
GPH10_ananassa clonel9 AAAGCCGATGAGTAAAATAATAACAAACTCGTAACCTAAAAGCGGCTTCA 682


GPH10ananassa clone TATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 676
GPH10ananassa clone20 TATCATCCGCTTGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 792
GPH10ananassa clonel8 TATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 732
GPH10_ananassa clonel9 TATCATCCACTGGATCATATATGCGGGTGTGATTCGAAAACCAAAGTTAA 732






167







































CGAATTCC


996














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





































































TCCTTAAGC
TCCTTAAGC
TCCTTAAGC
TCCTTAAGC



vGGAATTCAC
vGGAATTCAC
vGGAATTCAC
\GGAATTCAC














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














GPH10ananassa clone AACAACCCCTTCAGAAGATC TCCTGTAAAATTGACATCTTGATG 287822
GPH10ananassa clone20 AACAACCCCTTCAGAAGATCAAACTCCTGTAAAATCTACAATCTTGATG 2963
GPH10_ananassaclonel8 AACAACCCCTTCAGAAGATCAAACTCCTGTAAAATCTTTGATG 2963
GPH10ananassaclonel9 CAGCCCTTCAGAGATC CTCCTG TCTACTCTTGATG 2961


GPH10ananassa clone ATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGAT 2928
GPH10ananassaclone20 ATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGAT 3072
GPH10ananassa clonel8 ATTTGGTTACTGCTGAGCTGTTAAAACTTT-ACTCAGAGATCCCAAGGAT 3012
GPH10_ananassa clonel9 ATTTGGTTACTGCTGAGCTGTTAAAACTTTTACTCAGAGATCCCAAGGAT 3011


GPH10ananassa clone ATGGTTGCCAGGCACAAAAGCTATGATTCATCTTCTCAAGCATCTGATCC 2978
GPH10ananassa clone20 ATGGTTGCCAGGCACAAAGCTATGATTCATCTTCTCAAGCATCTGATCC 3122
GPH10ananassa clonel8 ATGGTTGCCAGGCACAAAAGCTATGATCCATCTTCTCAAGCATCTGATCC 3062
GPH10_ananassa clonel9 ATGGTTGCCAGGCACAAAAGCTATGATCCATCTTCTCAAGCATCTGATCC 3061


GPH10ananassa clone TGGATGTGAAGGCTTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3028
GPH10ananassa clone20 TGGATGTGAAGGCTTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3172
GPH10ananassa clonel8 TGGATGTGATGGCTTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3112
GPH10ananassa clonel9 TGGATGTGATGGCTTACTTCAGAAATAATAGTTCGAGAGTATCCTTTCA 3111


GPH10ananassa clone TTTCTCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3078
GPH10ananassa clone20 TTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3222
GPH10ananassa clonel8 TTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3162
GPH10_ananassa clonel9 TTTATCAGTTGATCGTTTTATTTTCTTTTATACTATGCATAATCAATTCT 3161


GPH10ananassa clone ACTTTAATGCTATGTAAACTTTGCCCCTTGTTAGTGTTACACTTTTCCTT 3128
GPH10ananassa clone20 ACTTTAATGCTATGTAAACTTTGCCCCTTGTTACTGTTACACTTTTCCTT 3272
GPH10ananassa clonel8 ACTTTAATGCTATGTAAACTTTGCCCCCTGTTACTGTTACACT--TCCTT 3210
GPH10_ananassa clonel9 ACTTTAATGCTATGTAAACTTTGCCCCCTGTTACTGTTACACT--TCCTT 3209


GPH10ananassa clone CACTAGCACAAAGATATGAATTACAGATACTTTTCCGGATGGAGATTTTA 3178
GPH10ananassaclone20 CACTAGCACAAAGATATGAATAACAGATACTTTTCCGATGGAGATTTTA 3322
GPH10ananassa clonel8 CACTAGCACAAAGATATGAATTACAGATACTTTTCCGGATGGAGATTTTA 3260
GPH10_ananassa clonel9 CACTAGCACAAAGATATGAATTACAGATACTTTTCCGGATGGAGATTTTA 3259


GPH10ananassa clone CAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGT 3228
GPH10ananassa clone20 CAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGT 3372
GPH10ananassa clonel8 CAATCAGAAGTTGGAGCAAGTATCAAAGATGCTGTGAAACAGAAGTTTGT 3310
GPH10_ananassa clonel9 CAATCAGAAGTTGGGGCAAGTATCAAAGATGCTGTGAAACAGAGTTTGT 3309


GPH10ananassa clone GAAACATATTTGCACGCTTTTGGAGACCATTCG-TGCTCGGTGTCATCTG 3277
GPH10ananassa clone20 GAAACATATTTGCACGCTTTTGGAGACCATTCG-TGCTCGGTGTCATCTG 3421
GPH10ananassa clonel8 GAAACATATTTGCACGCTTTTGGAGACCATTCG-TGCTCAGTGTCATCTG 3359
GPH10_ananassa clonel9 GAAACATATTTGCACGCTTTTGGAGACCATTCGGTGCTCAGTGTCATCTG 3359


GPH10ananassa clone GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3327
GPH10ananassa clone20 GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3471
GPH10ananassa clonel8 GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3409
GPH10_ananassa clonel9 GAGGGAGGCTTCTTTGGTGACTGGACCCTAGAAAATTATGCTGGAAAGAT 3409


GPH10ananassa clone TATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAAC 3377
GPH10ananassa clone20 TATAAAAAGCAGGTAGATGAGTCACATGTATAAATCTAATTACCCATAAC 3521
GPH10ananassa clonel8 TATAAAAAAGCAGGTAGATAGTCACATGTATAAATCTAATTACCCATAAC 3459
GPH10ananassa clonel9 TATA AGGTAGATAGTCACATTATAAATCTAATTACCCAT 3459






172




























































OCTTAAGG
OCTTAAGG
OCTTAAGG
OCTTAAGG
































































Polymorphic fragments of GPH1O: 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 AACGGAGAAGACTGTCGACATTTCTAGAGAAAGCTTTCAGCTTTGAA 50
10PPR1AB22 nilgerrensis AACGGAGAAGAAGACTGTCGACATATCTAGAGAAAGCTTTCAGCTTTGAA 50
10PPR1AB22 ananassa clonel8 AACGGAGAAGACTGTCGACATTTTTAGAGAAAGCTTTTTTCTTTGAA 50
10PPR1AB22 ananassa clone20 AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCGGCTTTGAA 50
10PPR1AB22 ananassa clonel9 AACGGAGAAGACTGTCGACATTTTTAGAGAAAGCTGCTTTCTTTGAA 50
10PPR1AB22 ananassa clone2 AACGGAGAAGAAGACTGTCGACATTTTTGGAGAAAGCTTTCATCTTTGAA 50
10PPR1AB22 iinumae AACGGAGAAGAAGACTGTCACATTTTTAGAGAAAGCTTTCAGCTTTGAA 50





174















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

















andshurica AAAAAAAATCGATATACAGTATATT--TTTTTTTGAATTAATTAAATGA 22(
esca AAAAAA--TCGATATACAGTATATT---TTTTTTGAATTAATTAAATGA 22
iridis AAATAAA-TCGATATACAGTATATT---TTTTTGAAGTAATTAAATGA 22'
ilgerrensis AAAT----TCTATATACA------------TTTTCGAAAGAATT-TTTG 19;
nanassa clonel8 ATATATTTTCGATATACA-TTT----TTTTTTTAAAGTAACTAAATGA 26


tandsh
esca
iridi
ilger


landsh
esca
iridi
ilger
nanas
nanas
nanas
nanas
inuma




ubico
landsh
esca
iridi
ilger


iridi
ilger

















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



























iridi
ilger


11D02














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














11D02 viridis AACAGTCACTAGAATCGAAATTGTTTGTACAAGTTTTGCTGGGCAGACATAGACCCCA 1061
11D02 nubicola AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACATAGCACCCCA 1022
D02 vesca AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACATAGCACCCCA 1023
11D02iinumae AACAGTCACTAGAATCGAAATTGTTTGTCACAAGTTTTGCTGGGCAGACGTAGCACCCCC 1020


11D02 viridis TAAATCATATCAGATGGGGTTAATGCTACCCAGGTGTACATATTTGTACAGTTAAACCT 1121
11D02 nubicola TAAATCATATCAGGTGGGGTTAATGCTACCCAGGTGTGACATATTTGTACAGTTAAACCT 1082
11D02 vesca TATATTAT ATCAGATGGGGTTAATGCTACCCAGGTGTACATATTTGTACAGTTAAACCT 1083
11D02 iinumae TAAATCATATCAGATGGGGTTAATACTACCCAGGTGTGACATATTTGTACAGTTAAACCT 1080


11D02 viridis AATTTTGTCTAAAGAATGCTAAAATCGAA--CTCCCAAGCAACCAAATCTTCTGTTCCCC 1179
11D02 nubicola AATTTTGTCTAAAGAATGCTAAAATCGAACACTCCCAAGCAACCGAATCTTCTGTTCCCC 1142
11D02 vesca AATTTTGTCTAAAGAATGCTAAAATCGAA--CTCCCAAGCAACCGAATCTTCTGTTCCCC 1141
11D02 iinumae AATTTTGTCTAAAGAATGCTAAAATCGAA--CTCCCAAGCAACCGAATCTTCTGTTCCTC 1138
***************************** ************* *************C

11D02 viridis TGCTTTAGTATGTTGTGATTATGCCTCTGCTTCCCCAGCAGCATGAATCCGCTCGTCTGG 1239
11D02 nubicola TGCTTTAGTATGTTGTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGG 1202
11D02 vesca TGCTTTAGTATGTTGTGGTTATGCCTCAGCTTCCCCAGCAGCATGAATCCGCTCGTCTGG 1201
11D02 iinumae TGCTTTAGTATGTTGTGGTTATGCCTCAGCTTCCCAGCAGCATGAATCCGCTCGTCTGG 1198


11D02 viridis AGTTACAGCATGAAGCAGTTCGTCTCT---------------------------TGTTGC 1272
11D02 nubicola AGTTACAGCATGAAGCAGCTCGTCTCT---------------------------TGTTGC 1235
1D02 vesca AGTTACAGCATGAAGCAGCTCATCTCT---------------------------TGTTGC 1234
11D02 iinumae AGTTACAGCATGAAGCAGCTCGTCTCTAGTTGCAGCATGAGGTAGCTCGTCTCTTGTTGC 1258


11D02 viridis AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCGTCTGGCATTGCAGCATG 1332
11D02 nubicola AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATG 1295
11D02 vesca AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTGAGGTAGCTCATCTGGCATTGCAGCATG 1294
11D02 iinumae AGCATGAGGTAGCTCGTCTCTTGTTGCAGTTTAGGTAGCTCGTCTGGCATTGCAAGATG 1318


11D02 viridis AAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCTAG 1392
11D02 nubicola AAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1355
11D02 vesca AAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAG 1354
11D02 iinumae AAGCTGCTCGTCTGGAGTTGCAGCATTAAGTAGTCCTTCTGGAGTTGCAGCAGGATCCAG 1378


11D02 viridis GTCCCAACACTNNNINNNNNN TqNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1452
11D02 nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1415
11D02 vesca GTCCCAACACTTACCAGGTAGGTTAGTCTCTTCTGCGTCGAGTAACCATGCGGGCACCTG 1414
11D02 iinumae GTCCCAACACTTACCAGGTAGGTTAGTCTCTTCTGCGTCGAGTAACCATGCGGGTACCTG 1438


11D02 viridis NNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1512
11D02 nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1475
11D02 vesca GTGAGAAAAGCGTAACATCTCTCTTCTCGGAATCCATAGAATGGCGCTTCTGTCCGTATC 1474
11D02 iinumae GTGGGAAAAGCGTAACATCTCTCTTCTCGGAATCCATAGAATGGCGCTTCTGTCCGTATC 1498


11D02 viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1572
1D02 nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1535
11D02vesca AGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGGTGAGACACAAACACCAAT 1534
11D02 iinumae AGTCCGGTATACTGACCTAAATCCAGCCAACTTCACAAGGGGTGAGACACAA-CACCAAT 1557


11D02 viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1632
11D02 nubicola NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1595
11D02 vesca CTCTTCAGAGTAATCATCAAGAACCTCCACCATTTGATATTGGTGCCTCACTTCATCTGG 1594
11D02 iinumae CTCTTCAGAGTAATCATCAAGAACCTCCACCATTTGATAT-GGTGCCTCACTTCATCTGG 1616






180




















17022


andsh
iridi


















andsh
iridi


-andshl
iridi
ubico
inuma




esca
andshl
iridi;


andshl
iridi;












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















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














27F10 vesca CGCTAGTGATTTTGATTATGAATTCTATAAATTCTATAAAAA-TTTATTTCATTTCTTAA 593
210 mandshurica CGCTAGTGATTTTGATTATAATTCTATAATTCTAAAAA-TTTATTTCATTTCTTAA 593
F10 nubicola CGCTAGTGATTTTGATTATAATTCTATAAATTCTACAAAAAATTTATTTCATTTCTTAA 592
F10 iinumae CGCTAGTGATTTTGATTATGAATTCTAT TTCTACAAAAA-TTTATTTCATTTCTTAA 583
2710 ananassa CGCTAGTGATTTTGATTATGAATTCTATAAATTCTACAAAAA-TTTATTTCATTTCTTAA 576
2710 viridis CNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNN 596




27F10vesca TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACTCTTCTGTGCTTCAGT------TTTG 647
27F10 mandshurica TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACTCTTCTGTGCTTCAGT------TTTG 647
27F10 nubicola TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACTCTTCTGTGCTTCAGT------TTTG 646
2710 iinumae TTCTTACTCTGTTTCGGTGTTGGCCAGATTACTCTTCTGCTTCAGTCATCTTTG 643
27F10 ananassa TTCTTACTCTGTTTCGGTGTTGGCCAGATTTGACACTTCTGTGCCTCAGTCATGACTTTG 636
27F10 viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 656




27F10 vesca ACCATTTACTTTTATAACCTCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTC- 706
F10 mandshurica ACCATTTATTTTTATATCCTCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTC- 706
F10 nubicola ACCATTTACATTTATAACCCCGGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTC- 705
F10 iinumae ACCATTTACTTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTCT 703
2710 ananassa GCCATTTACTTTTATAACCCCAGGAAGGGTTCAAGCGCGGCCTGCCACGTGGTGAATTCT 696
2710 viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 716




27F10 vesca -----------AAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 755
27F10 mandshurica -----------AAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 755
27F10 nubicola -----------AAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 754
27F10 iinumae GGTTCGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 763
27F10 ananassa GGTTCGTCCGGAAAAGAGTCTGGAAGCAAAGCCTTGACCTCGTGGAATTCGTCTCTCCCC 756
2710 viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 776


2710 vesca TCCCGGTAACAGTAACTTTATCGACAAAACGCTTCTTATTTTATTTTATTTTTTTTGGCG 815
F10 mandshurica TCCCGGTA-CAGTAACTTTATCGTTTTACCGCTAGTATGTCTCTGTCAGTACTCT--GTC 812
F10 nubicola TCCCGGTAACAGTAACTTTATCGTTTTACCGCTAGTATGTCTCTGTCT -----GTC 805
F10 iinumae TCCGGGTAACAGTAACTTTATCGTTTTACCGCTAGCATGTCTCTGTCTGTC----- GACA 818
2710 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
2710 viridis NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 896


27F10 vesca CAATCACTAGCAGGA-------AGACTTGCGTAT------------------------- 902
27F10 mandshurica CAATCACTAGCAGGT-------AGACTTGCGTAT------------------------- 899
27F10 nubicola CAATCACTAGCAGGA -------AGACTTGCGTAT------------------------- 892
27F10 iinumae CAATCACTTGAAACT ------------------------- 893
27F10 ananassa CAATCACTTGAAACATAGCAGGAAGACTTGCATAT------------------------- 885
2710 viridis NNNNNNNNNNNNNNN--------NNNNNNNNNTATGCAAAAATACACTCATATTTATGTA 948


27F10 vesca ------------------------------------------------------------
27F10 mandshurica - - - - - - -
27F 10 n ub ic ola - - - - - - - -
27F 10 i inum ae - - - - - - -
27F 10 an an a ssa - - - - - - -
2710 viridis GAAAACGAGAATTGAACCTCTAACCTCTTAC CACCTATGAAATGTATAATATATGT 1008





185



































mandshl
nubico
iinumas
ananas;
viridi;




vesca
mandshl
nubico
iinumas
ananas;
viridi:




vesca
mandshl
nubico
iinuma
ananas
viridi




vesca
mandshl
nubico
iinuma
ananas
viridi




vesca
mandshl
nubico
iinuma
ananas
viridi1















iridi


29G10












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
29G10nilgerrensis AATTTCAGATCCACTTGGGTGCTGCCTTCTTCT 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 AGTGATGCTATATCTTGGGCTCACTCTGGGACTCATGTCTTTGGGCCTCGTCGACCTCGAG 360
32L07 viridis ------------------------------------------------------------


32L07 vesca GTGCTCATGAAGTCCGGCCGTCCTCAGGGTCGAAACACCGCGGTACTACTGACTACTGT 420
3 2L 07 v i r id i s - - - - - - - -


32L07 vesca GTCATCGCTTTAGAATTTCATTAATTGGCTTTGCGAGCTATAAATAATTGTGATTTGGTT 480
3 2L 07 v i r id i s - - - - - - - -


32L07 vesca TGAATTTAGGTAAGTTTTAGTATTAGTATTTATCACGGGATTGCGGAGATGAGAAG 540
32L07 viridis ------------------------------------------------------------


32L07 vesca TTGAGGTTGATTTGGGGGAGTTTAGTTAGTTGAATTATTAGAAACGAAAAA 600
32L07 viridis ------------------------------------------------------------


32L07 vesca ATAACAGAAGAATATAAATGTGGATGGATTATTGGATTAAGATTTGATTCAACGGAAGAA 660
32L07 viridis ------------------------------------------------------------


32L07 vesca GGAGGCGTGGTGTGTGTTTTGATAGTCTAATTTGAACTGTTTTGCTTCTGACAGCTAAAA 720
32L07 viridis ------------------------------------------------------------


32L07 vesca TCTATCCGGTGGT-GA ATCAGCATCGGCTACTATGTACACTTTTATCGGCACGCAT 780
32L07 viridis ------------------------------------------------------------





188















iridi:



esca
iridi:



esca
iridi:



asc
iridi:



esca
iridi:



asc
iridi:



- a
iridi:



asc
iridi:




iridi:



esca
iridi:



esca
iridi:



esca
iridi:



esca
iridi:



asc
iridi:






asc
iridi:



asc



iri 'i


900




960




1020
224



1080
284



1139
344



1199
404



1259
464



1319
524



1379
584



1439
644



1499
704



1559
764



1619
824



1679
884



1739
944



1799














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-- - - - - -









34D20














34D20 vesca TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480
3D20 mandshurica TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480
34D20 nilgerrensis TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGACAGAAAATGGATTTAGTACTTCCA 479
3D20 iinumae TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 480
34D20 ananassa TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGACAGAAAATGGATTTAGTACTTCCA 480
34D20 viridis TTCCCTCACAAATGCGTGCTGTGTGACACAGAAGTACAGAAAATGATTTAGTACTTCCA 480
34D20 nubicola TTCCCTCACAAATGCGTGCTGGTGACACAGAAGTACAGAAAATGGATTTAGTACTTCCA 477




34D20 vesca TTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGACTGCA 540
3 D20 mandshurica TTAAGTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCACAGGAGAACTGCA 540
34D20 nilgerrensis TTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTCCATGCATAGGAGACTGCA 539
3D20 iinumae TTAACTAGTAACTGAGGAATCCAATTGCACTCTGTGTTTAC-ATGCACAGGAGAACTGCA 539
34D20 ananassa TTAACTAGTAACTGAGGAATACAATTGCACTCTGTGTTTTCCATGCACAGGAGACTGCA 540
34D20 viridis TTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGCTTTTCATGCACAGGAGAACTGCA 540
34D20 nubicola TTAACTAGTAACTGAGGAATCCAATTGCAATCTGTGTTTTCCATGCACAGAGAGCTGCA 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 GTGGATGAACTATACGTAGAACACCCAGAAAACCAAAAAAGAAAGAGGAACTGCGGGT 660
34D20mandshurica GTGGATGAACTATACGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 660
34D20 nilgerrensis GTGGATGAACTATACGTAGAACACCCAGAAAACCAAAAAAGAAAGAGGAACTGCGGGT 659
3D20 iinumae GTGGATGAACTATAAGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 659
3 D20 ananassa GTGTATGAACTATAAGTAGAACACCCAGAAAACCAGAAAAAGTAAAGAGGAACTGCGGGT 660
34D20 viridis GTGGATGAACTATACGTAGAGCACCCAAAAACCAAAAAAGTAAAGAGGAACTGCGGGT 660
34D20 nubicola GTGGATGAACTATACGTAGAACACCCAGAAAACCAGAGAAAGTAAAGAGGAACTGCAGGT 657


D20 vesca TGGGTATGAATCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 720
D20 mandshurica TGGGTATGAATCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 720
34D20 nilgerrensis TGGGTATGAGTCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 719
34D20 iinumae TGGGCATGAGTCTCCCTCCCGGCCACACTAGACCAC------------------------ 695
3 D20 ananassa TGGGCATGAGTCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 720
D20 viridis TGGGCATGAATCTCCCTCCCGGCCACAGTAGACCACACTTTTGAACTGGCGGATTCCATC 720
34D20 nubicola TGGGCATGAATCTCCCTCCCGGCCACACTAGACCACACTTTTGAACTGGCGGATTCCATC 717
e**e e*e* ***************** ********

34D20 vesca CGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCTCCTAGCCATA 780
3 D20 mandshurica CGTCCTAGATTTTGTGCCGACTATCACAATAGTGTAATTAAGTTGGTCCTCCTAGCCATA 780
34D20 nilgerrensis CGGCCTAGATTTTGTGCCGACTATCACAATAGTGTA ---AGTTGGTCCTCCTAGCTATA 775
34D20iinumae ----CTAGATTTTGTGCCGACTATCACAATAGTGAA ---AGTTGGTCCTCCTAGCTATA 747
34D20ananassa CGGCCTAGATCTTGTGCCGACTATCACAATAGTGTA--- -AGTTGGTCCTCCTAGCTATA 776
34D20 viridis CGGCCTAGATTTTGTGCCGACTATCACAATAGTGTA--- -AGTTGGTCCTCCTAGCTATA 776
34D20 nubicola CGGCGTAGATTTTGTGCCGACTATCACAATAGTGTA--- -AGTTGGTCCTCCTAGCTATA 773


3D20vesca GTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATATGA 840
D20 mandshurica GTTTCTAGTACTATTCTACTGATATCATGTATTGCCTCAGCTTTTGACAATGGAATGA 840
34D20 nilgerrensis GTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAATATGA 835
D20 iinumae GTTTCTAGTACTATTCTACTGATATCATGTTTCGTCTCAGCTTTTGACAATGGAATGA 807
34D20ananassa GTTTCTAGTACTATTCTACTGATATCATGTTTCGTCTCAGCTTTTGACAATGGAATATGA 836
34D20 viridis GTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAAATGA 836
34D20 nubicola GTTTCTAGTACTATTCTACTGATATCATGTTTTGTCTCAGCTTTTGACAATGGAA ATGA 833






192














34D20 vesca TGAATTTGGAATGAATACAAAAACTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCA 900
34D20 mandshurica TGAATTGGTGTACA GACTGCTTTGTCCATCTATTAGCATTTTCTGA CCCTA 900
34D20 nilgerrensis TGAATATGGAAC-----AAAGCTGCTTTGTCCATCTATTAGCATTTTCTGAAACCCA
34D20 iinumae TGAATATGGAATGA--ACAAAACCTGCTTTGTCCATCTATTAGCATTTCTGAACCCAA 865
34D20 ananassa TGGATA TGGAATGA--ACAAAACCTGCTTTGTCCATCTATTAGCATTTTCT GAAACCCA894
34D20 viridis TGAATATGG-ATGA--ACA GCTGCTTTGTCCATCTTTTAGCATTTTCTG- CCCA 894
34D20 nubicola TGAATATGGAATGA--ACAAAAGCT CTTTGTCCATCTGTTAGCATTTTCTGAAACCCAA 891




D20 vesca AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAGTTC 960
3 D20 mandshurica AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 960
34D20 nilgerrensis AAGATGGGTACATGTTTGCTTATTCTCTTTATCTAGTGCATCATGTGAGTTATCAAGTTC 948
3 D20 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 ATGTTTATGTATTCTGCTGATTTAGGATTAGGATTGCAGTACTAGG TATGTATAGTTGTATTGA 1014
34D20 nubicola ATGTTTATGCATTCTGCTGATTTAGGAATTAGGATTGCAGTACTTGTATAGTT--ATTGA 1009
4 4 4 4 4 4 4 4 4 4 4 1 4 4 4 4 4 4 4*4*4*4* 4 ******* ***** *****



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-CCTATTAACAGAGGATA 1043
34D20 viridis TCTGATATAACTCCCATT-AATGAATCTAATATAAATTTTTTCCTATTAACAGAGGATA 1073
34D20 nubicola TCTAATATAACATAAATTTAATGAATCTAATATAAATTTTT-CCTAGTT -------- 1060


D20 vesca GGTCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACAC 1139
D20 mandshurica GGCCTCCGGCTGACCTTATCCTACAAGGAAATAGAAACGTACAATTAACGCATTATACAC 1139
34D20 nilgerrensis ----- CCAAAAGACCTTATCCTACAAGGAAACAGAAACGTACAATTAACGCATTATACAC 1114
34D20 iinumae GGTCTCCGGCTGACGTTATCCTACAGGACAGAAACGTACAATTAACGGATT---CAC 1071
34D20 ananassa GGACTCCGGCTGACCTTATCCTACAAGGAAACAGAAACGTACAATTAACGGATT---CAC 1100
D20 viridis GGTCTCCGGCTGACCTTATCCAACAGGAAACAGAAACATACAATTAACGCATTATACAC 1133
34D20 nubicola -----TCAAAAGACCTTATCCTACAAGGAAACAGAAACGTACAATTAACGCATTATACAC 1115


34D20 vesca AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCT 1199
4D20 mandshurica AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGTTTCATTGATCATATTGTCCT 1199
D20 nilgerrensis AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT ----------------- 1154
D20 iinumae AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT ----------- 1111
4D20ananassa AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT ----------- 1140
D20 viridis AAGACTGGTCTATATAAGGCATCAAATTCTCTTTATCTGT ----------- 1173
D20 nubicola AAGACCGGTCTATATAAGACATCAAAATCTCTTTATCTGT ----------- 1155


3D20vesca CTTTATCTGTTTCATACTTTCAT--TGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1257
D20 mandshurica CTTTATCTGTTTCATACTTTCAT--TGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1257
D20 nilgerrensis ------------------TTCAT--TGATCATATTGTCTAGTACTGGAAGAGTTATATTT 1194
34D20 iinumae ------------------TTCAT--TGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1151
D20ananassa ------------------TTCAT--TGATCATATTGTCCAGTACTGGAAGAGCTATATTT 1180
34D20iridis ------------------TTCAT--TGATCATATTGTCTAGTACTGG,3GAGCTATATTC 1213
34D20 nubicola ------------------TTCATATTGATCATATTGTCTAGTACTGGAAGAGCTATATTT 1197






193








































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.












D20 vesca GAAGCTAGGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1737
3D20 mandshurica GAAGCTAGG AATGTATACAGTATTCAGAACTTGTGGCTGCTCCAGCACACl GCA 1737
D20nilgerrenss ATGCTAGAAAATGTATACAGTATTCAAGAAACTTGTGGAGCTCCAGCAACAAGCA 1663
3D20 inumae GAAGCTAGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1631
34D20 ananassa GATGCTAGGAAAATGTATACAGCATTCAAGAAACTTGTGGCAGTTCCAGCACACGCA 1660
34D20 viridis GAAGCTAGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCAACACAAGCA 1693
34D20 nubicola GAAGCTAGAAAATGTATACAGTATTCAAGAAACTTGTGGCTGCTCCAGCACACAAGCA 1677


34D20 vesca CCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGACAC 1797
34D20 mandshurica CCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGACAC 1797
34D20 nilgerrensis CCAA---CTCCATCTGGGACTAGTAGTTTGGTTACCAACAACAGTTCTACAATGCGACAC 1720
3 4D20 inumae CCAATAACTCCAGCTGGGACTAGTAGTTTGGCTACCAACAACAGTTCTACAATGAGACAC 1691
34D20 ananassa CCAA---CTCCAGCTGGGAGTAGTAGTTTGGTACCAACAACAGTTCTACAATGGGACAC 1717
3 D20 viridis CCAATAACTCCAGCTGGGACTAGTAGTTTGGTTACCAACAACAGTTCTACAATGAGACAC 1753
3 D20 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


D20 vesca TATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTA---ATCATGA 1974
D20 mandshurica TATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTA---ATCATGGA 1974
4D20 nilgerrensis TATC---CTTCGAAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTAGTAATCATGGA 1897
3 D20 inumae TATCATCCTTCGGAAGTGTGCTATCCCATGCAAAAAATCACCAATAGTA---ATCATGGA 1868
4D20ananassa CATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAGATCACCAATAGTA---ATCATGGA 1894
34D20 viridis TATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAAATCACGAATGGTA---ATCATGGA 1930
34D20 nubicola TATCATCCTTCGGAAGTGTGTTATCCCATGCAAAAAATCACCAATAGTA---ATCATGGA 1914


34D20 vesca GATGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 2018
34D20 mandshurica GATGATGA---------------TGATGTTATGACCTTGGTATAGAGAAG-AGACTCT 2018
34D20 nilgerrensis GATGATGAAGAGCGTAATGATGATGATGTTAATGAACCTTGGTATAGAGAAG-AGACCGT 1956
34D20iinumae GATGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 1912
D20ananassa GATGATGAAGAGCTTA ATAATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 1953
D20 viridis GGTGATGA---------------TGATGTTAATGAACCTTGGTATAGAGAAG-AGACTCT 1974
D20 nubicola GATGATGA---------------TGATGTTATGACCTTGGTATAGAGAAGGAGACTCT 1959


34D20 vesca TATGCCTCAGCAGACGAATTTTTACGACTGCG 2050
3 D20 mandshurica TATGCCTCAGCAGACGAATTTT-ACGAGTGCG 2049
4D20nilgerrensis TATGCCTCAGCAGACGAATTT--ACGACTNNN 1986
4D20 inumae T-TGCCTCAGCAGACGAATTT--ACGACTNNN 1941
34D20ananassa TATGCCTCGGCAGACGAATTTT-ACGACTGCG 1984
34D20 viridis TATGCCTCAGCAGACGAATTT--ACGAC-GCG 2003
D20 nubicola TATGCCTCAGCAGACGAATT---ACGAC-GCA 1987











195














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













63F17


























andshl
iridi
c anal



esca
andshl
iridi;


























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














,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















nandsh
nilger
viridi


mandshl
nilger
viridi


mandshurica TCTTAAACCTAATTGCATTTCCCTAATTGCATTTTCATTTTAGTGCTGAGATCAATTGA 967
nilgerrensis TCTTAAACCTAAT--------------TGCATTTTCATTTTANTGCTTANATCAATTGA 932
viridis TCTNAANGCTNATNGCNTTTNCCNNANNGCATTTGNATTTNNGNGCTTCNATCAATNCGN 974
iinumae NNNNNNNNNNNN ------- NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1056
ananassa ACCTTTTTGCCCCT------------CATTCCCTTTCCCTCTTCTCCGATCCCAACTTTC 932


vesca TTAAGAAGCTTCATTTTGTCAACACAAGGCAACAAGGACA--CAGGGGAGCATTTCGATC 1032
mandshurica TTAAGAAGCTTCATTTTGTCAACACAAGGCAACAAGGACA--CAAGGGAGCATGTCGATC 1025
nilgerrensis TTAAGAAGCTCCATTTTGTCAACACAAGGCAACAAGGACN--TANGGGAGCATGTCGATC 990
viridis NNNNAANGCTNCGTNTTGTCNTNNCANGGNTNCAAGGANCCTNNGGGANGCNNGTTGATC 1034
iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1116
ananassa TCAACTCTTCTTAACCCACCCAGTTGCATTTTCATTGTAGTGCTTAAATCAATATGGTTA 992


vesca ATCGTTCCAGTCATTTTCGTATATAAT-TTGGGCTTGAAATGGTTGATC-GGTCGTAAAA 1090
mandshurica ATCGTTCCAGTCATTTTCGTATATAAT-TTGGGCTTGAAATGGTTAATC-AATCGTAAAA 1083
nilgerrensis ATCGTTCCGGTCACTTTCGTATATAAT-TTGGACTTAAAATGGTTGATC-GATCGTAAAA 1048
viridis GTCAANTNCGGCCTGGTNATANANAATATNNGACNTNAAATGGTTGATTGNNTCGTTAAA 1094
iinumae NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 1176
ananassa AGAAGCTTCATTTTGTCAACACAAGGCAACAAGGACATAGGGGAGCATGTGGATGATCGT 1052


vesca TTTAAAATGACGTTTTGAATTGATATCTATAAAGA-GGACATAATTTACTTTGTATGTCA 1149
mandshurica TTTAAAATGACGTTTTGATATGATATCTATAAGA-GACATAATTTACTTTATATGTCA 1142
nilgerrensis TTTGAAATGACGTTTTGGTATGATATTTGTAAAGA-GGACATAATTTACT---------- 1097
viridis TTNGAAATAANNNNNNNNNNNNNNNNNNNNNNAAAAGGNCNTAATTTANTNNGTANNTCA 1154

















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
















nandsh
nilger
viridi


mandshl
nilger
viridi


mandshl
nilger
viridi;


mandshl
nilger
viridi;


nandsh
ailger
viridi


nandsh
ailger
viridi





















































nandshl
gilger
viridi
iinumas
ananas:



vesca
mandshl
nilger
viridi
iinumas
ananas:



vesca
mandshl
gilger
viridi
iinuma
ananas



vesca
nandshl
nilger
viridi









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BIOGRAPHICAL SKETCH

Denise Cristina Manfrim Tombolato was born to Vadir and Marlene Tombolato on March

20, 1976, in Campinas, Sao Paulo, Brazil. She received her bachelor's degree in agronomic

engineering from the Escola Superior de Agricultura "Luiz de Queiroz," at the University of Sao

Paulo in 1998. While an undergraduate student in Brazil, Denise was granted the FAPESP and

CNPq fellowships to investigate molecular markers for disease resistance in maize, working

under the supervision of Dr. Luiz Eduardo Aranha Camargo at the Plant Pathology Department.

During the last year of her undergraduate studies, she was introduced to University of Florida's

professor Dr. Richard D. Berger, who was on sabbatical studies at ESALQ. Denise was invited

to spend one semester in Dr. Berger's laboratory, carrying out investigation on plant disease

epidemiology for the completion of her degree.

Dr. David Pete Weingartner granted her a research assistantship from 1999 to 2002, when

she earned her master's degree from the Plant Pathology Department at the University of Florida.

In 2002, she started her doctorate program at the Horticultural Sciences 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 Horticultural Company in West Chicago. She is very

excited about the upcoming changes in her life!





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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

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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

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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-

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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.

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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

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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

PAGE 23

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

PAGE 24

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

PAGE 25

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

PAGE 26

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

PAGE 29

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

PAGE 30

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.

PAGE 33

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.

PAGE 34

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

PAGE 35

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

PAGE 36

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.

PAGE 38

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

PAGE 39

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

PAGE 40

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.

PAGE 41

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

PAGE 100

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

PAGE 101

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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

PAGE 120

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

PAGE 121

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

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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 TTTTTTTTTTAAGTAACTAAATGACTATTCGATATAT