Group Title: BMC Genomics
Title: Homeolog loss and expression changes in natural populations of the recently and repeatedly formed allotetraploid Tragopogon mirus (Asteraceae)
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 Material Information
Title: Homeolog loss and expression changes in natural populations of the recently and repeatedly formed allotetraploid Tragopogon mirus (Asteraceae)
Series Title: BMC Genomics
Physical Description: Book
Language: English
Creator: Koh,Jin
Soltis, Pamela
Soltis, Douglas
Publisher: BMC Genomics
Publication Date: 2010
 Notes
Abstract: BACKGROUND:Although polyploidy has long been recognized as a major force in the evolution of plants, most of what we know about the genetic consequences of polyploidy comes from the study of crops and model systems. Furthermore, although many polyploid species have formed repeatedly, patterns of genome evolution and gene expression are largely unknown for natural polyploid populations of independent origin. We therefore examined patterns of loss and expression in duplicate gene pairs (homeologs) in multiple individuals from seven natural populations of independent origin of Tragopogon mirus (Asteraceae), an allopolyploid that formed repeatedly within the last 80 years from the diploids T. dubius and T. porrifolius.RESULTS:Using cDNA-AFLPs, we found differential band patterns that could be attributable to gene silencing, novel expression, and/or maternal/paternal effects between T. mirus and its diploid parents. Subsequent cleaved amplified polymorphic sequence (CAPS) analyses of genomic DNA and cDNA revealed that 20 of the 30 genes identified through cDNA-AFLP analysis showed additivity, whereas nine of the 30 exhibited the loss of one parental homeolog in at least one individual. Homeolog loss (versus loss of a restriction site) was confirmed via sequencing. The remaining gene (ADENINE-DNA GLYCOSYLASE) showed ambiguous patterns in T. mirus because of polymorphism in the diploid parent T. dubius. Most (63.6%) of the homeolog loss events were of the T. dubius parental copy. Two genes, NUCLEAR RIBOSOMAL DNA and GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, showed differential expression of the parental homeologs, with the T. dubius copy silenced in some individuals of T. mirus.CONCLUSIONS:Genomic and cDNA CAPS analyses indicated that plants representing multiple populations of this young natural allopolyploid have experienced frequent and preferential elimination of homeologous loci. Comparable analyses of synthetic F1 hybrids showed only additivity. These results suggest that loss of homeologs and changes in gene expression are not the immediate result of hybridization, but are processes that occur following polyploidization, occurring during the early (<40) generations of the young polyploid. Both T. mirus and a second recently formed allopolyploid, T. miscellus, exhibit more homeolog losses than gene silencing events. Furthermore, both allotetraploids undergo biased loss of homeologs contributed by their shared diploid parent, T. dubius. Further studies are required to assess whether the results for the 30 genes so far examined are representative of the entire genome.
Citation/Reference: BMC Genomics 2010, 11:97
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Bibliographic ID: UF00099887
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/
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url - http://www.biomedcentral.com/1471-2164/11/97
doi - M3: 10.1186/1471-2164-11-97

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Koh et al. BMC Genomics 2010, 11:97
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BM ics
Genomics


Homeolog loss and expression changes in natural

populations of the recently and repeatedly

formed allotetraploid Tragopogon mirus

(Asteraceae)

Jin Koh*, Pamela S Soltis2 Douglas E Soltisi


Abstract
Background: Although polyploidy has long been recognized as a major force in the evolution of plants, most of
what we know about the genetic consequences of polyploidy comes from the study of crops and model systems.
Furthermore, although many polyploid species have formed repeatedly, patterns of genome evolution and gene
expression are largely unknown for natural polyploid populations of independent origin. We therefore examined
patterns of loss and expression in duplicate gene pairs (homeologs) in multiple individuals from seven natural
populations of independent origin of Tragopogon mirus (Asteraceae), an allopolyploid that formed repeatedly
within the last 80 years from the diploids T dubius and T porrifolius.
Results: Using cDNA-AFLPs, we found differential band patterns that could be attributable to gene silencing, novel
expression, and/or maternal/paternal effects between T mirus and its diploid parents. Subsequent cleaved
amplified polymorphic sequence (CAPS) analyses of genomic DNA and cDNA revealed that 20 of the 30 genes
identified through cDNA-AFLP analysis showed additivity, whereas nine of the 30 exhibited the loss of one parental
homeolog in at least one individual. Homeolog loss (versus loss of a restriction site) was confirmed via sequencing.
The remaining gene (ADENINE-DNA GLYCOSYLASE) showed ambiguous patterns in T mirus because of
polymorphism in the diploid parent T dubius. Most (63.6%) of the homeolog loss events were of the T dubius
parental copy. Two genes, NUCLEAR RIBOSOMAL DNA and GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE,
showed differential expression of the parental homeologs, with the T dubius copy silenced in some individuals of
T mirus.
Conclusions: Genomic and cDNA CAPS analyses indicated that plants representing multiple populations of this
young natural allopolyploid have experienced frequent and preferential elimination of homeologous loci.
Comparable analyses of synthetic F1 hybrids showed only additivity. These results suggest that loss of homeologs
and changes in gene expression are not the immediate result of hybridization, but are processes that occur
following polyploidization, occurring during the early (<40) generations of the young polyploid. Both T mirus and a
second recently formed allopolyploid, T miscellus, exhibit more homeolog losses than gene silencing events.
Furthermore, both allotetraploids undergo biased loss of homeologs contributed by their shared diploid parent, T
dubius. Further studies are required to assess whether the results for the 30 genes so far examined are
representative of the entire genome.


* Correspondence' jinkoh@ufledu
'Department of Biology, University of Florida, Gainesville, Florida, 32611 USA


0 BioMed Central


2010 Koh et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Common,
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.






Koh et al. BMC Genomics 2010, 11:97
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Background
Polyploidy is a particularly important evolutionary
mechanism in flowering plants [1-4]. During the past 70
years, many plant biologists have estimated the frequency
of polyploidy in the angiosperms using analysis of base
chromosome numbers [5-8], as well as measurements of
stomatal size in fossil and extant taxa [9]. Based on these
approaches, researchers estimated that from 40% to 70%
of angiosperms have experienced polyploidy in their evo-
lutionary history [5-7,9]. Recent genomic studies indicate,
however, that polyploidy is even more prevalent in
angiosperm lineages than previously suspected. Sequen-
cing of the entire nuclear genome of Arabidopsis thali-
ana indicated two or three rounds of genome-wide
duplication [10-17]. Complete genome sequences also
indicate multiple ancient polyploidy events in Populus
trichocarpa and Vitis vinifera [18-20]. Genomic data
(including analyses of ESTs) indicate ancient polyploidy
for other angiosperms [21], including the basal angios-
perm Nuphar advena, the magnoliids Persea americana,
Liriodendron tulipifera, and Saruma henryi, the basal
monocot Acorus americanus, and the basal eudicot
Eschscholzia californica [22]. It now appears that all
angiosperms may have undergone at least one round of
genome duplication (reviewed in [23,24]).
Several outcomes for duplicated genes are possible at
the genomic and transcriptional levels. First, both mem-
bers of a duplicate gene pair may retain their original
function. Second, one copy of a duplicate gene pair may
retain the original function, but the other copy may
become lost or silenced [3,13,14,23-26]. Third, duplicate
genes may partition the original gene function (subfunc-
tionalization), with one copy active, for example, in one
tissue and the other copy active in another tissue
[25,27-31]. Fourth, one copy may retain the original
function, while the other develops a new function (neo-
functionalization) [32-38].
Recent studies have revealed varied consequences of
genome evolution and gene expression following poly-
ploidy in diverse angiosperms, including Arabidopsis
[39-44] and crops such as cotton [31,45,46], wheat
[1,47-50], and Brassica [51-55]. Several investigations
have shown that following polyploidy, rapid genomic
rearrangement [48,51,56], gene loss [1,49,53], or gene
silencing via DNA methylation [39,41,43,44,49,53] may
occur. However, few analyses have explored the genetic
and genomic consequences of allopolyploidy in natural
systems. Six natural allopolyploids are known to have
formed within the past 150 years, thus affording the
opportunity to examine the nearly immediate conse-
quences of polyploidization in nature: Cardamine schul-
zii [57], Senecio cambrensis [58-63], Senecio eboracensis
[60], Spartina anglica [64-68], and Tragopogon mirus


and T. miscellus [26,51,69-72]. Several studies of these
recently formed allopolyploids show evidence of either
genomic or expression-level changes, relative to their
diploid parents. For example, Salmon et al. [65] showed
that methylation patterns differ between the hexaploid
parents (Spartina maritima and S. alterniflora), the
independently formed hybrids (Spartina x townsendii
and S. x neyrautii), and the allopolyploid S. anglica
(formed from Spartina x townsendii). In Senecio, hybri-
dization of diploid S. squalidus with tetraploid S. vul-
garis forms a sterile triploid, S. x baxteri, and
subsequent genome duplication produced the allohexa-
ploid S. cambrensis. Through microarray analysis of
floral gene expression patterns in synthetic S. cambren-
sis lines, Hegarty et al. [62,73] observed that the syn-
thetic hybrid S. x baxteri showed immediate
transcriptional changes compared to the parental
expression patterns, and that this "transcriptional shock"
was "subsequently calmed" in allohexaploid S. cambren-
sis, suggesting that hybridization and polyploidization
have distinct effects on large-scale gene expression in
this system.
One of the best systems for the study of naturally
occurring polyploids is provided by the genus Tragopo-
gon (Asteraceae). Tragopogon comprises ca. 100 to 150
species distributed throughout Europe, temperate Asia,
and North Africa [74-76]. Three diploid species (T.
dubius, T. porrifolius, and T. pratensis) were introduced
from Europe into the Palouse region of eastern
Washington and adjacent Idaho, USA, in the early
1900s [69,70]. The introduction of these three diploid
species brought them into close contact, and as a result,
two allotetraploid species (T. mirus and T. miscellus)
formed [69]. First collected in 1949 [69], these recently
formed polyploids are less than 80 years old. Morpholo-
gical, cytological, flavonoid, isozymic, and DNA evidence
confirmed the ancestries of these two allotetraploids
[77-83]. Multiple lines of evidence suggest that T. mis-
cellus has formed recurrently, possibly as many as 21
times, including reciprocal formation, and T. mirus has
formed repeatedly perhaps 13 times (but not recipro-
cally) [70,84,85]. Therefore, T. mirus and T. miscellus
afford unique opportunities for the investigation of
recent and recurrent polyploid evolution. In fact, nearly
every population of these species may have formed inde-
pendently (V. Symonds et al., unpublished data).
Tate et al. [26,86] and Buggs et al. [87] studied geno-
mic changes and expression differences of homeologs
within natural populations of Tragopogon miscellus, as
well as in synthetic F, hybrids and first-generation poly-
ploids formed from the diploid parents T. dubius and T.
pratensis. Most of the genes analyzed show additivity in
T. miscellus at both the genomic (seven out of 23) and


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cDNA levels (12 out of 17). However, loss of one paren-
tal homeolog was observed at several loci (27 out of 46
homeologs), as were several examples of gene silencing
(nine out of 34 homeologs). Both homeolog losses and
silencing patterns vary among individuals in natural
polyploid populations of independent origin [26,87].
Changes were also detected in rDNA content [71] and
expression [72] in populations of T. miscellus. Although
T. miscellus has fewer rDNA repeats of T. dubius than
of T. pratensis [71], apparently due to concerted evolu-
tion, most of the rDNA expression derives from the T.
dubius repeats [72]. The same pattern of rDNA expres-
sion has been observed in populations of T. mirus com-
pared to its parents [71,72]; T. mirus has fewer repeats
of T. dubius than of T. porrifolius [71], but most of the
rRNA is produced by the T. dubius copies [72].
Although homeolog loss events and expression changes
were observed in natural populations of T. miscellus, no
such changes were observed in comparable analyses of
F1 hybrids between the diploid parents, T. dubius and T.
pratensis [26,87], or in first-generation synthetic lines
[87].
In this study we extend our examination of gene loss
and differential expression to the polyploid T. mirus. In
nature, T. mirus has formed repeatedly, but only when
T. dubius is the paternal parent and T. porrifolius is the
maternal parent [69,82]. However, T. mirus can be pro-
duced synthetically in both directions with about equal
frequency [88]. Tragopogon mirus provides an opportu-
nity to compare expression differences at the genomic
and transcriptional levels with the results obtained for
T. miscellus [26,87]. Our main objectives were to: 1)
investigate the genomic changes and expression differ-
ences of parental homeologs in T. mirus relative to its
diploid parents, 2) determine the identity of the genes
that exhibit those changes, and 3) assess whether indivi-
duals within and among recurrently formed natural
populations of T. mirus show similar patterns of genome
evolution and gene expression.

Results
cDNA-AFLP polymorphism and identification of putatively
differentially expressed genes
We used cDNA-AFLPs [26,89,90]as a first step toward
identifying genes with putative differential expression in
the allotetraploid T. mirus, relative to its diploid parents
(T. dubius and T. porrifolius). From our initial screen
with 37 primer pairs, 1,440 fragments were produced,
and of these, 504 were monomorphic (35.0%), and 936
were polymorphic (65.0%) among the three species.
Novel cDNA-AFLP bands in the polyploid plants com-
prised 0.4% (6 fragments) of all fragments, fragments in
the polyploids of maternal origin constituted 5.0% (72
fragments) of all fragments, while fragments having a


paternal origin in the polyploids made up 3.5% (51 frag-
ments) of all fragments. From this initial screening, we
selected for further study 21 of the 37 primer sets,
which produced an average of 50 different fragments
per primer pair. From the remaining 16 primer sets, we
obtained an average of 24 different fragments, but these
were too short (below 250 bp) for further analysis. We
then conducted an analysis on an expanded sample of
the Pullman-1 population and its progenitors (10 indivi-
duals of T. mirus, 10 individuals of T. porrifolius, and 6
individuals of T. dubius) to obtain a larger set of poten-
tially informative fragments. From the 21 primer pairs,
1,056 fragments were produced, and of these, 375 were
monomorphic (35.5%), and 681 were polymorphic
(64.5%) (Table 1). Novel cDNA-AFLP fragments in the
polyploids comprised 0.6% (6 fragments) of all frag-
ments. Shared fragments with a maternal or paternal
origin in the polyploids represented 6.3% (67 fragments)
or 4.6% (49 fragments) of the total fragments,
respectively.
For the Pullman-2 and Palouse sites, we selected four
primer sets (EcoRI-AA/MseI-CTT, EcoRI-AG/Msel-
CTT, EcoRI-AG/MseI-CAT, EcoRI-TG/MseI-CTT) that
showed high variation in populations from the Pullman-
1 site. At the Pullman-2 site, 234 fragments were scored,
and of these, 116 were monomorphic (49.6%), and 118
were polymorphic (50.4%) (Table 1). Novel cDNA-AFLP
bands in the polyploids accounted for 0.4% (1 fragment)
of all fragments, and fragments of maternal or paternal
origin in the polyploids made up 7.7% (18 fragments)
and 8.1% (19 fragments) of all bands, respectively.
At the Palouse site, 251 fragments were scored, and of
these, 79 were monomorphic (31.5%), and 172 were
polymorphic (68.5%) (Table 1). Fragments with a mater-
nal or paternal origin in the polyploids made up 6.8%
(17 fragments) and 5.2% (13 fragments) of all fragments,
respectively. No novel cDNA-AFLP bands were detected
in polyploid plants from Palouse. When we compared
20 individuals of T. mirus from the Pullman-1, Pullman-
2, and Palouse populations, we observed very similar
patterns in the Pullman-1 and Pullman-2 populations.
However, individuals of T. mirus from Palouse have
more complex patterns than individuals of T. mirus
from Pullman-1 and Pullman-2. Individuals 2602-1 and
2602-3 from Palouse shared an AFLP pattern, whereas
2602-2 and 2602-4 showed a different pattern. There
are at least three genotypes among the five individuals
of T. mirus from Palouse.
With 125 variable fragments (>350 bp) identified from
cDNA-AFLP analyses, we then searched for fragment
identity based on sequence similarity using BLAST
searches and identified 33 putative genes in T. mirus
(Table 2). Further comparison with the Arabidopsis gen-
ome indicated that these genes are involved in various


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F RUCTOSE-BIPHOSPHATE ALDOLASE


Genomic CAPS
Tdu


Trin


Pullman2
Tm


El


Oakesdale
Tdu Tm
miU


Tnn


Pullman l
Tm


Tdin


Tpo


Palouse
Tm


Rosalia
Tm


Tnn


Tekoa


cDNACAPS
Tdu


Pullman 1
Tm


I-
Pullman2


Tdu Tm


Em


Oakesdale
Tdu T


Tno


Rosalia
u Tm


Tm Too


Tekoa
Tdu


Arizona
Tdu T


Figure 1 Genomic and cDNA CAPS analyses for a putative homolog of B-fructosidase, an example of an additive pattern, from
multiple individuals from several populations of independent origin of T. mirus and the parental diploids T. dubious and T. porrifolius.
Tdu T dubius, Tm T mirus, Tpo T porrifolius.


Page 4 of 16


Arizona


U


U"


Tpo


Palouse






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cellular processes, such as carbohydrate metabolism, sig-
nal transduction, protein transport and degradation, and
cell division (Table 2). However, we could not reliably
identify the remainder of the fragments because of their
short length (-150 bp).
Rapid loss of parental homeologs
The genes, enzymes, and sizes of digested genomic and
cDNA amplifications for CAPS analysis of T. mirus and
its parents are listed in Additional file 1.
For the genomic CAPS analysis, 20 of 30 genes
showed additivity, with both parental copies maintained
in all allopolyploid individuals (Figure 1). Nine of 30
genes showed that at least one allotetraploid individual
was missing one parental homeolog (Figure 2, Table 3).
To determine whether these losses were due to true
homeolog loss or simply loss of a restriction site (due to
sequence polymorphism), we sequenced the PCR frag-
ments of all genes exhibiting putative losses. Sequencing
revealed that all individuals exhibiting apparent loss
events have only one parental homeolog, confirming
that these inferred homeolog losses are not due to
restriction site divergence and loss of a CAPS marker.
Two genes exhibited homeolog losses of one parental
copy or the other in at least one individual, whereas
seven genes showed loss of only the T dubius homeolog.
For the putative homolog of Thioredoxin M-type 1, one
Pullman-1 T. mirus plant (2601-10) showed loss of the T.
porrifolius band, while one plant of Palouse T. mirus
(2602-4) exhibited loss of the T. dubius band (Figure 2,
Table 3). For the putative Nucleic acid binding homolog,
eight T. mirus plants from the Pullman-2 (2678-3 and
2678-11), Oakesdale (2673-4), and Arizona (1747-1,
1747-2, 1747-3, 1747-6, 1747-9) populations showed loss
of the T. porrifolius band, while one Palouse T. mirus
individual (2602-25) lost the T. dubius band. In contrast,
preferential loss of the T. dubius parental homeolog was
observed in several individuals for seven genes putativelyy
identified as Myosin heavy chain CLASS xl, LRR protein,
Prenyltransferase, NADP/FAD oxidoreductase, Tetratrico-
peptide repeat protein, RNA binding, and Glyceraldehyde-
3-phosphate dehydrogenase). In addition, variation was
observed among populations; losses of the T. dubius
homeolog occurred at more loci in the Pullman-1, Pull-
man-2, and Palouse populations than in the Oakesdale,
Rosalia, Tekoa, and Arizona populations. For example,
individuals from the Palouse population showed loss of
the T. dubius homeolog for four genes, while individuals
from the Oakesdale population exhibited gene loss for
only one gene (Table 3). Therefore, the Pullman-1, Pull-
man-2, and Palouse populations of T. mirus show higher
levels of, and greater variation in, homeolog loss than do
populations from Oakesdale, Rosalia, Tekoa, and Ari-
zona. However, one putative gene (Adenine-DNA glycosy-
lase) was polymorphic in both T. mirus and T. dubius


(Figure 3). Most individuals of T. dubius have a single
copy of this gene, but five T. dubius individuals have an
extra copy that corresponds to the PCR amplicon pro-
duced in T. porrifolius. Also, six T. dubius individuals
only have the "T. porrifolius" type (Figure 3). This poly-
morphism observed in T. dubius can affect interpretation
of the expression patterns of T. mirus, making it hard to
distinguish loss from polymorphism. As a result of this
polymorphism, this gene was not employed in our ana-
lyses of loss events.
cDNA CAPS analysis was performed for 15 of 30 genes.
The remaining 15 genes analyzed above for genomics
CAPS could not be amplified (see Materials and Meth-
ods). Eleven of the genes included in the cDNA analyses
showed additivity, whereas four genes (putative homologs
of Thioredoxin M-type 1, Myosin heavy chain CLASS XI,
Nuclear ribosomal DNA, and Glyceraldehyde-3-phos-
phate dehydrogenase) showed expression differences in
some polyploid individuals relative to the diploid parents.
However, the apparent expression differences from the
Thioredoxin M-type 1 and Myosin heavy chain CLASS XI
result from genomic losses (see above; Figure 2, Addi-
tional file 2). For the other two genes, true expression dif-
ferences were detected. For the putative homolog of
Nuclear ribosomal DNA, cDNA CAPS showed absence
of the T. dubius homeolog in one individual of T. mirus
(2602-3) from Palouse, while genomic CAPS found addi-
tive patterns in all tetraploid individuals (Additional file
3). Also, a putative homolog of Glyceraldehyde-3-phos-
phate dehydrogenase showed silencing in T. mirus in six
individuals from Pullman-1 (2601-5, 2601-10, 2601-12,
2601-14, 2601-45, and 2601-47), three individuals from
Pullman-2 (2678-1, 2678-2, and 2678-11), one individual
from Palouse (2602-1), and one individual from Oakes-
dale (2673-5) (Figure 4, Table 4).
In summary, 27 of 40 individuals sampled of T. mirus
showed loss of at least one homeolog, and 12 individuals
exhibited true loss of expression of one parental homeo-
log (Tables 3, 4).
Diploid F, hybrids are additive of their parental genomes
Genomic CAPS analyses for six synthetic Fi hybrids from
two independent crosses between T. dubius and T. porri-
folius were also performed for the same 30 genes sur-
veyed in natural populations of T. mirus. Significantly, all
genomic CAPS analyses of F, hybrids exhibited additivity
of the parental homeologs. cDNA CAPS analysis for all
15 genes investigated in natural populations of T. mirus
showed that both parental homeologs were expressed in
all F, hybrids examined (Additional file 4).

Discussion
cDNA-AFLP variation in populations of T. mirus
As cDNA-AFLPs reveal potentially differentially
expressed genes, the results can provide useful initial


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THIOREDOXIN M-TYPE 1
Genomic CAPS
Pullmanl
Tdu Tm


Tdu


Pullman2
Tm


Too Tdu


Oakesdale Rosalia Tekoa
Tdu Tm Tdu Tm Tdu


MMEB:HaMI


cDNA CAPS
Tdu


Pullman2


Tdu


Arizona
Tdu Tm


HM


Pullman1


Tpo


Tpo


Oakesdale


Palouse
Tdu T


Rosalia
Tdu


Tpo


Tekoa
Tdu


Arizona


Figure 2 Genomic and cDNA CAPS analyses illustrating homeolog loss in a putative homolog of Thioredoxin M-type I from multiple
individuals from several populations of independent origin of T. mirus; also shown are the parental diploids, T. dubious and T.
porrifolius. Tdu T. dubius, Tm T mirus, Tpo T. porrifolius. Arrows indicate homeolog loss.


Page 6 of 16


Tpo


Palouse
Tm


TDo






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information on the genetics of polyploids, especially
those that lack developed genomic resources, such as
Tragopogon. Thus, cDNA-AFLPs provide numerous can-
didate genes relatively quickly and inexpensively. How-
ever, cDNA-AFLP analysis must be followed by other
approaches, such as CAPS analysis, because cDNA-
AFLP fragment differences may result from true expres-
sion differences, sequence polymorphism, or gene or
homeolog loss [26,91].
From cDNA-AFLPs, we identified 33 putative genes
that were not additive of the parental bands in T. mirus;
most of these (21 out of 33) exhibited maternal banding
patterns (Table 2). However, subsequent analysis of 23
of these genes using genomic and cDNA CAPS analyses
showed that only four of these genes exhibited an


expression difference; two genes, THIOREDOXIN M-
TYPE 1 and MYOSIN HEAVY CHAIN CLASS XI,
showed homeolog losses, while NUCLEAR RIBOSOMAL
DNA and GL YCERALDEHYDE-3-PHOSPHA TE DEHY-
DROGENASE exhibited true silencing (Figure 4, Table
4). However, most of the genes exhibiting apparent
maternal, paternal, or novel banding patterns in cDNA-
AFLPs actually showed additive patterns in genomic and
cDNA CAPS analyses, indicating that the cDNA-AFLP
fragment differences observed in T. mirus may be
derived from sequence polymorphism and are not indi-
cative of homeolog loss or silencing.
Rapid genomic changes in T. mirus
Analyses of CAPS markers provide evidence of rapid,
frequent, and preferential elimination of homeologous


Page 7 of 16


ADENINE-DNA GLYSOSYLASE

Genomic CAPS Pullmanl

Tdu Tm Tpo








Pullman2 Palouse

Tdu Tm Tpo Tdu Tm Tpo









Oakesdale Rosalia Tekoa Arizona

Tdu Tm Tdu Tm Tdu Tm Tdu Tm








Figure 3 Genomic CAPS analysis of Adenine-DNA glycosylase, which exhibits a polymorphic pattern in the parental diploid T. dubious
(see arrows). An additive pattern is consistently seen in the polyploid T. mirus. Tdu = T. dubius, Tm T mirus, Tpo = T. porrifolius,





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GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE


Genomic CAPS
Tdu


Pullmanl
Tm


Tdu


Pullman2
Tm


-E


Tpo
H III


-------- --P------


Palouse
Tdu Tm


Oaksdalle
Tdu 71


Ma


Rosalia
Tdu


Il


Tekoa
Tdu


El


Arizona
Tdu Tm

i^^W^^^^H


cDNACAPS


Pullmanl
Tm


Pullman2


TDO


Palouse


Tdu Tm Tpo Tdu Tm Tpo
-gWMy^# i fle44aj i IIIH* P


Oaksdalle


Rosalia


Tekoa


Tm Tdu Tm Tdu Tm Tdu Tm


L


Figure 4 Genomic and cDNA CAPS analyses illustrating homeolog loss as well as silencing in a putative homolog of Glyceraldehyde-3-
phosphate dehydrogenase from multiple individuals from several populations of independent origin of T. mirus; also shown are the
parental diploids, T. dubius and T. porrifolius Tdu T dubius, Tm T mirus, Tpo T porrifolius. White arrows indicate homeolog loss and red
arrows show silencing.


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loci and changes in gene expression in allotetraploid
individuals of T. mirus. Most of the genes examined
showed genomic CAPS patterns that are additive of the
parental genes, but nine genes of the 30 examined
showed homeolog loss, fewer than observed for T. mis-
cellus, in which 16 loci of the 23 examined showed
homeolog loss [26,86,87].
In T. mirus, the T. dubius parental homeolog has been
lost at more loci (12.1%; 7 out of 58 homeologs) than
the T. porrifolius homeolog (6.9%; 4 out of 58 homeo-
logs). Likewise, in T. miscellus, T. dubius parental home-
olog has been lost at more loci (32.6%; 15 out of 46
homeologs) than the T. pratensis homeolog (26.1%; 12
out of 46 homeologs) [26,86,87]. However, the T. porri-
folius homeolog has actually been lost from more indivi-
duals of T. mirus (25) than has the T. dubius homeolog
(10), in contrast to data for T. miscellus [26,87]. This
difference between T. mirus and T. miscellus results
from the extensive loss of the T. porrifolius homeolog
for NADP/FAD OXIDOREDUCTASE. Furthermore, each
homeolog absence in a polyploid should not necessary
be viewed as a unique loss, as a single loss may subse-
quently be transmitted throughout a population.
The preferential loss of T. dubius homeologs also
agrees with the biased rDNA homogenization of T.
dubius repeats to the other diploid parental type in both
T. miscellus and T. mirus [71]. That is, the number of
T. dubius rDNA units has typically been greatly reduced
in the genomes of the allotetraploids T. mirus and T.
miscellus relative to the other diploid parent (either T.
porrifolius or T. pratensis) due to apparent concerted
evolution [71,72]. The single exception is the Palouse
population of T. mirus, in which individuals have a rela-
tively high number of T. dubius rDNA repeats relative
to T. porrifolius repeats (compared to the diploid paren-
tal plants from the Palouse) [71], illustrating variation in
rDNA repeat composition among populations of T.
mirus of independent origin. In contrast, allopolyploids
in Arabidopsis and Brassica apparently have not under-
gone loss or concerted evolution of rDNA units [92,93].
However, rapid concerted evolution of rDNA units in
just a few generations [94,95] has occurred in some syn-
thetic hybrids and allotetraploids, including synthetic
hybrids between maize and Tripsacum [96], somatic
hybrids of Medicago sativa [97], synthetic Nicotiana
allotetraploids [94,98], and synthetic allotetraploid Ara-
bidopsis suecica [99].
Despite being fewer in number, the rDNA units of T.
dubius origin dominate rDNA transcription in most
populations of T. mirus [72]. rDNA gene reduction by
concerted evolution in allopolyploids can therefore be
countered by high levels of expression controlled by epi-
genetic regulation. We obtained similar results for
rDNA expression here in our cDNA CAPS study. Based


on visual comparison of banding intensity, there is
higher rDNA expression of T. dubius-origin units than
of T. porrifolius-origin units in all plants from all popu-
lations examined, except for individuals from the
Palouse population (Additional file 3). Interestingly, for
the Palouse population, cDNA and genomic CAPS indi-
cate the silencing of the T. dubius rDNA unit in one
plant of T. mirus. Hence, these results further highlight
the importance of populational surveys, by indicating
some stochasticity for rDNA expression in the young
polyploid T. mirus.
Some synthetic and natural allopolyploids show
remarkable genomic restructuring (e.g., Arabidopsis sue-
cica [56], Brassica napus [51,54,55], Nicotiana lines
[94,95,100], Primula [101], and wheat [48]). For exam-
ple, synthetic Brassica napus allopolyploids exhibit
many chromosomal translocations and transposition
events during the S2 to S5 generations, based on RFLP
analysis of synthetic lines [51,55]. Genome evolution in
the natural polyploids Tragopogon mirus and T. miscel-
lus appears most similar to the results obtained for
these synthetic Brassica allopolyploids. Homeolog loss
appears frequent in both systems. Recent cytogenetic
studies using FISH and GISH indicate that both T.
mirus and T. miscellus show evidence of rapid genomic
rearrangement, including translocations and inversions
[102]. Genetic changes observed in synthetic Brassica
napus as well as natural populations of Tragopogon
mirus and T. miscullus may be related, in part, to chro-
mosomal rearrangements in these polyploids [51,55,102].
Plants of the synthetic and naturally occurring allopo-
lyploid Arabidopsis suecica also exhibit chromosomal
rearrangement [56], as well as many changes in gene
expression [40,41]. Genes from one parent, A. thaliana,
have often been silenced epigenetically by DNA methy-
lation [41]. Through microarray analysis, Wang et al.
[40] showed that approximately 65% of nonadditively
expressed genes in the synthetic allotetraploids were
repressed, and more than 94% of them matched the
genes that are highly expressed in one parent, A. thali-
ana. Tragopogon allopolyploids have undergone many
losses of homeologous loci, often eliminating copies of
one parent, T. dubius, but fewer instances of gene silen-
cing. Additional studies of Tragopogon are needed to
similarly examine gene expression on a genomic-level
scale.
F1 diploid hybrids and early synthetic allotetraploids
(S1 to S3 generation) between Aegilops sharonensis and
Triticum monococcum ssp. aegilopoides showed both
gene loss and silencing by DNA methylation
[1,47,48,103-105]. However, such immediate changes
have not been detected in Tragopogon. Genomic and
cDNA CAPS data for synthetic Fi hybrids between T.
dubius and T. porrifolius showed additivity rather than


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gene loss or silencing (Additional file 4). Similarly, no
homeolog loss was observed in synthetic F, hybrids
between T. dubius and T. pratensis [26], or in newly
produced (So) or first-generation (S1) synthetic T. mis-
cellus [87]. Therefore, in contrast to wheat, both T.
mirus and T. miscellus exhibit genome evolution, not
immediately following hybridization or allopolyploidiza-
tion, but apparently shortly thereafter, given that the
species are probably less than 80 years old (or 40 gen-
erations; these plants are biennials) [69,70]. Also, these
results suggest that loss of homeologs and gene expres-
sion changes, while still rapid in evolutionary time, may
be slightly more gradual in Tragopogon, occurring over
several generations, but further studies are required to
assess the speed and magnitude with which genomic
changes have occurred in Tragopogon.
A major question centers on the mechanisms respon-
sible for the loss of homeologs in these young allopoly-
ploids. Ownbey [69] observed the formation of complex
multivalents during meiosis in both T. mirus and T.
miscellus shortly after their formation and in F, hybrids
between T. dubius and T. porrifolius, and between T.
dubius and T. pratensis. Furthermore, multivalents have
also been observed in synthetic T. mirus and T. miscel-
lus [88,106]. In addition, rare patterns observed in analy-
sis of allozyme variation in Tragopogon are consistent
with non-homologous recombination [84]. Non-homolo-
gous recombination could provide a mechanism of
homeolog loss in T. mirus and T. miscellus, as in Bras-
sica [55]. Recent cytogenetic data provide additional
insights into potential mechanisms for gene loss in Tra-
gopogon. GISH studies have revealed that chromosomal
rearrangements and other changes may be common in
natural populations of T. mirus and T. miscellus [102].
Intergenomic translocations, inversions, as well as
apparent monosomy and reciprocal trisomy occur in fer-
tile individuals of both polyploids [102]. Such rearrange-
ments provide a potential mechanism for the homeolog
losses observed in both T. mirus and T. miscellus.
Genomic changes versus differential expression in T.
mirus
For most of the genes examined here, homeolog losses
appear to be responsible for the cDNA-AFLP fragment
differences observed in individuals of T. mirus relative
to its diploid progenitors. However, in two genes (puta-
tively Nuclear ribosomal DNA and Glyceraldehyde-3-
phosphate dehydrogenase), we found true expression dif-
ferences in the allopolyploid relative to its parents.
Nuclear ribosomal DNA encodes ribosomal RNA, and
Glyceraldehyde-3-phosphate dehydrogenase encodes an
enzyme that participates in multiple processes, including
transcription activation, initiation of apoptosis, and ER
to Golgi vesicle shuttling [107], so both of these genes
are crucial for cell function. The pattern of Nuclear


ribosomal DNA from genomic and cDNA CAPS ana-
lyses is consistent with Matydiek et al.'s [72] study: all
individuals from the Palouse population showed additiv-
ity with genomic CAPS and in the Southern blot rDNA
study of Matydiek et al. [72]. However, in one individual
from the Palouse population, the T. dubius Nuclear
ribosomal DNA homeolog was completely silenced in
both our cDNA CAPS analysis and in the rDNA tran-
script study of Matydiek et al. [72].
Most of the T. mirus individuals examined here,
except those from Tekoa and Arizona, show no expres-
sion of the T. dubius homeolog for Glyceraldehyde-3-
phosphate dehydrogenase (Figure 4). Genomic CAPS
data indicate homeolog loss in two individuals, but addi-
tivity of parental homeologs in the remaining indivi-
duals. cDNA CAPS analyses therefore exhibit gene
silencing in 13 individuals (Figure 4). Recent studies
have shown that rapid epigenetic gene silencing follow-
ing allopolypoid formation can be reversed by chemical
demethylation in allopolyploid Arabidopsis suecica
[41,43,44,108]. Therefore, silencing of these two genes
might result from epigenetic phenomena such as DNA
methylation or histone acetylation [109,110].
In this study, gene silencing of T. dubius homeologs
occurred in only two cases. In addition, expression stu-
dies of T. miscellus [26,87] showed that seven out of 17
genes exhibited silencing of the T. dubius homeolog,
while for two other genes, the T. pratensis homeolog
was silenced. These biased patterns in T. mirus and T.
miscellus indicate that T. dubius homeologs might be
more susceptible to silencing than the alternative paren-
tal homeologs.
When we compare gene silencing with gene loss with
respect to the number of individuals examined, the pre-
vious studies of T. miscellus [26,87] show that silencing
events are slightly more frequent than homeolog loss,
while in this study of T. mirus, homeolog losses are
slightly more frequent than silencing events. These
expression patterns result from biased expression of
only a few genes. For example, 14 out of 20 T. miscellus
individuals have silencing events in Leucine-rich repeat
transmembrane protein kinase, and 11 out of 40 T.
mirus individuals have silencing events in glyceralde-
hyde-3-phosphate dehydrogenase. Therefore, comparing
gene loss with silencing events in T. mirus and T. mis-
cellus seems to be affected by specific genes, with a few
of the genes examined here especially prone to
silencing.
However, when we consider the number of genes
examined in this study of T. mirus and in the previous
T. miscellus studies [26,86,87], homeolog losses in T.
mirus (18.97%; 11 out of 58 homeologs) and in T. mis-
cellus (58.7%; 27 out of 46 homeologs) are more fre-
quent than silencing events (6.7%; two out of 30


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homeologs in T. mirus; 26.4%; nine out of 34 homeo-
logs in T. miscellus). Nevertheless, we investigated only
a small portion of the genome, so further studies are
required to assess whether the results for the ~30
genes so far examined are representative of the entire
genome.
Genomic CAPS analysis of a putative Adenine DNA
glycosylase gene showed polymorphism in the popula-
tions of the diploid parent T. dubius surveyed here
(Figure 3). Although previous studies in Tragopogon
diploids using allozymes and other markers indicated
that genetic variation within populations is quite low
[83,84], T. dubius is the most genetically variable of
the three diploids; allozyme variation within popula-
tions of T. porrifolius and T. pratensis was limited or
absent. Therefore, polymorphism among T. dubius
individuals for one of the genes analyzed here is not
surprising. A recent survey of Tragopogon diploids and
polyploids from the Palouse using microsatellite mar-
kers similarly revealed low levels of genetic variation
within populations of T. dubius, but none within either
T. pratensis or T. porrifolius (V. Symonds et al.,
unpublished data).

Conclusions
Recently formed Tragopogon allotetraploids (<80 years;
40 generations for these biennial plants) exhibit various
consequences of genome evolution and gene expression
following polyploidy. In this study, using cDNA-AFLPs,
we found differential banding patterns, possibly attribu-
table to gene silencing, novel expression, and/or mater-
nal/paternal effects between T. mirus and its diploid
parents. Most of the banding patterns subsequently
investigated with genomic and cDNA CAPS analyses
revealed additivity. Most of the differences observed in
T. mirus result from homeolog loss, rather than gene
silencing; the latter was detected only infrequently (in
two genes in some individuals). Genomic and cDNA
CAPS analyses indicated that plants of T. mirus have
experienced frequent and preferential elimination of the
T. dubius homeolog, whereas comparable analyses of
synthetic F, hybrids between the parents (T. dubius x T.
porrifolius) of T. mirus showed only additivity.
These same results were also obtained for the recently
and repeatedly formed allotetraploid Tragopogon miscellus
[26,87]. Both T. mirus and T. miscellus undergo biased
loss of homeologs contributed by their shared diploid par-
ent, T. dubius. Furthermore, both allotetraploids exhibit
more homeolog losses than gene silencing in terms of the
number of genes undergoing change. Taken together, our
results suggest that in Tragopogon loss of homeologs and
gene silencing are not immediate consequences of hybridi-
zation or polyploidization, but are processes that occur fol-


lowing polyploidization, occurring over a relatively small
number of generations. These results further support the
idea of polyploidy as a dynamic evolutionary process
(reviewed in 117), with abundant and rapid genomic
changes occurring within a short time period following
polyploidization. Further studies of homeolog loss, nonad-
ditive expression patterns, and subfunctionalization of
homeologs are needed to explore the roles of genetic and
epigenetic phenomena in the evolution of allotetraploid
Tragopogon species.

Methods
Plant materials
For populations Pullman-1 and -2, Palouse, and Rosalia,
seeds were collected from natural populations and grown
in the greenhouse at Washington State University (Pull-
man, WA, USA) and allowed to self-fertilize. Seeds from
these greenhouse-grown plants were collected, germi-
nated, and grown under controlled conditions in the
greenhouse at the University of Florida (UF; Gainesville,
FL, USA). Seeds from the Oakesdale, Tekoa, and Arizona
populations were collected and then grown at UF without
a round of selling. Each population of T. mirus is inferred
to be of separate origin (V. Symonds et al., unpublished
data) and was analyzed, along with samples of the diploid
progenitors from each location (Table 5; [70,83,84]). How-
ever, only the paternal parent, T. dubius, was investigated
for populations from Oakesdale, Rosalia, Tekoa, and Ari-
zona because T porrifolius was not found at those sites.
Diploid F1 hybrids used in this study were generated
by J. Tate, who crossed T. dubius (2611-11, Pullman-1;
the paternal progenitor) and T. porrifolius (2613-24,
Pullman-1; the maternal progenitor) using plants grown
from seed in the greenhouse[88].
cDNA-AFLP display and identification of polymorphic
fragments
Here, following Tate et al. [26], we initially employed
cDNA-amplified fragment length polymorphisms
(cDNA-AFLPs) to identify potentially differentially
expressed genes [111]. This approach has proven to be
useful in systems without well-developed genetic
resources [1,26,43,91,112-114]. However, the weakness
of this approach is that fragment differences observed
on a cDNA-AFLP gel may result from true expression
differences, sequence polymorphisms, or gene or home-
olog loss [26,40]. Due to this limitation, we subsequently
examined the expression patterns of genes isolated from
cDNA-AFLPs using genomic and cDNA CAPS analysis
[26,115]. This approach can determine whether apparent
expression differences observed at the transcriptional
level result from genomic changes such as gene loss or
from true differences in expression. Putative homeolog
losses were further tested via DNA sequencing.


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Leaf segments less than 30 cm in length were collected
from young plants six weeks after germination. Due to
heavy latex in the leaf tissue, we extracted total RNA from
leaf tissue using the method of Kim et al. [116], which
combines a CTAB extraction protocol [117] and subse-
quent use of the RNeasy Plant Mini Kit (Qiagen, Valencia,
CA, USA). cDNA was synthesized from total RNA using
SuperScript Double-Stranded cDNA Synthesis Kit (Invi-
trogen, Carlsbad, CA, USA), and the cDNA-AFLP techni-
que was performed as previously described [43], except
that we replaced isotope-based signal detection with silver
staining [118].
To investigate the utility of cDNA-AFLPs in T. mirus,
we conducted an initial survey of the Pullman-1 popula-
tion with 37 primer combinations. Following the success
of this survey, expanded cDNA-AFLP analyses were
conducted on 5-10 individuals each from the Pullman-1,
Pullman-2, and Palouse populations of T. mirus and
from the diploid progenitors that occurred with the tet-
raploid populations. We employed the same methods,
using the primer sets that were the most variable in our
initial screen (21 for Pullman-1 and four each for Pull-
man-2 and Palouse; Additional file 5). We analyzed 56
individuals: 20 of T. mirus, 16 of T. dubius, and 20 of T.
porrifolius from the Pullman-1, Pullman-2, and Palouse
populations, respectively (Table 5). The expressed bands
on cDNA-AFLP gels were scored as monomorphic (pre-
sent in all individuals) or polymorphic (present in at
least one individual/absent in at least one individual)
(Table 1).
From the expanded cDNA-AFLP work, 125 variable
fragments exhibiting novel, maternal/paternal, or other
polymorphic patterns were identified from the Pull-
man-1, Pullman-2, and Palouse populations. To deter-
mine the putative identity of these fragments, we
excised and sequenced fragments over 250 bp in size,
as described in Lee and Chen [43], Wang et al. [91],
and Tate et al. [26]. The polymorphic bands were cut
from the polyacrylamide gels, and the fragments were
re-amplified using the same set of selective amplifica-
tion primers and cloned using a Topo TA Cloning Kit
(Invitrogen). Sequencing was performed with the CEQ
DTCS-Quick Start Kit (Beckman Coulter, Fullerton,
CA, USA). To identify the sequences obtained above,
we used BLAST searches against the NCBI database
http://www.ncbi.nlm.nih.gov, and the sequence identity
was rechecked against the Compositae Genome Project
database http://compgenomics.ucdavis.edu. Identified
sequences have been deposited in the EMBL/GenBank
database under accession nos (Additional file 6).
CAPS analyses
To determine if cDNA-AFLP fragment polymorphisms
resulted from genomic changes or expression differ-
ences, we conducted both genomic and cDNA CAPS


analyses. In CAPS analyses, amplified PCR products are
digested with diagnostic restriction enzymes that distin-
guish the diploid parental sequences, and the fragments
are separated by agarose gel electrophoresis.
For this study we included populations of T. dubius
and T. mirus from Oakesdale, Tekoa, and Rosalia,
Washington, and from Arizona, in addition to the Pull-
man-1, Pullman-2, and Palouse populations. Therefore,
100 individuals were examined for genomic CAPS ana-
lyses (Table 5): 40 of T mirus, 40 of T. dubius, and 20 of
T. porrifolius, with 5-10 individuals per population. For
cDNA CAPS analyses, we analyzed 40 individuals of T.
mirus, 36 of T. dubius, and 20 of T. porrifolius, with 5-10
individuals per population (Table 5). We also analyzed 6
F1 hybrid plants using both genomic and cDNA CAPS to
determine whether genomic changes and expression dif-
ferences appear in the first-generation hybrids.
For CAPS analyses, we used 30 primer sets: 23 primer
sets were designed based on the sequences that were
variable in the cDNA-AFLP analysis of the Pullman-1,
Pullman-2, and Palouse populations, and 7 primer sets
were from studies of T. miscellus [26,87]. To design pri-
mer sets, we first BLASTed our fragments against Lac-
tuca or Helianthus ESTs and then used the "hit" ESTs
for primer design because the ESTs are likely longer
than the isolated fragments, with a greater chance that
the expressed sequence spans the introns in genomic
DNA. With those primer sets, we amplified fragments
from T. dubius and T. porrifolius and then confirmed
their sequences. From those sequences, we redesigned
the primer sets to be more specific for analysis of Trago-
pogon CAPS. All primers were designed using the web
interface program, Primer3 (v. 0.4.0; http://frodo.wi.mit.
edu/). Primer sequence information is given in Addi-
tional file 6.
For each of the 30 gene regions, we aligned DNA
sequences from the diploid parents using Sequencher v.
4.1.4 (Gene Codes Corporation, Ann Arbor, MI, USA)
and identified diagnostic restriction sites between the
species.
Genomic CAPS analyses
We isolated genomic DNA from 100 individuals of the
allotetraploid and its two progenitors using a modified
CTAB protocol [117]. Genomic fragments were ampli-
fied in a 25 pl volume with 20 ng template, 5x buffer,
1.5 mM MgCl2, 0.2 mM dNTPs, 0.1 mM each primer,
and 0.5 unit Taq polymerase (Promega, Madison, WI,
USA). Thermal cycling conditions were as follows: 95C
for 2 min, followed by 35 cycles of 95oC for 30 sec, 54-
56oC for 30 sec, 72oC for 1 min 20 sec, and a final 7-
min extension at 72oC. Products were separated on a
1.5% agarose gel, stained with ethidium bromide, and
visualized using a UV transilluminator. Genomic digests
were performed in a 12 Pl volume, containing lx buffer,


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3 Ipl PCR product, 2 units of restriction enzyme (New
England Biolabs, Ipswich, MA, USA), and 100 ptg/ml
bovine serum albumin (when required), and incubated
at the appropriate temperature for 9 hr. Digested pro-
ducts were separated on 2-4% agarose gels, stained with
ethidium bromide or SyberGold (Invitrogen), and visua-
lized using a transilluminator.
To determine whether putative homeolog losses
observed in genomic CAPS analyses were due to true
homeolog loss or simply a loss of a restriction site (due
to sequence polymorphism in one parental fragment in
the polyploid), we sequenced the initial PCR product. A
homeolog loss would yield only one parental diploid
sequence, whereas loss of a restriction site in one parent
would still yield both parental DNA sequences.
cDNA CAPS analyses
We isolated total RNAs from 96 of the individuals ana-
lyzed for genomic CAPS (see above) using the method
of Kim et al. [116] and the RNeasy Plant Mini Kit
(Qiagen). The first-strand cDNA was synthesized with
5 pg total RNA using Superscript II reverse transcrip-
tase (Invitrogen) with a poly-T (T17) primer. Using the
same primer sets as for 15 of the loci in the genomic
CAPS analyses, RT-PCR was carried out using 50 ng
of template from the first-strand cDNA in a 25 Ipl
volume with 20 ng template, 5x buffer, 1.5 mM
MgCl2, 0.2 mM dNTPs, 0.1 mM each primer, and 0.5
unit Taq polymerase (Promega). The remaining 15
genes could not be amplified from these cDNAs; the
reasons for this are unclear, but perhaps these genes
were not expressed in the leaf tissue sampled for
cDNA CAPS. For amplification of fragments and diges-
tion of RT-PCR products, we employed basically the
same approach as described for the genomic CAPS
analyses. In addition, for NUCLEAR RIBOSOMAL
DNA, the relative PCR band intensities of the two
homeologs were measured using KODAK 1D Image
Analysis Software (Kodak, Rochester, NY, USA).

List of abbreviations
CAPS: cleaved amplified polymorphic sequence; cDNA-
AFLPs: cDNA-amplified fragment length
polymorphisms.
Organisms: T. dubius: Tragopogon dubius; T. mirus:
Tragopogon mirus; T. miscellus: Tragopogon miscellus; T.
porrifolius: Tragopogon porrifolius; T. pratensis: Tragopo-
gon pratensis.


Additional file 1: Supplementary Data Homeologous loci and
restriction enzymes examined in T mirus with genomic and cDNA CAPS
analyses
Click here for file
[http'//wwwbiomedcentral com/content/supplementary/1471-2164- 11-
97-51 DOC]


Additional file 2: Supplementary Data Genomic and cDNA CAPS
analyses illustrating homeolog loss in a putative homolog of MYOSIN
HEAVY CHAIN CLASS XI from multiple individuals from several populations
of independent origin of T mirus; also shown are the parental diploids, T
dubious and T porrifolius Tdu T dubious, Tm T mirus, Tpo T
porrifolius Arrows indicate missing homeologs
Click here for file
[http'//wwwbiomedcentral com/content/supplementary/1471-2164- 11-
97-52 PPT]
Additional file 3: Supplementary Data Genomic and cDNA CAPS
analyses of NUCLEAR RIBOSOMAL DNA, which exhibits silencing pattern in
one plant of T mirus from Tekoa (see red arrow) This plant is not
expressing the homeolog of T dubious Tdu = T dubious, Tm = T mirus,
Tpo = T porrifolius
Click here for file
[http'//wwwbiomedcentral com/content/supplementary/1471-2164- 11-
97-53 PPT]
Additional file 4: Supplementary Data Genomic and cDNA CAPS
analyses for 15 candidate genes from Tragopogon Fi hybrids and their
porgenitors Tdu = T dubius(2611-11, Pullman, WA), Tpo = T porrifolius
(2613-24, Pullman, WA)
Click here for file
Shttp//www biomedcentral com/content/supplementary/1471-2164- 11-
97-54 PPT]
Additional file 5: Supplementary Data Primer combination for
selective amplification and used in cDNA-AFLP analyses Asterisk indicates
primers used in expanded study
Click here for file
[http'//wwwbiomedcentral com/content/supplementary/1471-2164- 11-
97-55 DOC]
Additional file 6: Supplementary Data Primer
Click here for file
Shttp//www biomedcentral com/content/supplementary/1471-2164- 11-
97-56 DOC]



Acknowledgements
We thank Drs Zhongfu Ni, Z Jeffrey Chen, and Jennifer A Tate for technical
assistance This work was funded by National Science Foundation (NSF)
grants MCB-0346437 and DEB-0614421

Author details
]Department of Biology, University of Florida, Gainesville, Florida, 32611 USA
2Florida Museum of Natural History, University of Florida, Gainesville, Florida,
32611 USA

Authors' contributions
JK carried out all experiments described above, and with P55 and DES wrote
the manuscript PSS and DES designed and supervised the project All
authors read and approved the final submission

Received: 12 July 2009
Accepted: 8 February 2010 Published: 8 February 2010

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O loMed Central


doi:10.1186/1471-2164-11-97
Cite this article as: Koh et ail Homeolog loss and expression changes in
natural populations of the recently and repeatedly formed
allotetraploid Tragopogon mirus (Asteraceae). BMC Genomics 2010 1197


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