Group Title: BMC Plant Biology
Title: On the road to diploidization? Homoeolog loss in independently formed populations of the allopolyploid Tragopogon miscellus (Asteraceae)
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Title: On the road to diploidization? Homoeolog loss in independently formed populations of the allopolyploid Tragopogon miscellus (Asteraceae)
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Language: English
Creator: Tate, Jennifer
Joshi, Prashant
Soltis, Kerry
Soltis, Pamela
Soltis, Douglas
Publisher: BMC Plant Biology
Publication Date: 2009
 Notes
Abstract: BACKGROUND: Polyploidy (whole-genome duplication) is an important speciation mechanism, particularly in plants. Gene loss, silencing, and the formation of novel gene complexes are some of the consequences that the new polyploid genome may experience. Despite the recurrent nature of polyploidy, little is known about the genomic outcome of independent polyploidization events. Here, we analyze the fate of genes duplicated by polyploidy (homoeologs) in multiple individuals from ten natural populations of Tragopogon miscellus (Asteraceae), all of which formed independently from T. dubius and T. pratensis less than 80 years ago.RESULTS:Of the 13 loci analyzed in 84 T. miscellus individuals, 11 showed loss of at least one parental homoeolog in the young allopolyploids. Two loci were retained in duplicate for all polyploid individuals included in this study. Nearly half (48%) of the individuals examined lost a homoeolog of at least one locus, with several individuals showing loss at more than one locus. Patterns of loss were stochastic among individuals from the independently formed populations, except that the T. dubius copy was lost twice as often as T. pratensis.CONCLUSION:This study represents the most extensive survey of the fate of genes duplicated by allopolyploidy in individuals from natural populations. Our results indicate that the road to genome downsizing and ultimate genetic diploidization may occur quickly through homoeolog loss, but with some genes consistently maintained as duplicates. Other genes consistently show evidence of homoeolog loss, suggesting repetitive aspects to polyploid genome evolution.
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Research article

On the road to diploidization? Homoeolog loss in independently
formed populations of the allopolyploid Tragopogon miscellus
(Asteraceae)
Jennifer A Tate*1, Prashant Joshi1, Kerry A Soltis2, Pamela S Soltis3,4 and
Douglas E Soltis2,4


Address: 'Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand, 2Department of Biology, University of Florida,
Gainesville, Florida, USA, 3Florida Museum of Natural History, University of Florida, Gainesville, Florida, USA and 4Genetics Institute, University
of Florida, Gainesville, Florida, USA
Email: Jennifer A Tate* j.tate@massey.ac.nz; Prashant Joshi p.joshi@massey.ac.nz; Kerry A Soltis kerryl@ufl.edu;
Pamela S Soltis psoltis@flmnh.ufl.edu; Douglas E Soltis dsoltis@botany.ufl.edu
* Corresponding author


Published: 27 June 2009
BMC Plant Biology 2009, 9:80 doi:10.1 186/1471-2229-9-80
This article is available from: http://www.biomedcentral.com/1471-2229/9/80


Received: 5 May 2009
Accepted: 27 June 2009


2009 Tate et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons 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.



Abstract
Background: Polyploidy (whole-genome duplication) is an important speciation mechanism,
particularly in plants. Gene loss, silencing, and the formation of novel gene complexes are some of
the consequences that the new polyploid genome may experience. Despite the recurrent nature
of polyploidy, little is known about the genomic outcome of independent polyploidization events.
Here, we analyze the fate of genes duplicated by polyploidy (homoeologs) in multiple individuals
from ten natural populations of Tragopogon miscellus (Asteraceae), all of which formed
independently from T. dubius and T. pratensis less than 80 years ago.
Results: Of the 13 loci analyzed in 84 T. miscellus individuals, I I showed loss of at least one parental
homoeolog in the young allopolyploids. Two loci were retained in duplicate for all polyploid
individuals included in this study. Nearly half (48%) of the individuals examined lost a homoeolog
of at least one locus, with several individuals showing loss at more than one locus. Patterns of loss
were stochastic among individuals from the independently formed populations, except that the T.
dubius copy was lost twice as often as T. pratensis.
Conclusion: This study represents the most extensive survey of the fate of genes duplicated by
allopolyploidy in individuals from natural populations. Our results indicate that the road to genome
downsizing and ultimate genetic diploidization may occur quickly through homoeolog loss, but with
some genes consistently maintained as duplicates. Other genes consistently show evidence of
homoeolog loss, suggesting repetitive aspects to polyploid genome evolution.


Background
Allopolyploidy combines the processes of hybridization
with genome doubling, and together, these provide a
potential avenue for instantaneous speciation [1-3].


Whole-genome sequencing efforts have revolutionized
our thinking about the significance of polyploidy, as it is
clear that paleopolyploidy is a common phenomenon.
Ancient whole-genome duplications have been detected


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in many eukaryotic lineages, including angiosperms, ver-
tebrates, and yeast [4-12]. Polyploidy has been particu-
larly prevalent in flowering plants, where previous
estimates indicated that 30-70% of angiosperm species
had polyploidy in their ancestry [reviewed in [13]]. In the
last decade, the view of polyploidy in angiosperms has
changed, and it is now appreciated that perhaps all
angiosperm lineages have experienced at least one round
of polyploidy, with many lineages undergoing two or
more such episodes [14-18]. On more recent timescales,
molecular data have also revealed that most extant poly-
ploid plant species have formed recurrently [1,19-28]. In
fact, very few examples of a single unambiguous origin of
a polyploid species have been documented; these include
peanut, Arachis hypogaea, the salt marsh grass Spartina
anglica, and Arabidopsis suecica [29-32].

Following allopolyploidization, several evolutionary out-
comes are possible for the genes duplicated by polyploidy
(homoeologs). Both copies may be retained in the poly-
ploid and remain functional, one copy may accumulate
mutations and either diverge in function or become
silenced, or one copy may be physically lost [8,33,34]. The
fate of these duplicated gene pairs seems to vary depend-
ing on the system under investigation and the loci
involved [35-41]. Over longer evolutionary timeframes,
gene loss, genome downsizing, and, ultimately, genetic
'diploidization' appear to be common phenomena [8,42-
45]. Homoeologous recombination appears to play an
important role in the loss of small genomic fragments
during the early stages of polyploid formation [46-50],
which contributes to gene loss and genome downsizing in
allopolyploids [39,43,51,52]. Wolfe (2001) pointed out
that within a species, some loci may remain tetraploidd',
while others are diploidized; evidence from whole-
genome analyses supports this idea [e.g., [36,40]].
Although polyploidy is clearly a recurrent process on both
recent and ancient timescales, we know very little about
the evolutionary fate of genes duplicated by polyploidy in
independently formed allopolyploid populations. Specif-
ically, are homoeologs consistently retained or lost in a
repeated manner among individuals from independently
formed polyploid populations?

The allopolyploids Tragopogon mirus and T. miscellus
(Asteraceae) are textbook examples of speciation follow-
ing polyploidy and provide an ideal system to investigate
the evolutionary fate of duplicated genes in independ-
ently formed populations. These allopolyploids formed
recently in the Palouse region of the western United States
(eastern Washington and adjacent Idaho) following the
introduction of three diploid species (T. dubius, T. porrifo-
lius, and T. pratensis) from Europe in the early 1900s [53].
Tragopogon mirus formed independently several times
from T. dubius and T. porrifolius, while T. miscellus formed


multiple times from T. dubius and T. pratensis [53-57].
Only T. miscellus has formed reciprocally in nature, and
these reciprocally formed individuals can be distin-
guished morphologically. The 'short-liguled' form has T.
pratensis as the maternal progenitor, while the 'long-
liguled' form has T. dubius as the maternal parent (Figure
1). Today, only one long-liguled population exists (in


Washington


i --- -&" "s ss i
"^ Spokane Veradale Liberty Lake


Spangle

Freedom


Rockford

Fairfield


Idaho


Fernan Lake
Village


y Coeur D'Alene
Lake

Harrison


Worley


Chatcolet


Plaza
Latah
Malden Rosalia |
Tekoa


tle yOakesdale
Farmington

Garfield I


Diamond


Plummer


Tensed


Palouse Potlatch


Colfax Risbeck


Almota


I Albion |

SPullman r Moscow aI y
I


T. pratensis i. miscellus I. miscellus I. dubius
2n = 12 (short-liguled) (long-liguled) 2n = 12
2n = 24 2n = 24

Figure I
Tragopogon populations sampled. Populations of
Tragopogon sampled (boxed) in the United States and repre-
sentative capitula of the diploid (T. dubius and T. pratensis) and
allotetraploid (T. miscellus) species. Map modified from
Google Maps.


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Pullman, Washington; Figure 1); all other populations of
T. miscellus are short-liguled [58]. Molecular data have
confirmed that the populations of T. mirus and T. miscellus
in the Palouse have arisen independently (reviewed in
Soltis et al. 2004; Symonds et al. in prep.).

In this study, we examine ten populations of T. miscellus
for 13 loci to investigate the fate of homoeologous loci in
natural populations of Tragopogon miscellus. Our previous
study [59] revealed that T. miscellus individuals from two
populations (one each of short- and long-liguled forms)
had experienced loss of one parental homoeolog for seven
of ten loci examined. This loss was not fixed within or
between populations, nor was homoeolog loss 'fixed' for
any particular locus examined. Three of these loci were
retained in duplicate for all individuals examined.
Another recent study has also demonstrated loss of
homoeologous loci for five T. miscellus populations for a
different set often genes [60]. In addition to loss, homoe-
olog silencing has also occurred in these recent allopoly-
ploids [59,601. Because multiple origins typify most
allopolyploid species [27], we extended these previous
studies of T. miscellus to examine the extent to which
parental homoeologs might be lost from individuals from


several natural populations and to assess if recurrent pat-
terns of homoeolog loss or retention could be detected in
these independently formed populations.

Results
Genomic CAPS analyses
Two hundred individuals (83 T. dubius, 33 T. pratensis,
and 84 T. miscellus) from 10 populations (Table 1) were
screened for 13 markers Table 2). Variation in the restric-
tion digestion patterns of Tragopogon dubius was evident
for a single marker (TDF72.3) (Figure 2). No variation was
observed in T. pratensis based on the present sampling.
Two individuals, each grown from a seed collected from a
T. dubius plant in the field (one each from Troy and
Albion), apparently were hybrids, as the individuals pos-
sessed both T. pratensis and T. dubius fragment patterns in
the genomic restriction digests for all markers screened
(data not shown). Because T. pratensis does not occur in
either locality, these individuals likely represent hybrids
between T. miscellus and T. dubius.

Combining the new data generated here with data from
Tate et al. (2006), for the 13 loci examined, 11 showed
loss of a homoeolog in at least one of the T. miscellus


Table I: Populations of Tragopogon analyzed. Populations are ordered geographically from north to south.


Number of individuals


Spokane, WA T. pratensis
T. miscellus
T. miscellus
T. dubius
Spangle, WA T. pratensis
T. miscellus
T. dubius
Rosalia, WA T. pratensis
T. miscellus
T. dubius
Oakesdale, WA T. pratensis
T. miscellus
T. dubius
Garfield, WA T. pratensis
T. miscellus
T. dubius
Albion, WA T. pratensis
T. miscellus
T. dubius
Pullman, WA** T. pratensis
T. miscellus
T. dubius
Moscow, ID T. pratensis
T. miscellus
T. dubius
Troy, ID T. pratensis
T. miscellus
T. dubius
T. dubius

* Soltis and Soltis collection numbers; **long-liguled population


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Population


Species


Population ID*


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Table 2: Loci analyzed.


Gene abbreviation


CKINS
UBQ
THIOR
LTR2
PP2C
AUX
ADG
TDRC
BFRUCT
GTPB
PSBO
cryl
nrDNA


individuals surveyed (See Additional file 1; Figure 2). For
two genes (TDF46 and TDF85), both parental homoe-
ologs were retained in all individuals examined. Some
genes showed loss more frequently than others. For exam-
ple, a T. pratensis homoeolog ofTDFl17.4 was lost in only
one individual, while for TDF90, 18 individuals lost either
the T. dubius or T. pratensis copy. For the genes that
showed losses, 20 losses were from the T. pratensis
genome, while 40 were from the T. dubius genome (Z2 =
6.667, df = 1, P < 0.001). Considering the genic patterns
across populations, none of the genes showed loss in


Oakesdale


Locus ID

TDF7
TDF17.4
TDF36.3
TDF44
TDF46
TDF62
TDF72.3
TDF74
TDF85
TDF90
TDF27.10
cry I
nrDNA


Garfield


A TDF85

300 bp
200 bp
100 bp


T pratensis T miscellus T dubius


T pratensis T miscellus T dubius


B TDF72.3
300 bp *a r
200 bp o


U.mm.. m. -
b
A AA


C cry1

400 bp
200 bp


t t


Figure 2
Loss and retention of homoeologous loci in Tragopogon miscellus. Representative genomic CAPS results for three loci
from two populations of Tragopogon miscellus. An arrow indicates a loss, and an arrowhead indicates allelic variation. A. TDF85
showed no losses in any of the populations examined. B. Allelic variation was present in T. dubius from Garfield for TDF72.3. A
'missing' fragment in T. miscellus from Oakesdale may be interpreted as a loss, or the pattern may result from the polyploid indi-
vidual arising from a T. dubius individual with an allele that is similar in its digest pattern to T. pratensis. C. cryl showed loss in
some individuals and some populations, but not others.




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Putative protein/gene

Casein kinase
Polyubiquitin
Thioredoxin M-type I
Leucine-rich repeat transmembrane protein kinase
Protein phosphatase 2C family protein
Auxin conjugate hydrolase
Putative adenine-DNA glycosylase
Transducin family protein
P-fructosidase
Small GTP-binding protein
Oxygen-evolving enhancer
Cryptochrome I
Nuclear ribosomal DNA

every population. One gene, TDF36.3, showed loss in at
least one individual from all but one population (Troy).

Forty of the 84 T. miscellus individuals surveyed showed
loss of a homoeolog for at least one locus, with 15 of these
showing loss for multiple loci (See Additional file 1). For
example, individual 2693-14 from Spangle lost the T.
dubius homoeolog for both TDF7 and TDF90 and lost the
T. pratensis homoeolog for TDF62; individual 2625-3
from Albion lost the T. pratensis copy for TDF7, TDF44,
TDF74, TDF36.3, TDF27.10, and cry1. For individuals that


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lost a homoeolog at more than one locus, the same paren-
tal homoeolog was lost more often than different homoe-
ologs (i.e., 11 individuals lost homoeologs from the same
parent, while four individuals lost alternative homoe-
ologs). Of those that lost the same parental homoeolog,
nine cases were losses of T. dubius, while two were losses
of T. pratensis. Considering all populations, regardless of
the parental origin, the loss of one homoeolog was the
most common scenario (25 cases), followed by homoe-
olog losses at two loci in eight individuals, three loci in six
individuals, and six losses in one plant (individual 2625-
3 from Albion, mentioned above). For these multiple
losses, no clear pattern emerged (i.e., when two or more
loci were lost from multiple individuals, they were not the
same pairs of loci).

At the population level, differences in the number of
losses were also evident, but without a clear genomic,
genic, or geographical pattern (See Additional file 1). The
Albion and Moscow populations showed the greatest
number of total homoeolog losses (12 losses in three and
seven individuals, respectively), followed by Oakesdale
(nine losses in five individuals), Pullman (eight losses in
six individuals), Spokane-2617 (six losses in five individ-
uals), Garfield (six losses in five individuals), Spangle (six
losses in four individuals), Troy (four losses in two indi-
viduals), Rosalia (one), and Spokane-2664 (one). The
number of individuals (within a population) that showed
the same pattern of loss varied by population. The
Spokane-2664, Spangle, Rosalia, Garfield, Moscow, and
Troy populations did not have any individuals that shared
patterns of loss. Spokane-2617 and Pullman each had
three individuals with shared patterns, while Oakesdale
and Albion each had two individuals. Shared losses
among individuals within a population may represent
inheritance of a loss that occurred in a common ancestor.

Discussion
Homoeolog loss in independently formed populations
Our extended survey of 13 loci for ten populations of
Tragopogon miscellus indicates that some genes are main-
tained in duplicate in all populations, while others show
loss among some individuals from the independently
formed populations. This result is consistent with our pre-
vious finding of loss in two populations (Moscow and
Pullman) for ten of these same genes [59]. Although
homoeolog loss is not unique to Tragopogon, the present
study represents the largest survey of individuals from nat-
ural populations conducted thus far. Homoeolog loss
appears to be a common phenomenon in polyploids and
may occur rapidly following their formation. For exam-
ple, synthetic polyploids of wheat [471 and Brassica
[46,48,611 show loss of homoeologous loci in early gen-
erations. In Tragopogon, we have not detected loss in F,
hybrids or first-generation synthetic polyploids [59,60].


Thus, homoeologous loss does not appear to occur instan-
taneously upon hybridization or polyploidization in
Tragopogon, at least based on the loci examined thus far.

Given that genome downsizing and other processes may
ultimately contribute to genetic 'diploidization' in poly-
ploid organisms [8,43], what impact does homoeolog
loss have on recently and independently formed poly-
ploid populations? Our data indicate that homoeolog loss
in Tragopogon miscellus is stochastic among individuals
from polyploid populations that are less than 80 years old
(<40 generations as these are biennials). Almost half
(48%) of the individuals surveyed here showed loss of a
homoeolog for at least one locus, with some populations
showing loss more frequently than others. Five of these
same populations were examined for a different suite of
ten genes by Buggs et al. (2009), and a similar result was
found. The Moscow population showed the greatest
number of losses (eight), followed by Oakesdale (five),
Garfield (three), Spangle (one), and Pullman (one). Some
of the same individuals were examined here and as part of
that study, but again, no clear pattern of loss among indi-
viduals and populations could be identified. Given the
ecological success of T. miscellus, which is widespread in
the Palouse and whose range is expanding [58], this loss
does not appear to negatively affect the individuals or
populations. When Ownbey [53] first described T. miscel-
lus and T. mirus, he found that fertility (seed set) averaged
52-66% in the natural populations, but with a great deal
of variation outside this range among individuals. More
recent surveys of the natural populations indicate that fer-
tility (based on pollen stainability) is high, averaging 95-
100% (P. Soltis and D. Soltis, unpublished data), which
suggests that following their initial formation the poly-
ploid individuals experience some genomic instability,
but eventually become more stabilized. Recently, we
resynthesized allopolyploids of both T. miscellus and T.
mirus [62]. The initial S, plants exhibited slightly reduced
pollen stainability and fruit set; but successful lineages
that have survived to the S2 generation exhibit high fertil-
ity. Through homoeolog loss, perhaps the polyploid indi-
viduals from natural populations are still sorting out
potential genomic incompatibilities resulting from
hybridization and genome doubling [63]. It will be
important to follow the synthetic polyploids through suc-
cessive generations to determine when homoeolog loss
occurs and if this loss contributes to increased fertility.

One consistent pattern among the populations that has
emerged from the present study is that T. dubius homoe-
ologs appear to be lost more often than those ofT. praten-
sis, particularly when two or more loci undergo loss, and
this difference in losses is statistically significantly differ-
ent (P < 0.001) (See Additional file 1). Combining the
data presented here with those from Tate et al. (2006), we


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find that loss of a T. dubius homoeolog represents 67% of
the total losses, while loss of the T. pratensis copy repre-
sents 33% of the total losses. Why the T. dubius copy is
eliminated more frequently is not known. Significantly,
other allopolyploids, including wheat [49,641 and
Brassica [61], also show biased elimination of one paren-
tal genome over the other. The loss of T. dubius homoe-
ologs is especially evident for nrDNA (See Additional file
1, Figure 3) and is consistent with previous studies of
nrDNA evolution in natural populations of T. mirus and
T. miscellus [65,66]. Matyasek et al. (2007) found that
although the allopolyploids had fewer T. dubius nrDNA
copies, these were preferentially expressed over the alter-
native parental copies (i.e., either T. porrifolius or T. praten-
sis for T. mirus and T. miscellus, respectively). In the present
study, we also identified a few individuals that have
reduced T. pratensis nrDNA copy numbers relative to T.
dubius. These individuals were from both short- and long-
liguled T. miscellus populations (See Additional file 1, Fig-
ure 3). This bi-directional concerted evolution of nrDNA
copies has also been demonstrated in more ancient allo-
polyploids, such as Gossypium (G. tomentosum, G. hirsutum,
G. darwinii, G. barbadense, and G. mustelinum) [67].

The loss or retention of certain classes of genes appears to
be a recurrent pattern when ancient whole-genome dupli-
cation patterns are examined, although the classes that are
retained in duplicate differ depending on the lineage
under study [35,36,41,68]. For example, in Asteraceae,
Barker et al. (2008) found that genes associated with struc-
tural components and cellular organization were retained
in duplicate, while genes involved with regulatory (e.g.,
transcription factors) and developmental functions lack
duplicates. In Arabidopsis (Brassicaceae) and rice
(Poaceae), however, genes involved with transcription
were retained in duplicate [36]. In Tragopogon miscellus,
the two genes that were retained in duplicate (TDF46 and
TDF85) in all individuals did not fall into the category of
significantly enriched (or reduced) when compared to the
Barker et al. (2008) study. Similarly, the genes that were
lost did not match gene ontology (GO) slim categories
that were significantly either underrepresented or
enriched. TDF46 is a putative protein phosphatase 2C
family protein that functions in the plasma membrane,
and TDF85 is a putative 3-fructosidase that acts in the vac-
uole. As additional genomic resources are developed for
Tragopogon and these genes are analyzed in the polyploid
species, it will be imperative to determine whether certain
gene classes are consistently lost or retained following
allopolyploidization.

Mechanism for homoeolog loss
Studies of Brassica [46,50,61] allopolyploids have
revealed a significant role for homoeologous recombina-
tion in DNA loss, although this process does not appear to


affect wheat allopolyploids [38]. A recent karyological
study using fluorescent and genomic in situ hybridization
(FISH, GISH) of natural and synthetic Tragopogon allopol-
yploids identified extensive chromosomal changes,
including monosomy and trisomy, intergenomic translo-
cations, and variation in nrDNA loci [69]. Importantly,
the same study [69] showed that some chromosomal
changes occurred in the first synthetic generation of T.
mirus (synthetics of T. miscellus have not yet been investi-
gated). Ownbey [53] observed multivalent formation in
individuals of T. mirus and T. miscellus from natural pop-
ulations and also noted univalents and a ring of four chro-
mosomes in F1 hybrids between T. dubius and T. pratensis.
We have also observed frequent multivalent formation in
synthetic lineages of T. mirus and T. miscellus [62]. These
prevalent meiotic irregularities suggest a mechanism for
the homoeolog losses observed here. That is, through
homoeologous recombination in early generations fol-
lowing polyploid formation, genome reshuffling and
gene loss could act to stabilize the new polyploid genome
[63]. Perhaps in Tragopogon a combination of factors acts
over time to stabilize the new polyploid genomes. For
example, some chromosomal changes could happen
immediately following polyploid formation, with
homoeolog loss acting gradually over successive genera-
tions. The study of additional genes and comparisons
with synthetic T. miscellus lineages [62] over several gener-
ations will be important for establishing the overall pat-
tern of genome change in this system.

Conclusion
Our survey of 13 homoeologous loci in individuals from
ten populations of Tragopogon miscellus represents the
most extensive survey of the fate of duplicate genes in allo-
polyploid genomes from independently formed natural
populations. In this species, loss of a parental homoeolog
has occurred for several loci in individuals from these
populations. Some loci are consistently maintained as
duplicates in all individuals from these populations.
Other genes consistently show evidence of homoeolog
loss across populations of independent origin; signifi-
cantly, the T. dubius homoeolog is typically lost. Hence,
some aspects of genome evolution appear to have been
repeated in these new polyploids. In these young (~40
generations) allopolyploids, genomic incompatibilities
may be resolved, in part, through loss of a parental
homoeolog for some loci. As polyploidy and genome
downsizing are recurrent processes in many lineages,
other polyploid groups should be investigated to deter-
mine if similar patterns emerge for the loss and retention
of genes duplicated by polyploidy.







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T pratensis T miscellus


Spangle






Oakesdale






Garfield


T dubius


t t


Moscow Pullman


Figure 3
nrDNA variation in populations of Tragopogon miscel-
lus. Most individuals show genomic digest profiles of T. prat-
ensis nrDNA copies > T. dubius nrDNA copies, although a
few individuals show the opposite pattern, and some individ-
uals have lost a parental locus entirely (indicated by an
arrow).


Methods
Plant material and population sampling
Mature fruiting heads of Tragopogon dubius, T. miscellus,
and T. pratensis were collected from multiple individuals
from several populations in Washington and Idaho, USA,
in July 2005 (Table 1). Seeds were germinated in 11.4 cm
pots in a glasshouse at the University of Florida (Gaines-
ville, FL, USA) under standard conditions. Material from
Pullman, Washington, and Moscow, Idaho, was utilized
from a previous study [59].

In total, we included ten populations of T. miscellus, four
populations of T. pratensis, and nine populations of T.
dubius (Table 1). Our sampling strategy was intended to
survey as many individuals and populations from the Pal-
ouse as possible. Similarly, we recognize that sympatric
diploid populations may not represent the progenitor
genotypes for a particular local polyploid population


(although they typically do; Symonds et al. unpublished).
Therefore, we wanted to survey as many diploid individu-
als as possible to screen for potential variation in the loci
examined. The number of populations and number of
individuals from the diploid populations included in the
study differed because of changes in population dynamics
since the formation of the Tragopogon polyploids [70]. For
example, while once locally common, T. pratensis has
become sparse in the Palouse over the last several decades
[58] and is not always found in the vicinity of T. miscellus
populations (Table 1). Nevertheless, data accumulated
from previous studies [23,57] indicate that very little
genetic variation exists within and between populations
of T. pratensis in the Palouse. On the other hand, T. dubius
is more widely distributed [58] and harbors more genetic
variation than does T. pratensis [57]. Of the two diploid
parents of T. miscellus, T. dubius is more likely to exhibit
variation in the genes examined.

Genomic CAPS analysis
To determine if parental homoeologs were maintained or
lost in the Tragopogon miscellus individuals from inde-
pendently formed populations, we used genomic cleaved
amplified polymorphic sequence (CAPS) analysis [71].
Leaf material was harvested from seedlings and DNA
extracted following a modified CTAB protocol [721. For
two of the three loci not previously analyzed (cryl and
TDF27.10), primers were designed from Tragopogon dubius
sequences using Primer3 [73]. The cryl sequence origi-
nated from an EST library of T. dubius (Tdu01-
6MS1_Dll.e), and the TDF27.10 (TDF stands for tran-
script-derived fragment) sequence was derived from a pre-
vious cDNA-AFLP study [59]. Primer sequences for these
two loci were cryl- F: 5'-AATGGTTCCCAGTITGACCA-3',
cryl-lR: 5-GGCAAAGTITTACCCGGTIT-3'; TDF27.10F:
5'-CATTCATGCAACCAACCAAG-3', TDF27.10R: 5'-
CITCGGACTTCCITCAGCAC-3'. These primers were used
to amplify genomic DNA from T. dubius and T. pratensis
with the aim of identifying sequence polymorphisms that
could distinguish the homoeologs in T. miscellus.
Genomic amplifications were conducted in a 25 iL vol-
ume with 50 ng template, 10x Thermopol buffer (New
England Biolabs, Ipswich, MA, USA), 0.4 mM dNTPs, 0.2
jiM each primer, and 0.5 unit Taq polymerase (New Eng-
land Biolabs). Thermal cycling conditions were as follows:
94 C for 2 min, followed by 35 cycles of 94 C for 30 sec,
52-540C for 30 sec, 720C for 1 min, and a final 5-min
extension at 720C. Products were separated on a 1.5%
agarose gel, stained with ethidium bromide, and visual-
ized by UV on a transilluminator. PCR products were pre-
pared for sequencing by adding 5 units of Exonuclease I
(Fermentas, Glen Burnie, MD, USA) and 0.5 unit Shrimp
alkaline phosphatase (Fermentas) and treating them at
37 C for 30 min followed by 80 C for 15 min. Cleaned
products were separated on an ABI 3770 following the


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manufacturer's recommendation (Applied Biosystems,
Foster City, CA, USA). Sequences were edited in
Sequencher version 4.7 (Gene Codes, Ann Arbor, MI,
USA) and deposited in GenBank under accession num-
bers FI770374-FI770377. To determine if sequence poly-
morphisms between the two diploid parental sequences
occurred at a restriction site, the sequences were analyzed
with dCAPS Finder 2.0 [74]. For cryl, the restriction
enzyme Acil cut the T. pratensis 382-bp product into two
fragments (232 and 150 bp), while the T. dubius product
remained uncut (385 bp). For TDF27.10, MseI cut the T.
pratensis PCR product into two fragments (216 and 83
bp), and the T. dubius product remained uncut at 299 bp.
Restriction digestions for both markers were conducted in
a 10-il volume with lx buffer (New England Biolabs), 1
iL PCR product, 5-10 units of enzyme (New England
Biolabs), and 100 lg/ml Bovine Serum Albumin (when
required). The reactions were allowed to incubate at the
temperature specified by the supplier for three hours.
Digested products were separated on a 2% agarose gel,
stained with SybrGold (Molecular Probes Inc., Eugene,
OR, USA), and visualized on a transilluminator. Once the
utility of these markers was established, the remaining
individuals of T. dubius, T. miscellus, and T. pratensis were
PCR-amplified and digested in the same manner. For
nrDNA repeats in T. miscellus, genomic CAPS analysis was
conducted as described in Kovarik et al. [65].

For the previously analyzed markers (TDF7, TDF17.4,
TDF36.3, TDF44, TDF46, TDF62, TDF72.3, TDF74,
TDF85, and TDF90), genomic amplification and restric-
tion digestion were conducted as described in Tate et al.
[59]. The Moscow and Pullman populations of T. miscellus
were the subject of a previous study [59]; those data are
combined here with data for three new loci (cryl,
TDF27.10, and nrDNA).

To verify that the observed homoeolog losses based on
CAPS analysis were not the result of a point mutation at
the diagnostic restriction site in T. miscellus post-polyploid
formation, PCR products were sequenced for all individu-
als of Tragopogon miscellus that showed loss of a homoeol-
ogous fragment. For a given individual, a homoeolog loss
was scored only when the sequence data verified the pat-
tern from the CAPS gel analysis (i.e., no sequence poly-
morphisms were detected in the chromatogram either at
the diagnostic restriction site or at other positions where
T. dubius and T. pratensis differ). These same criteria
applied for nrDNA loci. However, when the intensity of
the digested parental fragments differed in the CAPS gel,
the nrDNA patterns were scored as P > D or D > P to reflect
differing copy numbers in the allopolyploid individuals
[65,66]. For all loci, when a loss was determined, we
assumed that both alleles of a parental homoeolog were
lost. In cases where one allele of a homoeolog was lost,


CAPS analysis might not detect these losses. Furthermore,
identical patterns of loss in individuals from the same
population may be the result of shared ancestry. There-
fore, total losses from a single population were tabulated
as both minimum and maximum number of losses.

Authors' contributions
JAT designed the study, collected and analyzed genomic
CAPS data, and drafted the manuscript. PJ and KAS con-
ducted genomic CAPS analyses. PSS and DES helped to
design the study and contributed to drafting the manu-
script. All authors read and approved the final manu-
script.

Additional material


Additional file 1
Summary of homoeolog losses in Tragopogon miscellus. A '+'symbol
indicates that no losses were detected in a population for a particular gene.
'D' or 'P' 1..11.., ,,, an individual number indicates the parental homoe-
olog lost (D = T. dubius; P= T. pratensis) from that individual.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-9-80-Si.doc]


Acknowledgements
This work was supported by a grant from the National Science Foundation
(MCB0346437) to DS, PS, and JT and a grant from the Massey University
Research Fund toJT. We thank V. Symonds and two anonymous reviewers
for comments on the manuscript and the many individuals who have helped
with field work, including S. Brunsfield, B. Petersen, and R. Brooks.

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