Transcriptomic shock generates evolutionary novelty in a newly formed, natural allopolyploid plant.
ABSTRACT New hybrid species might be expected to show patterns of gene expression intermediate to those shown by parental species. "Transcriptomic shock" may also occur, in which gene expression is disrupted; this may be further modified by whole genome duplication (causing allopolyploidy). "Shock" can include instantaneous partitioning of gene expression between parental copies of genes among tissues. These effects have not previously been studied at a population level in a natural allopolyploid plant species. Here, we survey tissue-specific expression of 144 duplicated gene pairs derived from different parental species (homeologs) in two natural populations of 40-generation-old allotetraploid Tragopogon miscellus (Asteraceae) plants. We compare these results with patterns of allelic expression in both in vitro "hybrids" and hand-crossed F(1) hybrids between the parental diploids T. dubius and T. pratensis, and with patterns of homeolog expression in synthetic (S(1)) allotetraploids. Partitioning of expression was frequent in natural allopolyploids, but F(1) hybrids and S(1) allopolyploids showed less partitioning of expression than the natural allopolyploids and the in vitro "hybrids" of diploid parents. Our results suggest that regulation of gene expression is relaxed in a concerted manner upon hybridization, and new patterns of partitioned expression subsequently emerge over the generations following allopolyploidization.
- SourceAvailable from: Graham John King[Show abstract] [Hide abstract]
ABSTRACT: Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.Science 08/2014; 345(6199):950-3. · 31.48 Impact Factor
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ABSTRACT: Gene products from organellar genomes (chloroplast and mitochondria) can interact with nuclear gene products in a variety of pathways. Most protein complexes consisting of multiple subunits have proteins encoded by both nuclear and organellar genomes. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) is involved in carbon dioxide fixation in the Calvin cycle and has two subunits: a large subunit (rbcL) encoded by the chloroplast genome and a small subunit (rbcS) that is encoded by the nuclear genome. How these subunits interact in hybrids and allopolyploids is unknown. In order to explore the inheritance and expression of both maternal and paternal homeologs of the nuclear-encoded rbcS, and to determine which of the nuclear homeologs of rbcS is interacting with the chloroplast-encoded subunit rbcL, rbcS was amplified from three diploid species of Tragopogon (T. dubius, T. pratensis and T. porrifoilius), which are the parents of the allopolyploids T. mirus and T. miscellus. Sequence analysis of rbcS from the diploid parents showed the presence of seven single nucleotide polymorphisms (SNPs). Based on this variation among the parents, progenitor-specific markers are being designed, which will be used to amplify rbcS homeologs from reciprocally generated synthetic polyploid lines of T. mirus and T. miscellus. We expect that both parental copies of rbcS will be present in the genomes of the synthetic polyploids, but that the maternal homeolog of rbcS will be expressed and will interact with rbcL from that same progenitor.International Plant and Animal Genome Conference XXI 2013;
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ABSTRACT: Hybridization coupled with whole-genome duplication (allopolyploidy) leads to a variety of genetic and epigenetic modifications in the resultant merged genomes. In particular, gene loss and gene silencing are commonly observed post-polyploidization. Here, we investigated DNA methylation as a potential mechanism for gene silencing in Tragopogon miscellus (Asteraceae), a recent and recurrently formed allopolyploid. This species, which also exhibits extensive gene loss, was formed from the diploids T. dubius and T. pratensis.BMC Genomics 08/2014; 15(1):701. · 4.04 Impact Factor
Current Biology 21, 551–556, April 12, 2011 ª2011 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2011.02.016
Transcriptomic Shock Generates
Evolutionary Novelty in a Newly Formed,
Natural Allopolyploid Plant
Richard J.A. Buggs,1,2,3,* Linjing Zhang,1,2,4
Nicholas Miles,1,2Jennifer A. Tate,5Lu Gao,6Wu Wei,6
Patrick S. Schnable,6W. Brad Barbazuk,1,7
Pamela S. Soltis,2,7and Douglas E. Soltis1,7
1Department of Biology, University of Florida, Gainesville,
FL 32611, USA
2Florida Museum of Natural History, University of Florida,
Gainesville, FL 32611, USA
3School of Biological and Chemical Sciences,
Queen Mary University of London, London E1 4NS, UK
4School of Life Sciences, Shanxi Normal University,
1 Gongyuan Street, Linfen City 041000, Shanxi Province,
People’s Republic of China
5Massey University, Institute of Molecular Biosciences,
Palmerston North 4442, New Zealand
6Center for Plant Genomics, Iowa State University, Ames,
IA 50011, USA
7Genetics Institute, University of Florida, Gainesville,
FL 32610, USA
New hybrid species might be expected to show patterns of
gene expression intermediate to those shown by parental
species [1, 2]. ‘‘Transcriptomic shock’’ may also occur, in
which gene expression is disrupted; this may be further
modified by whole genome duplication (causing allopoly-
ploidy) [3–16]. ‘‘Shock’’ can include instantaneous partition-
ing of gene expression between parental copies of genes
among tissues [16–19]. These effects have not previously
been studied at a population level in a natural allopolyploid
plant species. Here, we survey tissue-specific expression
of 144 duplicated gene pairs derived from different parental
species (homeologs) in two natural populations of 40-gener-
ation-old allotetraploid Tragopogon miscellus (Asteraceae)
plants. We compare these results with patterns of allelic
expression in both in vitro ‘‘hybrids’’ and hand-crossed
F1 hybrids between the parental diploids T. dubius and
T. pratensis, and with patterns of homeolog expression in
synthetic (S1) allotetraploids. Partitioning of expression
was frequent in natural allopolyploids, but F1hybrids and
S1allopolyploids showed less partitioning of expression
than the natural allopolyploids and the in vitro ‘‘hybrids’’ of
diploid parents. Our results suggest that regulation of
gene expression is relaxed in a concerted manner upon
hybridization, and new patterns of partitioned expression
the generations following
Results and Discussion
Variation in Relative Gene-Copy Expression
Changes in patterns of parental gene expression are
frequently observed in hybrids and allopolyploids; this
phenomenon has been termed transcriptomic shock [3–13,
16]. Study of natural transcriptomic shock in the wild is
hampered by the rarity of known, recently formed polyploid
species that still co-occur with their parental species.
Tragopogon miscellus (Tm) (Asteraceae) is a young natural
allotetraploid species that formed multiple times during the
past 80 years in the NW USA from the diploids T. dubius (Td)
and T. pratensis (Tp), which were introduced from Europe
and remain extant in areas of polyploid formation . Allo-
polyploid populations formed reciprocally, with an immediate
and conspicuous phenotypic difference: populations with Td
as the maternal parent have inflorescences with long ligules
with short ligules (see Figure 1A).
To explore transcriptomic shock in the formation and early
generations of allopolyploidy in T. miscellus populations, and
unravel the effects of hybridization and whole-genome dupli-
sion in: the diploid parents Td and Tp, as 1:1 mixes of cDNA
from five pairs (designating these as in vitro ‘‘hybrids,’’
showing simple additivity of parental gene expression); true
synthetic diploid F1hybrids (n = 6); synthetic first-generation
(S1) allopolyploids (n = 6); and in two naturally occurring Tm
allopolyploid populations of reciprocal origin (n = 10+8). We
examined expression of 144 gene pairs: 126 with quantitative
Sequenom MassARRAY allelotyping assays previously devel-
oped using 454 and Illumina sequencing data , and 18
using previously developed qualitative cleaved amplified poly-
morphic sequence (CAPS) assays [22–24]. We examined the
expression of both copies of these genes in transcriptomes
of up to seven tissues of each of 40 plants. Example data for
four genes are shown in Figure 2.
For many genes, we found cases of lack of expression of
a homeolog in all tissues of individual plants (e.g., gene
07259_1424 in four short-liguled allopolyploid plants, Fig-
ure 2A). These cases of nonfunctionalization  were
excluded from our analysis of tissue-specific transcriptomic
shock. They are likely a consequence of a genomic change.
Consistent with this hypothesis a Sequenom analysis on
genomic DNA of a sample (n = 168) of these cases in Tm
showed that 89.9% had one homeolog missing from the
genome. This corresponds with previous studies showing los-
ses of homeologs in Tm [22–24].
To understand differences among the plant groups in the
variability of relative expression of Td and Tp genes, we
calculated the standard deviation of expression for every Se-
quenom gene assay within each tissue sample transcriptome
and used these statistics to generate the boxplots shown in
Figure 1B. The five plant groups showed a significant differ-
ence (Wilcoxon test X2= 62.49, p < 0.0001). Nonparametric
comparisons of each pair of plant groups using the Wilcoxon
method showed a small difference between the F1hybrids
and S1allopolyploids (Z = 3.07, p < 0.0021), no significant
difference between diploid in vitro ‘‘hybrids’’ and the two
populations of natural allopolyploids (p > 0.5 in all three
comparisons), and a significant difference in every other
comparison (p < 0.0001 in six comparisons). We also made
a similar comparison of the variation of tissue samples within
genes for each group (Figure 1C), following the same method
as that above, except that we standardized the standard
deviation for each gene within each plant group by the
average standard deviation shown by each gene across all
samples. This showed a similar pattern: the five plant groups
differed significantly (Wilcoxon test X2= 278.03, p < 0.0001),
due to highly significant differences (p < 0.0001) between all
pairs of groups except F1s and S1s (Z = 2.79, p = 0.0052)
and the two natural Tm populations (Z = 2.85, p = 0.0043).
The high standard deviation shown by diploid in vitro
‘‘hybrids’’ among tissues within genes (Figure 1C) may have
been increased by small pipetting errors that shifted the over-
all ratio of the two transcriptomes in each sample away from
1:1, but this factor would not affect the measurement of
standard deviation among genes within tissue samples
These results suggest that F1hybrids have lower variation of
is observed when cDNAs of the parental diploids are mixed (in
vitro ‘‘hybrids’’). Yet after 40 generations following allopoly-
ploidization, variation in relative Td:Tp expression is observed
among plants and tissues in natural Tm populations. This
difference in variation of expression between the groups
occurred despite the fact that the six F1and six S1plants
n = 33
n = 40n = 41
n = 69
n = 53
n = 126
n = 126
n = 126
n = 117
n = 120
Diploid in vitro
Figure 1. Transcriptomic Shock in Tragopogon
(A) Examples of inflorescences from the groups
of Tragopogon plants sampled. Identity of
plants is shown by abbreviations used in the
main text (Tp = T. pratensis, Td = T. dubius,
Tm = T. miscellus, F1= diploid hybrid between
Tp and Td, S1 = first-generation allopolyploid
produced between Tp and Td).
(B) Variation in quantitative results for relative
allele/homeolog expression (excluding cases of
apparent nonfunctionalization) from Sequenom
data, based on 126 genes; showing boxplots of
standard deviation among genes within samples.
Number of samples within each group is shown
above boxplots; box fill colors indicate plant
groups that differed with a significance greater
than p = 0.0001.
(C) Variation in quantitative results for relative
allele/homeolog expression (excluding cases of
apparent nonfunctionalization) from Sequenom
data,showing boxplots ofstandardized standard
deviation among samples within genes. Number
of genes analyzed in each group is shown above
differed with a significance greater than p =
0.0001. The standard deviation of relative gene
expression among samples in diploid in vitro
‘‘hybrids’’ may have been increased by pipetting
(D) Mean percentage of tissues showing tissue-
specific silence of alleles/homeologs in a series
of Tragopogon diploids and polyploids for 126
genes assayed using Sequenom and 18 genes
assayed using CAPS. Error bars show standard
were derived from a total of eight
unique crosses (i.e., between different
parental combinations, see Table S1
available online), whereas the 18 Tm
plants studied were from two natural
Tissue-Specific Silence in Gene-Copy Expression
Of particular interest are cases where relative expression of
gene copies is so skewed that one copy is not detected at all
in the transcriptome of a tissue; we call this tissue-specific
silence (TSS). Taking Sequenom and CAPS assays together,
the mean percentage of assays showing TSS per gene was
highest in the diploid in vitro ‘‘hybrids’’ (Figure 1D; in Wilcoxon
matched pair analysis for diploid data compared with the four
other groups, TSS was more frequent for diploids with p <
0.0001 in all four comparisons except for that with short-lig-
uled Tm, where p = 0.0243). F1hybrids and S1allopolyploids
had the lowest frequency of TSS and these two plant groups
did not differ significantly. The two natural allopolyploid
populations both had more frequent TSS than the F1s and
S1s (p < 0.0001), and TSS differed in frequency between the
two natural Tm populations (p = 0.0416). In no group was there
a significant difference in the frequency of TSS of Td versus Tp
homeologs. Thus, it appears that in diploid parental species it
tissues examined, but these genes are globally activated by
hybridization, such that copies from both parents are
Current Biology Vol 21 No 7
expressed. In many genes, tissue-specific silencing of one ho-
meolog occurs in the first 40 generations of allopolyploidy
(while total silencing of both copies of a gene in the same
tissue is rare). The Sequenom and CAPS assays gave the
ences in sample size and genes sampled (Figure 1D).
We then asked whether the same genes showed TSS in the
diploid in vitro ‘‘hybrids,’’ F1hybrids, S1allopolyploids, and
natural allopolyploids. There was a significant correlation
between the percentage of TSS shown by individual genes in
the diploid in vitro ‘‘hybrids’’ and in the natural allopolyploids
(R2=0.307, F = 48.23, p< 0.0001),for 111genes assayed using
Sequenom, which were expressed in at least one tissue in
every diploid in vitro ‘‘hybrid.’’ There was a weaker correlation
between F1s and natural allopolyploids (R2= 0.097, F = 11.73,
p < 0.0009) and between S1s and natural allopolyploids (R2=
0.106, F = 12.92, p < 0.0005). Therefore, the same genes
allopolyploids despite loss of TSS upon hybridization. It must
be emphasized that in the diploids TSS involves total nonex-
pression of that gene in a tissue, whereas in allopolyploids
exhibiting TSS, the expression of one homeologous gene
copy is retained.
While there are general trends across all genes, five genes
that we studied showed patterns that are found with some
regularity among plants after hybridization and/or whole-
genome duplication (Figures 2B–2D; Figure S2). For example,
gene15567_808 (Figure 2C),a putative haloacid
P S TOAC L
P S TOAC L P S TOAC L
no data Tp expression only
Td expression only
Equal expression of
Tp and Td genes
P S TOAC L
Figure 2. Examples of Results for Individual
(A–D) Tissue-specific relative expression of gene
copies derived from T. dubius and T. pratensis in
diploid in vitro ‘‘hybrids,’’ F1hybrids, S1synthetic
allopolyploids, and natural populations of T. mis-
cellus. Results for four example gene pairs are
shown, measured using Sequenom analysis (the
full data set of 126 Sequenom assays and 18
CAPS gene assays are shown in Figure S1).
Columns represent tissues, and lines represent
plants. Columns are grouped by gene, and rows
are in plant groups. Tissue abbreviations are as
follows: P = Phyllary, S = Style, T = Stigma, O =
Ovary, A = Pappus, C = Corolla, L = Leaf. Colors
show relative tissue-specific expression of Td
and Tp gene copies (see legend). Cells joined
by diagonal gray lines represent groups of
tissues that were assayed together. The Arabi-
dopsis thaliana homologs of the four genes
shown are: (A) Histidine kinase 3; (B) a remorin
family protein; (C) a haloacid dehalogenase-like
hydrolase family protein; (D) a metal ion binding
protein, showed zero TSS in all but one
F1 hybrid, but the pattern of tissue-
specific expression found in this one
hybrid – Td bias in the stigma, style
and corolla – was also found in most of
the synthetic and natural allopolyploids
examined. Two genes showed patterns
of TSS that are found across all groups
(diploid through natural allopolyploids)
(Figures S2D and S2E). Two genes
expression that were to some extent
found in diploid in vitro ‘‘hybrids,’’ F1hybrids, and S1allopoly-
ploids but absent in natural Tm populations (Figure 2D; Fig-
of the Td copy in the stigma and corolla of diploid in vitro
‘‘hybrids,’’ F1and S1plants, but this expression pattern was
not present in the majority of the natural 40-generation-old
Transcriptomic Shock as a Reduction in Tissue-Specificity
of Gene Expression
The general trends of our results suggest that transcriptomic
shock upon hybridization  includes the activation of allele/
homeolog expression in all tissues, causing a loss of tissue-
specific expression patterns seen in the diploid parents. Such
activation has been shown for repetitive and transposable
elements [26–29], but has seldom been considered in terms
of the tissue-specific activation of protein-coding genes. A
rare example is in the derepression of Polycomb group
proteins in hybrid endosperm . Activation of homeologs
hasalso been found occasionally incotton F1hybrids and allo-
polyploids by Chaudhary et al. , who termed it ‘‘transcrip-
tional neofunctionalization.’’ Here, we show this to be wide-
spread in Tragopogon.
Our findings may fit a newly proposed transcriptomic shock
scenario in which activity of small interfering RNA molecules,
which influence gene expression, is temporarily lost in F1
hybrids and early allopolyploids, but restored as subsequent
Transcriptomic Shock in Tragopogon miscellus
generations stabilize [31, 32]. Novel expression in hybrids
could also bedue to trans-activation between the two parental
genomes, whereby a regulatory element produced by one
parental genome activates gene expression in the other
Previous studies in domesticated cotton allopolyploids,
using methods that distinguish among tissues and between
homeologs, have led to the conclusion that allopolyploidiza-
of ancestral expression patterns’’ [7, 14–16, 33]. Alterations in
gene expression have also occurred upon allopolyploidization
in Arabidopsis [4, 5, 9, 12], wheat [3, 10, 13, 34, 35], and Bras-
sica . Our results shed new light upon these disrupted
expression patterns in a wild, nonmodel plant.
Our results might appear to contrast with those of a study in
the young natural allohexaploid Senecio cambrensis, where
hybridization was found to be the most influential step with
respect to the transcriptome . Gene expression changes
that occurred over five generations of a synthetic allopolyploid
line and over the w100-year existence of the natural allopoly-
ploid Senecio cambrensis were smaller than those that
occurred at the time of hybridization . However, the Senecio
experiment did not distinguish between homeologs or among
tissues, and we therefore do not know whether expression
changes occurring upon hybridization were due to loss of
tissue-specific expression patterns. Regardless, the results
for Senecio might be expected to differ from those reported
here because in Senecio the hybridization step was between
a diploid and a tetraploid, not between two diploids as in
Tragopogon. Hence, in Senecio the F1hybrids were triploids,
which are likely to have genome dosage effects .
Frequency of Expression Subfunctionalization
specific silence of homeologs, that may be indicative of rapid
subfunctionalization, the partitioning of multiple functions of
an ancestral gene between its duplicate descendents . In
the in vitro ‘‘hybrids’’ of Tragopogon diploid transcriptomes,
we found 26 cases of reciprocal TSS, where a gene was not
expressed by one diploid parent in a certain tissue but not ex-
pressed by the other diploid parent in another tissue (3.97% of
655 plant 3 gene combinations examined that did not show
apparent nonfunctionalization). In contrast, we found just six
cases of reciprocal TSS in F1hybrids (0.74% of 807 plant 3
gene combinations), zero cases in S1s (0% of 707), and eight
cases in natural allopolyploids (0.37% of 2152; for an example
identified using CAPS, see Figures S2F and S3). Thus, the acti-
vation of genes by transcriptomic shock seems to cause lower
expression were strictly additive of that in parental diploids.
Models of subfunctionalization involving tissue-specific
expression tend to assume identical expression patterns of
ancestral and newly duplicated genes . Under such a
condition, reciprocal TSS has been shown to occur in F1
domesticated cotton . However, the data presented here
sion are common in the diploid parents of Tm. The data also
suggest that the activation of genes by transcriptomic shock
seems to cause lower reciprocal TSS after hybridization than
we might expect if gene expression were strictly additive of
that in parental diploids. This suggests that instantaneous
subfunctionalization is the exception, not the norm, in the
evolution of gene expression in new allopolyploids.
The patterns of transcriptomic shock shown here are likely to
affect profoundly the evolutionary success of the natural pop-
ulations of allopolyploid Tragopogon miscellus. If genes in the
diploid parental species have finely tuned patterns of tissue-
specific expression, disruption of these patterns could have
negativefitnessconsequences inanunchanging environment,
but might be highly beneficial after a long-range migration,
such as that undergone by Tragopogon species when intro-
duced to the NW USA from Europe [37, 38]. Current models
for the evolution of genetic complexity and diversity rely
upon gene and genome duplication [19, 39–42]; divergence
in the location or timing of gene expression is likely to be
one possible early step in the functional divergence of dupli-
cated genes [17, 42, 43]. In light of these models, the data pre-
sented here suggest that rather than being a saltational leap to
a new fitness peak, allopolyploidization, for the majority of
genes, provides the genetic and transcriptomic resources for
novel trajectories of evolution, by activation of gene expres-
sion (as well as by genetic redundancy at all loci). Even though
allopolyploid formation inevitably involves a genetic bottle-
neck, subsequent generations display diverse patterns of
tissue-specific gene expression, whose phenotypic effects
may be exposed to natural selection and thus gradually lead
the new allopolyploid species to new adaptive peaks.
Seeds were collected from natural populations of T. miscellus (Tm) of inde-
pendent and reciprocal origin: the short-liguled form (with T. pratensis [Tp]
as the maternal parent) from Moscow, ID and the long-liguled form (with
T. dubius [Td] as the maternal parent) from the only known natural popula-
tion, which is found nearby in Pullman, WA (for Soltis and Soltis collection
numbers, see Table S1). Samples of Td were obtained from Pullman, WA;
Palouse, WA; and Spokane, WA, and those of Tp were obtained from Mos-
cow, ID and Spangle, WA (see Table S1). These seeds were grown in the
greenhouse (at Washington State University, Pullman, WA) and allowed to
self-fertilize for one generation. After self-fertilization, seeds were germi-
nated and grown under controlled conditions in a greenhouse at the Univer-
sity of Florida (Gainesville, FL). Five plants of each diploid parent and ten
plants of each Tm population were used in the experiment.
Six F1 hybrids and six first-generation synthetic allopolyploids (S1s)
formed between Td and Tp, were grown in the same greenhouse . The
crosses that gave rise to these synthetic lines are shown in Table S1. Five
of the F1s and S1s were Tp 3 Td crosses, and one F1and S1were a Td 3
Tp cross. The latter cross had a much lower success rate in terms of viable
progeny than the former . The synthetic allopolyploids were produced
using colchicine treatment of F1hybrids . These hybrids were different
individuals to those included as F1hybrids in this experiment. In only one
case was an F1from exactly the same parental diploid pair as an S1, due
to the difficulties of successfully inducing whole genome duplication with
colchicine and nurturing the new allopolyploid to flowering.
Tissue Dissection and RNA Extraction
Leaf and inflorescence tissue was collected from all plants and flash frozen
tissue types: corolla, pappus, ovary, stigma, style, and phyllary. Sometimes
the quantities of stigma and style tissues available were not sufficient to
provide the quantity of RNA needed, so the two tissues were combined.
Tissues were ground with a mortar and pestle at 280?C, and RNA was
extracted using the RNAeasy kit with on-column DNase digestion from
QIAGEN (Valencia, CA). First-strand cDNA synthesis was carried out on
500 ng of RNA using Superscript II reverse-transcriptase (Invitrogen, Carls-
bad, CA) and polyT primers. In the CAPS analyses, cDNAs from diploids
Current Biology Vol 21 No 7
the Sequenom analyses, cDNAs for specific tissues were combined from Tp
and Td to make in vitro ‘‘hybrids,’’ allowing quantification of the relative
expression levels of homologs.
We wished to characterize the expression of genes derived from Td and Tp
in tissues of the five plant groups. In diploid hybrids these genes are present
as alleles, butin allopolyploids they are present as homeologs. Allele/home-
olog expression was analyzed in the tissue transcriptomes isolated above,
using 18 CAPS markers (including one set of homeolog-specific PCR
primers) and 128 Sequenom MassARRAY assays. The CAPS assays were
carried out on tissue transcriptomes of all the plants mentioned above,
and the Sequenom assays were carried out on the same set of transcrip-
tomes with the omission of those from two short-liguled Tm plants. Below
we outline how the assays were implemented.
cDNA using 10 primer sets from Tate et al. , seven primer sets from
Buggs et al. , and one primer set from Tate et al. . Putative identities
of these genes are provided in the original papers cited. The PCR products
were digested using the enzymes and conditions specified in the above
publications, which cut only one of the two alleles/homeologs, due to a
single nucleotide difference in the enzyme cut site between the two
alleles/homeologs. CAPS and allele/homeolog-specific PCR products
were visualized on high-resolution 4% Metaphor agarose (Lonza, Allendale,
NJ) gels. Qualitative results were scored as 0 for expression of both alleles/
homeologs, 1 for expression of only the Tp allele/homeolog and 21 for the
expression of only the Td allele/homeolog.
Sequenom MassARRAY assays were developed as described in Buggs
et al. . In this technology, a short section of DNA containing a SNP is
extension reaction over the SNP being assayed, using nucleotides of modi-
fied mass. The different SNP-alleles therefore produce oligonucleotides
with mass differences that can be detected using Matrix-Assisted Laser
Desorption / Ionization Time-Of-Flight mass spectrometry. This provides
a trace in which peak heights correspond to the frequency of each different
oligonucleotide, allowing measurement of the relative frequency of two
SNP-alleles at one locus. This can be used to measure quantitative gene
mix if primers are designed to give unique mass ranges for each single-
base-extended primer. We identified single base-pair differences between
homologous genes in Td and Tp using 454 and Illumina sequencing and de-
signed Sequenom assays to measure the expression of 139 gene pairs as
alleles/homeologs in the plant groups in this study .
The Sequenom assays were carried out at Iowa State University. The
accuracy of our assays in measuring relative expression of alleles/
homeologs was checked by running three replicates of a T. miscellus leaf
transcriptome that had been Illumina deep-sequenced . Where the three
replicates gave a result in agreement with the Illumina count data, the
assays were deemed to be accurate. Where there were insufficient Illumina
count data, the accuracy of the assays was checked using genomic DNA
from six F1hybids between Td and Tp and mixes of Td and Tp genomic
DNA in ratios of 1:3, 1:1, and 3:1. Where the majority of these gave an ex-
pected result, the assays were deemed to be accurate. By the first method,
111 assays were found to be accurate and a further 17 by the second
method, giving a total of 128 working assays. Two of these were not used:
one because one diploid was polymorphic, and one because the assay
failed to work on a homeolog that had not been covered in the Illumina run.
Where one homeolog was found to be silenced in all tissues of an indi-
vidual Tm plant by Sequenom analysis, genomic DNA was extracted from
that plant using a modified CTAB protocol , and Sequenom analysis
carried out, to test for genomic loss of that homeolog. Only 73 of the Seque-
nom assays that had worked on cDNA worked consistently on genomic
DNA, probably due to intron splicing.
Raw Sequenom MassARRAY allelotyping data consist of areas under
verted the data to quantitative measures of relative expression of the two
alleles/homeologs, where 0 represents equal expression of both alleles/
homeologs, 1 represents expression of only the Tp allele/homeolog and
21 represents the expression of only the Td allele/homeolog. Results
from both CAPS and Sequenom gene expression analyses on tissue-
specific cDNAs were clustered separately using Cluster 3.0 , using a
hierarchical centered Pearson correlation with average linkage. The two
clustered datasets were visualized in Java Treeview 1.1.4r3  (Figure S1).
To compare variation in relative gene expression among the plant groups
(parental diploid in vitro ‘‘hybrids,’’ F1hybrids, S1allopolyploids, long-lig-
uled natural allopolyploids, and short-liguled allopolyploids), we calculated
the standard deviation of expression both among genes within tissue tran-
scriptomes (Figure 1B) and among tissue transcriptomes within genes (Fig-
ure 1C). The former statistics did not include variation caused by small
pipetting errors in the production of the diploid in vitro mixes, whereas the
latter inevitably did. Differences between the mean standard errors of
each group were tested in JMP using Wilcoxon tests among all groups
and nonparametric comparisons for each pair of groups using the Wilcoxon
For the data sets from both CAPS and Sequenom methods we calculated
the mean percentage of tissue-specific assays from each of the five plant
groups that displays tissue-specific silence (TSS; Figure 1D). Only a 1 or
21 in the Sequenom result for an assay was scored as nonexpression of
one gene copy. If all Sequenom assays for a plant yielded a result of all 1
or all 21, except for a single tissue, we counted this as a putative nonfunc-
tionalization, as comparison with results from assays on genomic DNA
showed that occasional false positives occurred, spuriously showing slight
expression of one homeolog in only one tissue where that homeolog had
been lost from the genome. The TSS data were analyzed in JMP using Wil-
coxon matched pair analysis and bivariate fits.
Supplemental Information includes three figures and one table and can be
found with this article online at doi:10.1016/j.cub.2011.02.016.
Funding for this research was provided by the University of Florida and NSF
grants DEB-0614421 to P.S. Soltis and D.S.S., MCB-0346437 to P.S. Soltis,
D.S.S., and J.A.T., DEB-0919254 to P.S. Soltis, D.S.S., and W.B.B., and
DEB-0919348 to P.S. Schnable. R.J.A.B. has been supported since March
2010 by NERC Fellowship NE/G01504X/1. We thank two anonymous
reviewers for helpful comments.
Received: January 6, 2011
Revised: February 10, 2011
Accepted: February 14, 2011
Published online: March 17, 2011
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