Patterns of homoeologous gene expression shown by RNA sequencing in hexaploid bread wheat

Abstract

Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoloci, each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat cytogenetic resources (chromosome substitution 'nullisomic-tetrasomic' lines) with next generation deep sequencing of gene transcripts (RNA-Seq), to directly and accurately identify homoeologue-specific single nucleotide variants and quantify the relative contribution of individual homoeoloci to gene expression.
We discover, based on a sample comprising ~5-10% of the total wheat gene content, that at least 45% of wheat genes are expressed from all three distinct homoeoloci. Most of these genes show strikingly biased expression patterns in which expression is dominated by a single homoeolocus. The remaining ~55% of wheat genes are expressed from either one or two homoeoloci only, through a combination of extensive transcriptional silencing and homoeolocus loss.
We conclude that wheat is tending towards functional diploidy, through a variety of mechanisms causing single homoeoloci to become the predominant source of gene transcripts. This discovery has profound consequences for wheat breeding and our understanding of wheat evolution.

Full text

R E S E A R C H A R T I C L E Open Access
Patterns of homoeologous gene expression shown
by RNA sequencing in hexaploid bread wheat
Lindsey J Leach
1,2
, Eric J Belfield
1
, Caifu Jiang
1
, Carly Brown
1
, Aziz Mithani
1,3
and Nicholas P Harberd
1*
Abstract
Background: Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D
homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoloci,
each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat
cytogenetic resources (chromosome substitution ‘nullisomic-tetrasomic’lines) with next generation deep sequencing of
gene transcripts (RNA-Seq), to directly and accurately identify homoeologue-specific single nucleotide variants and
quantify the relative contribution of individual homoeoloci to gene expression.
Results: We discover, based on a sample comprising ~5-10% of the total wheat gene content, that at least 45% of
wheat genes are expressed from all three distinct homoeoloci. Most of these genes show strikingly biased expression
patterns in which expression is dominated by a single homoeolocus. The remaining ~55% of wheat genes are
expressed from either one or two homoeoloci only, through a combination of extensive transcriptional silencing and
homoeolocus loss.
Conclusions: We conclude that wheat is tending towards functional diploidy, through a variety of mechanisms
causing single homoeoloci to become the predominant source of gene transcripts. This discovery has profound
consequences for wheat breeding and our understanding of wheat evolution.
Keywords: Wheat, Wheat transcriptome, mRNA-Seq, Diploidization, Homoeologues, Polyploidy
Background
Whole genome duplication (WGD) by polyploidy is a
major driving force in the evolution of eukaryotes, par-
ticularly plants and fungi [1,2]. All flowering plants have
undergone at least one round of WGD [3]. Between 30-
80% of species are currently polyploids [4], while the rest
exist as paleopolyploids [5,6], having undergone a grad-
ual process of “diploidization,”or reversion to a diploid
state over evolutionary time. This process involves ex-
tensive genomic rearrangements, including the physical
loss of a large fraction of duplicate regions, and the ac-
cumulation of mutations that distinguish the sequences
of the duplicate ‘homoeologous’copies and contribute to
their functional divergence [7-9].
Many of our most important crop species are currently
either autopolyploid, for example potato and coffee, or al-
lopolyploid, for example pasta and bread wheats, oat, cot-
ton and canola. Allopolyploid plants often undergo major
changes in genome structure and function induced by the
“genomic shock”[10,11] occurring when two divergent ge-
nomes combine. For example, newly formed synthetic
wheats undergo changes in DNA methylation and rapid,
non-random elimination of up to 14% of genomic DNA se-
quence, which differentiates the homoeologous chromo-
somes and fosters the transition to diploid-like meiotic
behaviour [12-15]. In addition, newly formed hexaploid
wheat lines are frequently unstable, leading to aneuploid
plants with missing or extra chromosomes [16]. Other
major crops are paleopolyploids, including barley, maize
and rice, whose diploid genomes have been reduced to-
wards their ancestral chromosome number and gene
content through removal of duplicate copies, called homo-
eologues or homoeoloci, in a process of fractionation. Far
from being a random process, genes are commonly re-
moved preferentially from particular homoeologues, in
a process termed “biased fractionation”[17]. This bias
in gene densities between homoeologous subgenomes
has been observed in several paleopolyploid plants
* Correspondence: nicholas.harberd@plants.ox.ac.uk
1
Department of Plant Sciences, University of Oxford, Oxford, UK
Full list of author information is available at the end of the article
© 2014 Leach 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 credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Leach et al. BMC Genomics 2014, 15:276
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including Arabidopsis [18], maize [19], Brassica [20] and
soybean [21,22].
A hallmark feature of polyploid genomes is rapid and
novel change in gene expression, ranging from slight
alterations in the expression of homoeoloci through to
the complete absence of expression caused by epigenetic
homoeolocus silencing [23,24]. Studies of both natural
and synthetic polyploids have shown that homoeoloci fre-
quently make unequal contributions to total gene expres-
sion levels. For example, they may become differentially
regulated in a tissue-specific manner [25,26], in response
to stress [27], during development [28] or during different
stages of fermentation in yeast [29]. In allotetraploid
plants of various ages, genes from one homoeologous sub-
genome, typically the less fractionated subgenome, make a
greater contribution to the transcriptome, a phenomenon
termed “genome dominance”. These include newly syn-
thesized Arabidopsis suecica [30], the natural allotetra-
ploid Tragopogon miscellus that originated within the last
80 years [31], and allotetraploid cotton [32,33], originating
naturally 1–2 million years ago or synthetic (newly synthe-
sized) [34]. Genome dominance has also been observed in
autotetraploid maize [35], even though polyploidization
occurred some 12 million years ago. However, it has not
been observed in the more ancient plant tetraploidies,
such as those in the Arabidopsis or rice lineages [6,36].
Bread wheat (Triticum aestivum) is an excellent model
system for studying the long-term responses of plant ge-
nomes to polyploidy. It is also a vital crop to food security,
accounting for almost 20% of the world’s daily food con-
sumption [37]. Hexaploid bread wheat evolved through
two distinct interspecies hybridization steps, involving
three diploid donor species each with 7 pairs of chromo-
somes. First, ~0.5 million years ago, tetraploid T. turgidum
arose from hybridization between a wild diploid species
closely related to extant T. urartu (AA chromosomes)
and another unknown wild diploid species related to
Aegilops speltoides [38], called the B genome donor. The
T. turgidum genome is symbolized by AABB. About
8,500 years ago, hexaploid T. aestivum (AABBDD) arose
from hybridization between cultivated T. turgidum and the
wild diploid species A. tauschii (DD). The 42 chromo-
somes of the large ~17 Gb bread wheat genome [39,40] are
therefore distributed in three diploid homoeologous sets
(A, B and D), each comprising seven chromosome pairs.
Within each chromosomal group (e.g., group 1), each dip-
loid chromosome pair (e.g., the 1A pair) is largely homozy-
gous due to the natural inbreeding of the polyploid wheats
and does not recombine with its homoeologues (e.g., the
1B and 1D pairs) [41]. Thus, every bread wheat gene is
potentially represented by at least six copies; there are
three homoeoloci (or homoeologues) carried on the three
(A, B and D) homoeologous chromosome pairs, with each
homoeolocus representing a diploid pair of homoeoloci.
Since the diploid progenitors of wheat originate from a
common ancestor, each trio of A, B and D homoeoloci
in hexaploid wheat originates from a common ancestral
gene. Hence a simple null hypothesis would be equal
expression of each homoeolocus, with A, B and D
homoeoloci contributing mRNA transcripts (‘A=B=D’).
There are two main reasons why deviations from this
1:1:1 ratio might be widespread. First, evolutionary di-
vergence of homoeoloci may have caused changes in
relative expression, from slight changes, to physical loss
(deletion) of a homoeolocus. Second, the complex suc-
cessive hybridization and domestication processes that
have shaped bread wheat may have influenced homoeo-
locus divergence, in particular the coordinate regulation
of relative homoeolocus expression levels. In synthetic
hexaploid wheat, differential expression of homoeoloci
is rapidly established [42,43], is stably inherited [44] and
is similar to the patterns observed in natural hexaploids
[44]. Some studies have also showed a parent-specific
expression dominance in the synthetic allohexaploid
wheat transcriptome, involving a biased contribution
from the AB (tetraploid) genome parent [43,45], though
others have not detected any genome dominance [44].
These studies have been somewhat limited by the inabil-
ity to discriminate gene expression from all three indi-
vidual (A, B and D) homoeoloci. While the differential
contribution from all three homoeoloci has been illus-
trated for a small number of individual genes [46-48],
little is known about the relative contributions of
individual homoeoloci to wheat phenotypes on a larger
scale, or about the existence of any discernible genome-
wide patterns.
Here we systematically determine the extent to which
bread wheat genes retained in three homoeologous copies
deviate from the null hypothesis of equal expression. This
determination presents an intricate challenge. Distinguish-
ing expression of a specific homoeolocus from expression
of other similar sequences is difficult, in part because the
sequences of homoeoloci are very similar [40]. The wheat
genome is also highly repetitive, having more than 80% re-
petitive DNA [49,50]. So, in addition to three potential
homoeologous copies of each gene, there may be multiple
“paralogues,”here defined as similar loci resulting from
gene duplication. Such paralogues may be present on
more than one chromosomal group (e.g., on both the
group 1 and group 5 chromosome groups). Several strat-
egies have been employed to assemble homoeologue-
specific bread wheat sequences. These include approaches
that reduce the sequence complexity, including the phys-
ical isolation and sequencing of specific chromosome
homoeologues [51-54], direct assembly of transcriptomic
sequences [55] and utilizing the sequence similarity be-
tween bread wheat subgenomes and those of its diploid
progenitors [40,56]. In a recent landmark study, Brenchley
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et al. [40] accomplished direct gene assembly from wheat
genomic sequences and assigned two thirds of gene se-
quences to their homoeologous chromosomes, based on
sequence similarity with diploid species related to the pro-
genitors of the A, B and D subgenomes.
Here we describe a novel approach to understanding
wheat gene expression patterns that combines wheat cyto-
genetic resources with next generation RNA sequencing.
This approach enables assessment of homoeologous gene
expression for a sample of ~2,300 genes located on the
group 1 and 5 chromosome groups. Our study shows
detailed global patterns of relative homoeologous contri-
bution through homoeolocus-specific characterization of
bread wheat gene transcripts. In particular, we find that
reduction in complexity, through the mechanisms of
homoeolocus loss, silencing and biased expression, is a
major feature of wheat genome evolution.
Results
Alignment of reads from euploid and nullitetra wheat
cDNA libraries to a partial reference transcriptome
The hexaploid nature of the wheat genome permits the en-
gineering of wheat lines lacking specific homoeologous
chromosomes, lines known as nullisomic-tetrasomics (‘nul-
litetras’) [57,58]. In each nullitetra line, lack (‘nulli’)ofone
homoeologue is compensated by an extra set of either of
the remaining homoeologous chromosomes, thus restoring
the hexaploid state. For example, N1AT1B lacks chromo-
some 1A but has two sets (‘tetra’) of chromosome 1B
(Figure 1A). The nullitetras have historically been used for
determining presence of wheat genes on specific homoeo-
logous chromosomes [59-61], and we reasoned that, in
combination with next generation sequencing, they could
be used to develop a systematic understanding of the
relative contributions of individual homoeoloci to overall
wheat gene expression.
We therefore obtained mRNA-Seq datasets [62] from
non-normalized cDNA libraries created from shoot and
root tissues of the euploid bread wheat cultivar Chinese
Spring, from which the nullitetra lines are derived [58],
from complete sets of chromosome 1 and 5 nullitetras,
and from extant relatives of the diploid A (Triticum
urartu) and D (Aegilops tauschii) genome donors, herein
referred to as A and D genome diploids (Figure 1A; see
Additional file 1: Table S1). In the then absence of a T.
aestivum genome sequence, we exploited an extensive
wheat ‘expressed sequence tags’(ESTs) resource to con-
struct a reference transcriptome sequence (see Additional
file 2: Table S2). We selected ESTs physically mapped to
segments (‘bins’) of wheat group 1 or group 5 chromo-
somes [58,63]. Concatenating ESTs from chromosomes 1
(1,123) and 5 (1,247), we created a wheat group 1 and 5
partial reference transcriptome consisting of 2,354 ESTs
(see Methods). Since the mean length of these ESTs
(hereafter referred to as ‘genes’) was 455 ± 133 bp (see
Additional file 3: Figure S1), the genes in our reference are
representative of fragments of sequence transcribed from
entire genes in their subgenome of origin. Assuming the
diploid wheat genome contains approximately ~32,000
genes [40], this reference set represents ~5-10% of the
total wheat gene content.
We next aligned our wheat RNA-Seq reads using
novoalign software [64] to the group 1 and 5 reference
transcriptome, using a number of quality filters (see
Additional file 4: Figure S2) to give high confidence read
alignments, with a deep average coverage of 700x enab-
ling gene expression analysis (Table 1). On average these
reads represented 5.2 ± 1.3% of the total reads from each
sample (see Additional file 1: Table S1), a figure roughly
comparable with the above estimation that the group 1
and 5 reference transcriptome represents ~5-10% of
wheat genes.
Homoeolocus-specific sequence analysis shows at least
45% of wheat genes are expressed from all three
homoeoloci
We next developed a novel strategy for the direct detec-
tion of wheat sequence variants and their assignment to
homoeologous chromosomes using nullitetra analysis (see
Additional file 4: Figure S2). We implemented this strat-
egy computationally using Perl and visually using the
Integrative Genomics Viewer [65]. Homoeologue-specific
variants (HSVs) are defined as single base differences be-
tween homoeolocus sequences, and candidate HSVs were
therefore identified at nucleotide positions (‘sites’)inthe
reference sequence where 2 or 3 different bases were de-
tected in aligned euploid wheat reads. True HSVs that
were ‘diagnostic’(i.e., specific) for a particular homoeolo-
gue were confirmed by the presence of the diagnostic base
at a particular site in all nullitetra lines except for those
lines lacking the corresponding homoeologous chromo-
some. For example, Figure 1A illustrates a notional ‘A/
T’homoeologue-specific variant at the tip of the short
arm of chromosome 1, where the A base is diagnostic
for the A homoeologue, as distinct from the T base
present on the B and D homoeologues. Such ‘diagnostic’
sequences were used to infer the expression (and there-
fore presence) of a particular gene on a specific homo-
eologous chromosome.
Genes were called as expressed if they had at least one
quality filtered mapped read in at least one euploid wheat
sample and at least one nullitetra line. Most genes in the
reference transcriptome were expressed, both for group 1
chromosomes: 1,096/1,114 (98%) genes, and group 5
chromosomes: 1,218/1,240 (98%) genes (Additional file 5:
Figure S3). The majority of such expressed genes on
chromosome group 1 (689/1,096 or 63% genes) and group
5 (812/1,218 or 67% genes) carry one or more HSVs
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(Table 1) identified in the sequence reads from shoot and/
or root tissues via nullitetra analysis. Overall, there were
15 HSVs per kilobase of reference transcriptome, with an
average number of HSVs per gene of 10.6 ± 8.8 (Figure 2A).
There was only a very weak correlation between gene
length and the number of HSVs (R
2
= .083; Figure 2B).
Most variant sites were two-allele HSVs, although we
didobservealowfrequency(~1%)ofsiteswith3
homoeologue-specific alleles (Table 1). This lower fre-
quency of three-allele versus two-allele HSVs is expected
because three-allele HSVs require two independent (rather
than a single) mutations at the same site for their gener-
ation. Intriguingly, we identified a significant deficit of D-
diagnostic compared with A- or B- diagnostic HSVs (chi-
square, p< .001) for genes on groups 1 and 5. This deficit
is more pronounced for group 1 compared with group 5
genes (Table 1), though the reason for this difference is
unclear. In addition, we observed an enrichment of B-
diagnostic HSVs on chromosome 1, but not on chromo-
some 5. The reasons for these deviations in relative
frequencies of homoeolocus-specific variants between the
two chromosome groups are unknown.
Figure 1 Analysis of hexaploid bread wheat homoeologous gene expression using nullisomic-tetrasomic lines. (A) Schematic illustrating
the group 1 homoeologous chromosomes in hexaploid bread wheat, the derived nullisomic-tetrasomic lines and the A and D genome diploids.
A single homoeologue-specific variant (HSV) diagnostic for each of A (red), B (blue) and D (green) homoeologous chromosomes is shown. (B)
Two sets of three homoeologue-specific haplotypes of BE497595, a gene encoding a chloroplast thylakoidal peptidase-like protein and which
has 24 HSVs (inverted triangles) diagnostic for either 5A (red), 5B (blue) or 5D (green) homoeologues. RNA-Seq reads are aligned to a degenerate
reference sequence shown in grey containing genetic ambiguity codes at HSV locations. Double ended arrows illustrate candidate sequence
regions for haplotype quantification. (C,D) RNA-Seq read frequencies from roots for the nullitetras and A and D genome diploids (C) and in two
biological replicates of euploid wheat (D) for each homoeologue-specific haplotype given in the boxed region shown in (B). The homoeologous
expression pattern in euploid wheat roots is indicated below the pie chart.
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We next combined HSVs co-locating within a region
of gene sequence less than the 51 base pair length of
an individual sequence read to produce haplotypes.
Figure 1B illustrates this process for BE497595,agene
with 24 HSVs distinguishing three homoeoloci on chro-
mosomes 5A, 5B and 5D. The boxed region shows three
homoeologue-specific haplotypes defined by two A-
diagnostic HSVs and a single B-diagnostic HSV. Hence,
three homoeolocus-specific haplotypes can be distin-
guished, despite the absence either of individual 3-allele
HSVs or 2-allele diagnostic HSVs for all three homoeo-
loci. The homoeolocus-specificity of each haplotype is
confirmed by its absence in the nullitetra line lacking
the respective homoeologous chromosome and by its
presence in all other samples (Figure 1C), allowing esti-
mation of the number of homoeoloci of an individual
gene that are detectably expressed (e.g., exactly three A,
B and D homoeoloci of gene BE497595;Figure1D).For
the majority of sequences, haplotypes in the diploids are
identical to the corresponding homoeologous haplo-
types in euploid wheat (Figure 1C).
To assess the qualitative patterns of expression of
wheat homoeoloci on chromosome groups 1 and 5, we
looked at genes expressed in both biological replicates
of each tissue. There were 1,023 (1,058) such group 1
genes expressed in shoots (roots) and 1,164 (1,181) such
group 5 genes expressed in shoots (roots). Systematic
analysis of homoeolocus-specific sequences allowed us
Table 1 Summary statistics for homoeologue-specific variants (HSVs) in euploid wheat
Chrom
group
Reference transcriptome
length (bp)
RNA-Seq
coverage
a
Number (%) of 2-allele
HSVs diagnostic for
homoeolocus
P value
b
Number (%) of
3-allele HSVs
Total HSVs HSV density
c
ABD
1 505,132 540 X 2,611 2,830 1,883 1.89x10
−44
97 7,421 0.015
(35.2) (38.1) (25.4) (1.3)
5 567,044 860 X 2,905 2,837 2,710 0.031 94 8,546 0.015
(34.0) (33.2) (31.7) (1.1)
Total 1,072,176 700 X 5,516 5,667 4,593 1.21x10
−28
191 15,776 0.015
(34.5) (35.5) (28.8) (1.2)
a
Coverage is given as the average number of quality filtered reads aligned per site using novoalign software, from all euploid Chinese Spring wheat samples.
b
pfor a chi-square test of equal numbers of 2-allele HSVs diagnostic for each homoeolocus.
c
HSV density is given as the average number of variants per site.
Figure 2 Distribution of variants for wheat genes on chromosome groups 1 and 5. HSVs are homoeologue-specific variants detected by
nullitetra analysis of wheat RNA-Seq reads. 1,501/2,314 (65%) genes on chromosome groups 1 and 5 have at least 1 HSV expressed in shoots
and/or roots. (A) Distribution of the number of HSVs per gene for genes with at least 1 HSV. (B) Correlation between gene length and number of
HSVs, with linear regression fit (red line). The Pearson correlation coefficient r = 0.287 (R squared = .083, p< .001).
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to assign reads, i.e., expression, to specific homoeolo-
gous chromosomes. The vast majority of these genes
were expressed from the same homoeologues in the two
replicates and were selected for further analysis: (956/
1,023 (1,004/1,058) group 1 genes expressed in shoots
(roots) and 1,093/1,164 (1,129/1,181) group 5 genes
expressed in shoots (roots); see Figure 3A). We discov-
ered that at least 45% of such group 1 and group 5 genes
whose expression was assigned to homoeoloci are un-
equivocally expressed from all three possible homoeo-
loci (833/1,781 genes (47%) in shoots and 872/1,854
genes(47%)inroots;Figure3A).Thisfigureisanunder-
estimate because expressed homoeoloci are detectable only
if expressed at sufficient levels and because homoeologue-
specific sequences are required to distinguish homoeolocus
expression. Genes expressed from all three group 1 or 5
Figure 3 Expression of up to three homoeoloci for wheat genes on chromosomes 1 and 5. (A) Genes may be expressed from one, two or
three homoeologous chromosomes of group 1 or 5, inferred using nullitetra analysis of RNA-Seq data to detect homoeologue-specific variants
(HSVs) or haplotypes in shoots or roots. Genes expressed from 3 homoeologous chromosomes (yellow) have diagnostic sequences for all 3
homoeoloci (A, B and D). Genes expressed from at least two homoeologous chromosomes either have diagnostic sequences for a single
homoeolocus, leaving the identity of the second expressed homoeolocus uncertain (light green) or diagnostic sequences for two homoeoloci
(dark green). Genes expressed from at least one homoeolocus (orange) have no diagnostic sequences. Genes without evidence for expression
from all three homoeoloci are classified as expressed from at least one (≥1) or at least two (≥2) homoeoloci, since they may also be expressed
from additional homoeoloci with identical sequences, which cannot be identified. (B) Total expression level (RPKM) from all expressed homoeoloci for
group 1 and 5 genes expressed from one (orange), two (green) or three (yellow) homoeoloci in shoots (left hand boxes) or roots (right hand boxes).
P values are given for a Mann–Whitney test and significant values indicated with an asterisk. (C) The number of group 1 (upper panel) or group 5
(lower panel) genes expressed from each combination of homoeologous chromosomes in shoots (left panel) or roots (right panel). Coloured dashed
circles indicate genes expressed from the corresponding homoeologous chromosome plus at least one other chromosome whose identity cannot be
distinguished. Grey dashed circles indicate genes expressed from a single homoeolocus (A, B or D), whose identity cannot be distinguished. Grey filled
circles represent ‘unassigned’genes whose expressed homoeoloci remained uncertain (see Additional file 15: Table S6).
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homoeoloci have a significantly higher total expression
level than genes expressed from only one or two homo-
eoloci, in either shoots or roots (Figure 3B). This is not
surprising since genes expressed at high levels were
preferentially retained and expressed from multiple
copies after polyploidization in other species, including
maize [35] and soybean [22]. However, it is difficult to
confirm a similar effect in bread wheat, since genes
expressed at higher levels will also have a higher prob-
ability for detection of all three homoeoloci by nullite-
tra analysis of homoeolocus-specific sequences in the
RNA-Seq data.
Genes expressed from all three homoeoloci are
predominantly expressed from a single homoeolocus
Having established that at least 45% of genes are expressed
from all three A, B and D homoeoloci, we next determined
the relative contribution of transcripts from each homoeo-
locus to total gene expression, to test the ‘A=B=D’(1:1:1)
null hypothesis. While we could not be certain that each
homoeolocus exists in single copy on each homoeologous
chromosome, we could assess the relative contribution
from each homoeologous subgenome. To do this we
aligned reads using novoalign [64] to a degenerative ref-
erence sequence (shown in grey in Figure 1B). For se-
quence regions with exactly three homoeologue-specific
haplotypes, for example those marked by arrows in
Figure 1B, reads with A, B and D sequences were quan-
tified and compared using chi-square to a 1:1:1 expect-
ation. Regions with more than three haplotypes were
ignored as they most likely contained additional sequence
alignments from paralogous genes. Over 93% of the haplo-
typed regions from all genes showed only two or three
homoeologue-specific haplotypes (see Additional file 6:
Figure S4), therefore extra sequence alignments from re-
lated genes did not significantly constrain our analysis.
Genes expressed from all three homoeoloci in both bio-
logical replicates of a given tissue were classified into
those not diverging from the ‘A=B=D’null hypothesis
versus those exhibiting differential expression (Figure 4A).
A minority of group 1 and 5 genes showed no sequence
regions with evidence of differential expression at p<.01
and were classified as equally expressed from all three
homoeoloci (‘A=B=D’). This group included 225/833
genes (27%) in shoots and 206/872 genes (24%) in roots
(Figure 4A). This estimate of ~1/4 of genes showing
‘A=B=D’is almost certainly an upper bound, since the
statistical power to detect differential expression is gov-
erned by the read depth, strength of differential expression
and number of sequence differences between homoeoloci.
Genes displaying the ‘A=B=D’pattern have reduced
probability for detection of differential expression (see
Additional file 7: Figure S5A), due to reduced total read
coverage or RPKM (reads per kb per million aligned reads)
compared to differentially expressed genes (p< .001) and a
lower per-site density of HSVs (p< .001).
Most genes showed evidence for differential expres-
sion of homoeoloci (608/833 genes (73%) in shoots, 666/
872 genes (76%) in roots; Figure 4A). These included all
genes with quantified regions passing the p< .01 thresh-
old. Those passing a Bonferroni-corrected threshold of
p< .05 were called as significantly differentially expressed,
while the remainder were only “suggestive”of differential
expression and classified as “unknown”(Figure 4A). We
were interested in the diversity and frequency spectrum of
different homoeologous expression patterns for genes with
significant differential expression. Differentially expressed
genes were therefore sorted into all 12 possible categories
(Figure 4B): 6 patterns with three distinct levels of expres-
sion from each of the three homoeoloci, for example ‘A>
B>D’) and a further 6 patterns with two distinct levels of
expression, for example ‘A>B=D’. Each pattern with
three distinct expression levels ranks the expression of
each homoeolocus and is ‘consistent’with two patterns
with just two different expression levels (Additional file 8:
Figure S6). For example, ‘A>B>D
’is consistent with ‘A>
B=D’and ‘A=B>D.’We used a conservative approach
to classify genes into each of these categories, requiring
the same or a consistent pattern of differential expression
in two biological replicates from the same tissue and a
consistent pattern across sequence regions (see Methods).
For such genes, we combined the two replicates for ana-
lysis of the significance of differential expression in each
tissue (see Additional file 9: Table S3). For example, in
Figure 1D, BE497595 showed a ‘B>A=D’pattern in roots
for both replicates in both illustrated regions with three
homoeolocus-specific haplotypes. This pattern was signifi-
cant at p< .001, with both replicates combined or indi-
vidually (Additional file 9: Table S3). Around half of genes
expressed from all three group 1 or group 5 homoeoloci
showed such ‘consistently biased’patterns of differential
homoeolocus expression in shoots (391/833 or 47% genes)
and in roots (417/872 or 48% genes). The remainder were
added to the “unknown”group of genes, which totalled
217/833 or 26% genes in shoots and 249/872 or 29% genes
in roots (Figure 4A). We observed highly similar homoeo-
locus expression patterns from the two replicates of each
tissue, with less than 7% of ‘unknown’cases in either
shoots (14/217 genes) or roots (15/249 genes) caused by
conflicting patterns of expression. Note that while we
do not focus on the expression of homoeoloci in the
nullitetras here, Figure 1C shows that for BE497595,all
the nullitetras except for N5BT5A show an expression
pattern consistent with a dosage response to the extra
copies of the ‘tetra’genome. N5BT5A shows the oppos-
ite response, a negative dosage compensation in which
the contribution from the ‘tetra’genome (A) is lower
than that of the other ‘diploid’genome (D).
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All twelve patterns of differential homoeolocus expres-
sion occur in both shoots and roots, though not with
equal frequency (Figure 4B; Additional file 8: Figure S6).
Less than 10% of differentially expressed genes had three
distinct expression levels (e.g., ‘A>B>D’) in shoots (27/
391 genes) or roots (34/417 genes). This is an underesti-
mate since these genes are expressed at significantly
higher levels compared with genes having two distinct
expression levels (p< .001, Additional file 7: Figure S5C),
which improves the statistical power to detect differential
expression of homoeoloci. Cases where one homoeolocus
dominates expression (e.g., ‘A>B=D’), were significantly
more frequent than cases with co-upregulation of two
homoeoloci (e.g., ‘A=B>D’) (Figure 4B; chi-square,
p< .001 in shoot and roots). On average, among such
cases, the predominant homoeolocus contributes ~55%
of total expression, while the other two homoeoloci play
a subsidiary role and supply the remaining 45% (Figure 4C;
see Additional file 8: Figure S6). A small number of genes
exhibit extremely strong homoeolocus expression bias.
For example, the group 5 gene BF484913 encoding a pre-
dicted enolase shows ‘B>A=D’in shoots, with an average
of 86 ± 5% expression arising from the B homoeolocus
(see Additional file 8: Figure S6J). However, the extent
of expression bias shows no clear relationship with the
total gene expression level from all three homoeoloci
(see Additional file 10: Figure S7).
There is no global bias for preferential expression of
homoeoloci from a particular A, B or D homoeologous
subgenome (Figure 4B). In contrast, when two of the three
expressed homoeoloci dominate total gene expression,
there is a striking difference in the frequencies of the three
possible co-upregulated pairs (Figure 4B), with A and B
homoeoloci significantly underrepresented compared with
Figure 4 Biased patterns of expression for homoeoloci on wheat group 1 and 5 chromosomes. (A) The number of genes expressed from
all three homoeoloci in euploid wheat showing either equal expression (‘A=B=D’), a consistent and significant pattern of differential expression
bias (e.g., ‘B>A=D’) or classified as unknown (i.e., lack of consistent pattern of significant expression bias) for genes on chromosome groups 1
and 5, in shoot and root tissues. (B) The number of genes in roots (light blue) and shoots (dark blue) showing each pattern of differential
homoeolocus expression. (C) The mean percentage of total transcripts arising from A, B and D homoeoloci for 12 distinct patterns of differential
homoeolocus expression in roots (for shoots see Additional file 8: Figure S6). Error bars (−) are given at one standard deviation above and below
the mean.
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‘A, D’or ‘B, D’combinations (chi-square, p<.001in shoots
and in roots). However, we do not observe a similar deficit
for the alternative pattern in which A and B homoeoloci
are expressed at the same level (‘D>A=B’) compared
with either ‘B>A=D’or ‘A>B=D’.
To explore the factors underlying differential homoeo-
logous expression patterns in euploid wheat, we looked
at the possible role played by biological function. We used
the FatiGO functional analysis tool [66] to explore the re-
lationship between biological function and homoeologous
gene expression patterns. We found that genes expressed
from all three homoeoloci in either shoots or roots were
significantly enriched for only a single term, cellular com-
ponent GO:0005739 (mitochondria), compared with genes
expressed from only one or two homoeoloci (Fisher exact
test, p< .001). However, we did not find any functional
trends (significant GO terms) to explain specific patterns
of expression of homoeoloci (e.g.,‘A>B=D’).
We experimentally validated, using two independent ap-
proaches, our computationally determined homoeologue-
specific variants (HSVs), and the differential homoeolocus
expression patterns observed in roots, for six genes
expressed from all three A, B and D homoeoloci. First,
semi-quantitative RT-PCR and second, PCR product
cloning into a bacterial vector followed by Sanger sequen-
cing, using the same root RNA samples used for RNA-
sequencing (Additional file 11: Figure S8 and Additional
file 12: Table S4). All HSVs were validated for these six
genes, giving a zero rate of false positives. Some HSVs in
the amplified sequences were missed by our nullitetra ana-
lysis strategy, with an average false negative rate of 18%.
HSVs were missed for various reasons, including insuffi-
cient coverage across all nullitetra samples in the region of
the HSV, absence of homoeologue-specificity in one or
more nullitetras, or sequence divergence between Chinese
Spring and EST sequences, which originated from a var-
iety of Triticum species (Additional file 2: Table S2). All
differential expression patterns were validated for each
gene using one or both of these methods. Therefore, our
strategy can successfully determine the homoeolocus ex-
pression patterns of wheat genes using fragments of gene
sequence, rather than full gene models.
Similar patterns of differential homoeologous expression
in two wheat tissues
We next determined the degree to which expression pat-
terns for genes expressed from all three A, B and D
homoeoloci are shared between shoot and root organs
of the wheat plant, based on 787 genes expressed from
all three group 1 or 5 homoeoloci in both tissues. A
comparison of homoeolocus expression patterns was
possible despite significantly higher average total ex-
pression levels in roots compared with shoots, of un-
known cause (p= .014; Additional file 13: Figure S9). Of
the above 787 genes, 373 displayed either significant
differential expression or equal expression of all three
homoeoloci (‘A=B=D’) from both tissues. 218/373
(58%) of these genes showed agreement, defined where
both tissues showed either the same pattern of expression,
or two ‘consistent’patterns of differential expression (see
Additional file 8: Figure S6). Most disagreements (121/155
genes) were cases where the three homoeoloci were differ-
entially expressed in one tissue, but equally expressed in
the other (‘A=B=D’). For most such cases (77/121 or
64% genes), the tissue showing equal expression also had
lower coverage, suggesting that some of these disagree-
ments may be false positives caused by the statistical diffi-
culty in confirming differential expression of homoeoloci
for genes expressed at low levels. Of more interest are
those 34 genes with conflicting patterns of differential
homoeolocus expression (see Additional file 14: Table S5).
For example, BE606302 is a highly expressed uncharacter-
ized protein similar to the Arabidopsis gene TET3,which
shows a ‘B>A=D’pattern in shoots but has a signifi-
cantly higher contribution from the A homoeolocus in
roots, showing ‘A=B>D’(35% ± 1% of expression from
the A homoeolocus in roots versus 24 ± 2% in shoots).
The biological relevance of these tissue-specific homoeo-
locus expression patterns warrants further investigation.
Detecting the presence and expression of homoeoloci
The remaining ~55% of wheat genes on chromosome
groups 1 and 5 were not detectably expressed from all
three possible homoeoloci (948/1,781 genes in shoots
and 982/1,854 genes in roots; Figure 3A). Such expression
patterns may be generated by physical loss (deletion) of
homoeoloci, absence of transcripts from a particular
homoeolocus (due to epigenetic transcriptional silencing
or transcript-null mutation), and includes genes without
diagnostic homoeolocus-specific sequences. Distinguish-
ing these causes is a challenge because lack of evidence for
presence or expression of a homoeolocus on a particular
homoeologous chromosome may not mean it is absent or
not expressed. Nevertheless, we proceeded to categorize
the presence and expression of each gene based on several
sources of evidence (see Additional file 15: Table S6).
To infer presence of a gene on a particular homoeo-
logous chromosome, we focused on group 1 chromo-
somes and combined three sources of evidence. First, the
deletion bin mapping dataset of Qi et al. [58,63], which
assigned genes to homoeologous chromosomes according
to the presence or absence of a restriction fragment in
Southern blots from a series of aneuploid and deletion
wheat lines [58,63]. Second, nullitetra analysis of our shoot
and root RNA-Seq data from euploid wheat and its de-
rived nullitetra lines to identify transcribed homoeologue-
specific sequences (individual HSV sites or haplotypes).
Third, the group 1 homoeologous chromosome specific
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DNA sequence assemblies of Wicker et al. [52], generated
by sequencing flow sorted chromosomes from double
ditelosomic lines of Chinese Spring wheat (Additional
file 16: Table S7). This dataset included assemblies from
the short and long arms of chromosomes 1A and 1B,
and from the long arm of 1D, but lacked assembled
sequences from the 1D short arm due to chromosomal re-
arrangement in the corresponding wheat line. We identi-
fied matches for 642 (58%) of our 1,114 group 1 genes
using BLAST comparison with our homoeologue-specific
sequences. The absence of sequences from chromosome
1DS [52] means we have most likely underestimated the
number of genes present on the 1D homoeologue.
We discovered evidence for substantial gene loss dur-
ing the evolutionary history of bread wheat (Figure 5A).
Among 1,104 group 1 genes, (excluding 10 group 1 genes
with inconsistent expression of homoeoloci between repli-
cates), we found 708 (64%) genes present in three copies
(1A, 1B and 1D; Figure 5A). There are more genes on
chromosome 1B (943 genes) than on 1A (915 genes),
which in turn exceeds the number present on 1D (862
genes) (Figure 5A). We estimate that most (at least 60%)
homoeolocus loss events occurred after the two poly-
ploidization events that created bread wheat. For ex-
ample, there are 44 genes present on chromosomes 1B
and 1D (and expressed from one or both), but absent
from 1A, 25 (57%) of which are detectably expressed
(with at least one quality filtered read) in the A genome
diploid. Similarly, 73 genes are present on 1A and 1B
but absent from 1D, of which 47 (64%) are expressed in
the D genome diploid.
Widespread homoeolocus loss and silencing mean ~1/3
wheat genes are expressed from only a single homoeolocus
For the majority of genes from chromosomes 1 and 5,
we confirmed the identity of all expressed homoeoloci in
both shoots (635/956 (66%) group 1 genes; 766/1,093
(70%) group 5 genes) and roots (654/1,004 (65%) group
1 genes; 793/1,129 (70%) group 5 genes), as indicated by
the solid circles in Figure 3C; see also Additional file 15:
Table S6. For the remaining genes, the identity of one or
more homoeoloci was uncertain, as indicated by dashed
circles in Figure 3C. For example, genes with diagnostic
sequences (HSVs) for a single homoeologous chromosome
(e.g., 1A) must be expressed from this chromosome, plus
at least one other chromosome whose identity cannot be
confirmed. Genes present on more than one homoeolo-
gous chromosome and with no HSVs are likely to be
expressed from a single indistinguishable homoeologue
(e.g., 1A or 1B homoeologues for genes present on 1A
and 1B chromosomes). We found that 1,772/1,979 (90%)
of group 1 and 5 genes showed evidence for expression
from the same homoeoloci in both shoots and roots, al-
though a small number showed evidence for organ-
specific transcript absence for one or more homoeoloci;
Additional file 17: Table S8).
We observe frequent loss of expression (transcript ab-
sence) of group 1 homoeoloci (Figure 3A). For example,
Figure 5 Presence and expression of up to three homoeoloci for wheat genes on group 1 chromosomes. (A) Presence of a gene on 1A,
1B, and 1D homoeologous chromosomes is inferred using nullitetra analysis of RNA-Seq data combined with deletion bin mapping data [58,63]
and chromosome 1 arm-specific assemblies from Wicker et al. [52]. The pie chart shows the number of genes present on one, two or three
homoeologous chromosomes, while the Venn diagram shows the number of genes present on each combination of chromosomes. Dashed
circles indicate genes present on the corresponding homoeologous chromosome plus at least one other chromosome whose identity cannot
be distinguished. (B) Genes with evidence for presence on all three homoeologous chromosomes of group 1 are expressed from at least one,
at least two, or all three homoeoloci in shoots (left panel) and roots (right panel) according to the presence of group 1 homoeologue-specific
sequences. Grey circles show the number of genes with expression classified as ‘unassigned’(see also Additional file 15: Table S6).
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among genes with evidence for the presence of three dis-
tinct homoeoloci, 12% appear to be expressed from only
a single homoeolocus in both shoots and roots, while
another 14% are expressed from only two of the three
homoeoloci (Figure 5B). Overall, around one third of
wheat genes are expressed from just one homoeolocus,
having lost or silenced the other two copies (566/1,781
(32%) genes in shoots and 565/1,854 (30%) genes in
roots; Figure 3A). If homoeoloci are lost or lose tran-
scription function randomly, genes expressed from two
homoeoloci should be more frequent than those expressed
from one. Intriguingly, however, we found that genes on
group 1 are significantly more likely to be expressed from
just one homoeolocus than from two homoeoloci in
shoots (251 genes versus 145 genes, chi-square, p<.001;
Figure 3A) and in roots (264 genes versus 152 genes,
p< .001; Figure 3A). The same is true for group 5 genes
expressed in shoots (315 genes versus 237 genes, p<.001),
though the difference is not significant at cutoff p<.05
for roots (p= .083). We conclude that in terms of its
gene expression, the bread wheat genome is tending to-
wards diploidization in two ways; first, we estimated
that only ~64% of group 1 genes are present in three
homoeologous copies, indicating substantial gene loss
has occurred (Figure 5A). Second, while there is still
more than one homoeologous copy for at least 84% of
group 1 genes (Figure 5A), only 69% of these genes are
expressed from more than one copy in either shoot or
root tissues (Figure 3A), which could indicate substan-
tial homoeolocus silencing.
Assuming we have correctly identified all present and
expressed homoeoloci, we found a comparable frequency
of genes showing physical absence (loss) and transcript
absence (silencing) of homoeoloci. In the absence of evi-
dence for expression from a particular homoeologous
chromosome, we identified cases of potential homoeolo-
cus loss where there was no evidence for its presence, or
of transcript absence where there was evidence for its
presence. Among 805 genes whose expression in shoots
was assigned to one or more group 1 homoeologous
chromosomes, 211 (26%) have lost one or two homoeo-
loci, while 193 (24%), have silenced one or two homoeo-
loci (Additional file 15: Table S6). Similarly, for genes
expressed in roots, 227/846 (27%) have lost one or two
homoeoloci, while 201/846 (24%) have silenced one or
two homoeoloci. We conclude that physical loss and
transcript absence have both played an important role in
the loss of homoeologous gene expression in hexaploid
bread wheat.
B-subgenome homoeoloci contribute disproportionately
to wheat gene expression
B subgenome homoeoloci not only exceed A or D homo-
eoloci in number but also contribute more to the bread
wheat transcriptome (Figure 3C). Significantly more genes
are expressed from B homoeoloci of groups 1 or 5 than
from either A or D homoeoloci, both in shoots (A(1,134),
B(1,205), D(1,110), chi-square, p= .046) and in roots
(A(1,188), B(1,262), D(1,158), p= .036). Among genes
expressed from a single homoeolocus, significantly
more are expressed from B (6-10% genes) than from ei-
ther A or D chromosomes (4-6% genes), both in shoots
(Figure 3C, chi-square, p= .013 for group 1; p< .001 for
group 5) and in roots (Figure 3C, chi-square, p= .016
for group 1; p< .001 for group 5). It is possible that there
are more genes detectably expressed from 1B and 5B
homoeologous chromosomes than from A or D chromo-
somes due to a higher frequency of B homoeolocus-specific
variants. Indeed, it is plausible that B homoeologous
chromosomes are more polymorphic than A or D chro-
mosomes, given the differences in both mating systems
and genome ages of the parental diploid species [63,67,68];
the B homoeologous genome was acquired from a cross-
pollinating species related to Aegilops speltoides,whilethe
A and D homoeologous chromosomes trace their origins
to self-pollinating diploids [67,68]. However, this higher
frequency of B-specific HSVs is not common to our entire
dataset (not seen on chromosome 5 for example, see
Table 1). We therefore conclude that B-genome homoeo-
loci tend to contribute more to wheat gene expression than
do A or D genome homoeoloci.
A natural and long-standing question is whether A, B
and D homoeoloci are equally likely to be transcript-absent
[61,69-71]. It is possible that genes from particular wheat
homoeologues have been preferentially silenced during
polyploid evolution. Contradictory evidence exists for pref-
erential homoeologue silencing in wheat, with one study
showing D genome homoeoloci to be silenced twice as
frequently as A or B homoeoloci [72], and others showing
weak evidence for preferential silencing of D homoeoloci
[70], or indeed no preferential silencing at all [61,69]. Here
we focussed on genes with evidence for presence on all
three group 1 homoeoloci, but detectable expression for
only two homoeoloci (see Additional file 15: Table S6). In
shoots, there are more genes with a transcript-absent D
homoeolocus (23 genes) compared to transcript-absent B
(17 genes) or A (11 genes) homoeoloci, though the differ-
ence was not significant. In roots, there were significantly
more genes with a transcript-absent D homoeolocus (21
genes) than genes with a transcript-absent B (11 genes) or
A (12 genes) homoeolocus (chi-square, p=.043). Most of
the transcript-absent A homoeoloci (10/12) were expressed
in the A genome diploid. Similarly, most transcript-
absent D homoeoloci (17/21) were expressed in the D
genome diploid. While our results corroborate previous
work demonstrating preferential post-polyploidization
transcript absence for homoeoloci on the D genome
[70,72], it is nonetheless particularly unexpected given
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that the D genome was integrated into hexaploid wheat
only ~8,500 years ago. The mechanism(s) and reversibility
of such transcriptional silencing are not well understood,
and may involve methylation [14], mutation in the pro-
moter region, or other gene silencing mechanisms.
Discussion
Understanding the structure and function of the bread
wheat genome is a major challenge, due to its complex
evolutionary history of successive hybridization and allo-
polyploidization. In particular, it has remained unclear
on a large scale whether the three ancestral diploid ge-
nomes contribute equally or differentially to wheat gene
expression, and hence to wheat phenotypes. Our study
explores this question in detail, using an extensive, sys-
tematic approach for dissecting the relative contribu-
tions of the three potential homoeoloci (A, B and D) to
total wheat gene expression.
Several studies on the differential expression of homo-
eoloci in wheat have focussed on the expression of a
relatively small number of genes [46-48,61,71]. Subse-
quent genome-wide assessments have so far used micro-
arrays, which are difficult to interpret for the expression
of closely related genes [42-45]. Such studies have there-
fore been unable to separate the contributions of indi-
vidual homoeoloci, particularly the A and B homoeoloci
originating from the parental tetraploid species. Here we
have developed a novel analysis of euploid bread wheat
and its derived nullisomic-tetrasomic lines bearing specific
chromosome substitutions. Using high coverage RNA-
sequencing, we have successfully distinguished gene tran-
scripts from all three hexaploid wheat homoeoloci (A, B
and D). This direct approach alleviates the issue of
chimeric sequence assembly encountered in previous
work [40,55], for genes on those chromosome groups
for which nullitetra sequences are available (groups 1
and 5 in the present study). This allowed direct quantifi-
cation of gene transcripts for which homoeolocus speci-
ficity was established.
Combining our RNA-Seq data with published deletion
bin mapping data [58,63] and chromosome arm-specific
assemblies of group 1 chromosomes [52], we have assessed
the physical distribution and expression of a sample of
2,314 wheat genes present on groups 1 and 5 of the seven
homoeologous bread wheat chromosome groups. We esti-
mate that at least 45% of genes are expressed as three dis-
tinct homoeoloci (A, B and D) in both shoot and root
tissues, as would be expected based on the hexaploid na-
ture of the wheat genome. The majority of such genes
display a biased pattern whereby the output of a single
homoeolocus dominates total gene expression. Similar
studies of other allopolyploids including oilseed rape [73],
and cotton [28,32,74-76], have also shown that genomically
biased expression is common across a range of vegetative
and floral tissues. In addition, all possible patterns of biased
homoeolocus expression occur from various genes and tis-
sues, consistent with our results, which show all twelve
possible patterns of differential expression from three
wheat homoeoloci. Previous work on polyploid wheat [61]
and cotton [76], and on diploid Arabidopsis [77], has
shown frequent cell type-specific homoeolocus (or du-
plicate gene) expression patterns. Perhaps surprisingly,
we uncovered limited evidence for contrasting patterns
of differential expression between homoeoloci in two
wheat tissues. Extending our work to the expression
analysis of specific cell-types, developmental stages or
growth environments would likely show more variation
in the expression of homoeoloci. Our analysis strategy
involves simultaneously identifying homoeologue-specific
variants and quantifying their contribution to the tran-
scriptome. As such, it has focused on genes that are highly
expressed and highly polymorphic. It is possible that the
expression of homoeoloci is more variable for such genes.
As the international wheat community continues to make
rapid progress towards completion of an annotated refer-
ence wheat genome sequence [40,78], we will in the future
be able to assess the generality of the patterns we have ob-
served here for all wheat genes, including those expressed
at low levels and those with subtle or highly skewed ex-
pression of homoeoloci.
Genes expressed from all three homoeoloci in either tis-
sue were enriched for the mitochondrion cellular compo-
nent GO term. This supports a key role for mitochondria
in providing hexaploid wheat with more efficient energy-
generating mechanisms than its diploid and tetraploid
relatives [79]. However, there were no functional trends
(significant GO terms) to explain specific patterns of ex-
pression of homoeoloci (e.g., ‘A>B=D’). This may be due
to insufficient statistical power, but it is also consistent
with recent work [45] showing that while the gene expres-
sion pattern itself is heritable, the identity of genes show-
ing various patterns can be highly stochastic and involve
diverse gene functions. We did not find any global bias
towards preferential expression of homoeoloci from a
particular homoeologous subgenome, for those genes
expressed from all three homoeoloci. In contrast, bias in
favour of D genome homoeologues has been reported
in five cotton species [33,34]. In addition, the trans-
criptomes of two resynthesized hexaploid wheat lines
contained more genes showing tetraploid parental dom-
inance than diploid parental dominance [45], though
the contributions of A and B homoeoloci could not be
separated. Observations in other polyploid species, in-
cluding cotton [32,76], oilseed rape [73] and Spartina
[80], suggest there are both immediate and long term
alterations in homoeologous gene expression patterns
after polyploidization. We propose that the biased pat-
terns of homoeolocus expression we observed are
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attributable to coordinated (as opposed to independent)
divergence in the regulation of individual homoeolocus
expression levels following polyploidization. This possi-
bility is strengthened by our observation that in cases
where two homoeoloci equally dominate total gene
expression, dominance of A and B homoeoloci is less
likely than dominance of A and D or B and D homoeo-
loci. This may reflect the fact that A and B genome
homoeoloci have been associated in bread wheat (and
progenitors) for ~0.5 million years, whilst D genome
homoeoloci were only incorporated ~8,500 years ago.
We estimate that around 55% of wheat genes are no
longer expressed from all three potential A, B and D
homoeoloci, due to both extensive homoeolocus loss
and extensive transcript-absence of homoeoloci. Homo-
eolocus loss has been extensive since only 64% of genes
exist in A, B and D copies. This result is consistent with
wheat whole-genome sequencing [40], which showed
most wheat genes with orthologous copies in related
cereals have between one and five copies, with a peak of
two copies. In addition, assembly of sequences from
wheat chromosomes 7A, 7B and 7D showed that 54% of
genes remain present on all three homoeologues [81].
We also observed more B homoeoloci compared with A
or D homoeoloci, supporting earlier observations based
on bin mapping data [58,63]. This is likely to reflect, in
part, the accumulation of non-syntenic genes on the 1B
homoeologue [52], as well as differential loss of homoeo-
loci from particular homoeologous chromosomes, which
is commonly observed in paleopolyploid species [17-22].
This enrichment for B homoeoloci was not detected in
the genome-wide analysis of wheat gene assemblies [40],
perhaps due to poor discrimination of B genome homoeo-
loci. A different pattern has been observed for genes on
group 7 chromosomes, with homoeolocus loss greater
from chromosomes 7A and 7B than from chromosome
7D [81], which was attributed to two rounds of gene frac-
tionation following polyploidization during the evolution-
ary history of bread wheat. The causes of these differences
are unknown and might involve chromosome group-
specific patterns of homoeolocus presence/loss.
In both shoot and root tissues, an unexpectedly high
proportion (~1/3) of all genes appear to be expressed
solely from a single homoeolocus and we have no reason
to believe this does not apply to all wheat tissues and to
genes on chromosome groups other than 1 or 5. Among
genes present in three copies (1A, 1B and 1D), 26% have
silenced one or more homoeoloci, which agrees well
with a previous estimate of 29% of genes in the estab-
lished hexaploid [70]. In a study of synthetic wheat, only
5% of genes showed evidence for homoeolocus silencing,
suggesting that wheat homoeoloci are silenced gradually.
Overall, our results show that the bread wheat genome
has undergone extensive “diploidization”[5]. This is a
particularly interesting finding in the light of other ob-
servations. For example, hexaploid wheat can tolerate at
least a ten times higher mutation rate (mutated base
pairs per kb of sequence) than diploid wheat [82], which
has made hexaploid wheat particularly well suited for
TILLING (targeting induced lesions in genomes). This is
usually explained by the redundancy provided by homo-
eoloci in the hexaploid genome [82,83]. The fact that
nullisomic-tetrasomic lines of wheat can be produced
exhibiting only minor phenotypic effects [58,83] further
testifies to the buffering effects of polyploidy. This could
be explained at least in part by the reversibility of gene
silencing, as demonstrated previously [70], and by the
duplication of homoeologues across more than a single
chromosomal group.
Conclusions
The analysis strategy we have developed should be of
great use for the assembly of homoeologue-specific se-
quences in wheat genome and transcriptome sequencing
projects. Wheat displays substantial levels of variation at
the phenotypic, genetic and epigenetic levels, which is of
paramount importance to wheat breeding. Our work
gives a comprehensive understanding of the homoeolo-
cus expression patterns characteristic of bread wheat
genes, providing a key stepping-stone towards under-
standing the relationship of wheat variation with differ-
ential homoeolocus expression patterns. In addition, the
observation that bread wheat is tending towards func-
tional diploidy has important practical implications for
the strategies employed by breeders in the development
of improved wheat strains.
Methods
Plant material
Three spikelets of T. aestivum,T. urartu,Ae. speltoides
and Ae. tauschii and chromosomes 1 and 5 nullisomic-
tetrasomic lines were soaked on Whatman filter paper in
Petri dishes. The spikelets were stratified at 4°C for 2 days
in the dark before extracting the seeds from the spikelets.
The seeds were soaked on Whatman filter paper in Petri
dishes and stratified at 4°C for a further 2 days. Three rep-
licates for each plant were grown hydroponically in a
controlled environment room (16/8 h light/dark cycle at
21°C). Root and shoot tissue samples were collected when
the fifth leaf appeared. At this point, the fourth leaf was
taken as the shoot sample.
Construction of reference transcriptome from wheat EST
sequences
Sequences for 6,419 Chinese Spring wheat deletion
bin-mapped 5′ESTsfromabroadrangeoflibraries
[58,63] were acquired from the website of the wheat
EST project (http://wheat.pw.usda.gov/cgi-bin/westsql/
Leach et al. BMC Genomics 2014, 15:276 Page 13 of 19
http://www.biomedcentral.com/1471-2164/15/276

map_locus.cgi) and processed to remove polyA tails.
These sequences were already assigned to specific
homoeologous chromosomes according to the presence
or absence of a restriction fragment in Southern blots
from a series of aneuploid and deletion wheat lines
[58,63]. ESTs present on one or more homoeologues of
group 1 (1,123) and group 5 (1,247) chromosomes were
assembled into groups of similar, overlapping ESTs (con-
tigs) using CAP3 software [84] to reduce gene redun-
dancy. Using a stringency level of 95% sequence identity
over a 40 bp overlap, 17 ESTs on chromosome 1 were as-
sembled into 8 contigs and 14 ESTs on chromosome 5
were assembled into 7 contigs, giving a total of 15 contigs
for both chromosome groups. The final set therefore con-
sisted of 2,354 ESTs/contigs (‘genes’), with 1,114 on group
1 chromosomes and 1,240 on group 5 chromosomes.
Gene sequences were concatenated and spatially separated
with 200 ‘N’nucleotides to produce a consensus wheat
transcriptome with 505,132 and 567,044 bases of genic
sequence for chromosomes 1 and 5 respectively. Note that
a near-complete assembly of genome-wide wheat gene
sequences has subsequently become available [40].
Functional assignments
Genes were assigned functions using either 1) translated
nucleotide query (BLASTx) against the database of all
non-redundant proteins or 2) BLASTn against database
release 12.0 of TIGR TCs (tentative contigs) for Triticum
aestivum and extraction of GO assignments of the TCs
showing the best hit. All BLAST hits were filtered using
e-value 1e
−10
. Extracted GO terms were used in FatiGO
within the Babelomics suite [66] for functional over-
representation analysis.
Next generation RNA sequencing data
Total RNA was extracted using the TRIzol Reagent (Invi-
trogen) from leaf and root tissue of the following wheat
samples from the John Innes Centre Wheat Germplasm
collection: 1) Sears’Chinese Spring hexaploid (euploid
wheat), 2) the six chromosome 1 compensating nullitetras,
3) the six chromosome 5 compensating nullitetras, 4) D
genome diploid (accession code 2220007), and 5) A gen-
ome diploid (accession code 1010005). Non-normalized
cDNA libraries were created to show the realistic gene ex-
pression levels in each of the 32 wheat samples. Each li-
brary was paired-end sequenced (mostly 51 bp read length)
in at least four lanes of the Illumina Genome Analyzer II
(GAII) at the Wellcome Trust Centre for Human Genetics,
Oxford (see Additional file 1: Table S1). The average num-
ber of sequence reads per wheat sample was 232 million.
RNA sequencing read mapping
Sequence reads were mapped to our reference trans-
criptome using three mapping software tools. First, Maq
version 0.7.1 [85] was used with default parameter set-
tings to detect homoeologue-specific variants (HSVs) in
euploid wheat sequence reads using nullitetra analysis.
Two software tools, novoalign V2.07 [64] and gsnap [86],
were used for quantification of homoeologous gene expres-
sion. These tools are designed to reduce read mapping
biases [87], particularly preferential mapping of reads de-
rived from the homoeolocus matching the reference se-
quence. Novoalign uses a degenerate reference constructed
by replacing the allele in the reference sequence with the
corresponding genetic ambiguity code (IUPAC) at HSV
sites. For example, a ‘T/C’HSV was encoded with a ‘Y’in
the degenerative reference sequence. Gsnap supplements
the reference sequence with a list of all known HSVs and
their corresponding homoeoloci. It produced virtually
identical results (not reported here) to those from novoa-
lign. All read alignments were converted to the standard
format using SAMtools version 0.1.5c [88] and quality
filtered using custom Perl scripts (see Additional file 4:
Figure S2). Entire reads were excluded based on: 1)
non-unique mapping (mapping quality zero), 2) nucleo-
tide(s) inserted or deleted relative to the reference, 3)
mapping quality <30. Mapped pairs with an unexpect-
edly large insert size (>700 bp) were also excluded based
on an expected library insert size of 250 ± 50 bp. Within
each mapped read, bases with Phred quality score <20
were excluded. For each gene, the relative total expres-
sion level from all expressed homoeoloci was calculated
from the total number of reads mapping to the gene se-
quence, normalized by both the sequence length and
the total number of mapped reads in the sample, to give
a standard RPKM measure of expression (reads per kb
per million aligned reads). The Mann–Whitney test was
used to compare RPKM between groups of genes due to
lack of normality of the RPKM distribution.
Detection of homoeologue-specific sequences
Custom Perl scripts were used to identify homoeologue-
specific variants (HSVs), defined as single base differences
between homoeolocus sequences. Candidate HSVs were
identified at nucleotide positions (‘sites’) in the reference
sequence where 2 or 3 different bases (homoeoloci) were
detected in the aligned euploid wheat reads from the
combined shoot and root RNA-Seq datasets. Sites with
coverage less than 10 mapped reads were excluded. A can-
didate homoeolocus was defined for bases with either
frequency ≥2 (and at least 3% of mapped reads) or with
frequency ≥15 reads. True HSVs that were ‘diagnostic’
(i.e., specific) for a particular homoeologue (A, B or D)
were confirmed by the presence of the diagnostic base at a
particular site in all nullitetra lines except for those lines
lacking the corresponding homoeologous chromosome.
Homoeologous sequence variants (HSVs) co-locating
within a region of gene sequence less than the 51 base
Leach et al. BMC Genomics 2014, 15:276 Page 14 of 19
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pair sequence read length were combined to produce
haplotypes. Homoeolocus-specificity of each haplotype
was confirmed by its absence in the nullitetra line lack-
ing the respective homoeologous chromosome and by
its presence in all other samples. Haplotypes with read
frequency < 2 were discarded.
Expression patterns of genes expressed from all three
homoeoloci
For genes with homoeolocus-specific sequences from all
three homoeoloci, duplicate read pairs with the same se-
quence, orientation and start position were removed. The
chi-square test was used to identify differential expression
of homoeoloci. For each sequence region with exactly
three homoeolocus-specific haplotypes, and a minimum
coverage of 18 reads, the frequency of mapped reads with
each haplotype was tested against the null hypothesis of
equal expression from all three homoeoloci (1:1:1, ‘A=
B=D’). Unadjusted p< .01 were considered “suggestive”
of differential expression. Such regions were confirmed
as “significant”if passing a Bonferroni adjusted thresh-
old of p< .05 (accounting for the number of regions
tested), based on combined reads from two biological
replicates. Sequence regions indicating possible read align-
ments from paralogous genes were ignored, including
those with additional non-homoeolocus-specific haplo-
types in euploid wheat, or with more than one haplotype
in diploid wheat read alignments. Genes without any “sug-
gestive”evidence of differential expression were assigned
an equal expression pattern (‘A=B=D’).
For genes with “significant”differential expression of
homoeoloci, we assigned one of twelve patterns, includ-
ing six with all three homoeoloci expressed at distinct
levels (A > B > D, A > D > B, B > A > D, B > D > A, D > A >
B, D > B > A) and a further six with two homoeoloci
expressed at indistinguishable levels (A > B = D, B > A = D,
D > A = B, A = B > D, A = D > B, B = D > A). For each
quantified region, the three pairs of haplotypes (‘AB’,‘AD’
and ‘BD’) were compared to a null hypothesis of equal
expression from each homoeolocus (1:1), to determine
whether each pair of homoeoloci was expressed at equiva-
lent levels (e.g.,‘A=B’) or distinct levels (e.g., ‘A>B’), using
p< .01. Genes with evidence for a single pattern of differ-
ential expression in both biological replicates were called
“consistently biased”. For such genes, the expression level
from each homoeolocus was calculated by averaging the
proportions of aligned reads with each homoeolocus-
specific haplotype from all quantified regions, weighted by
the total number of aligned reads in each region. The
remaining genes were called “unknown,”and excluded
from further analysis. Genes in the “unknown”category in-
cluded those with evidence for multiple, inconsistent pat-
terns of differential expression, (e.g., both ‘D>A=B’and
‘A=D>B’) or inconsistent patterns between replicates.
This is explained by various factors, including low expres-
sion levels (leading to lack of statistical power to distin-
guish expression of homoeoloci), alternative splicing, or
RNA-Seq related biases [86]. We also assigned genes as
“unknown”if they showed differential expression only at
the “suggestive”level.
Assigning presence and expression of genes among
homoeologous chromosomes
A gene was defined as present on a specific homoeolo-
gous chromosome (A, B, or D) in the presence of evi-
dence from deletion bin mapping [58,63] or group 1
chromosome arm-specific assemblies [52]. The exist-
ence of homoeolocus-specific i.e., diagnostic sequences
(either individual sites (HSVs) or haplotypes) from RNA-
Seq enabled us to define both presence on, and expression
from, the corresponding homoeologous chromosome (see
Additional file 4: Figure S2). BLAST searches were used to
find the best match for each group 1 gene to sequences
from chromosome arms 1AL, 1BL, 1DL, 1AS and 1BS
[52]. No sequences were available for chromosome arm
1DS;seeAdditionalfile16:TableS7.Significanthitswere
defined using an e-value cut-off of 1e
−10
and required to
have a minimum stretch of 60 identical bases.
Experimental validation of homoeologue sequence
variants (HSVs)
Two micrograms of total RNA extracted from euploid
(CS) and nullitetra wheat root samples using the TRIzol
Reagent (Invitrogen) was reverse-transcribed to synthesize
first-strand cDNA using the SuperScript II First-Strand
Synthesis System for RT-PCR Kit (Invitrogen). cDNA was
treated with Qiagen RNase A to remove any residual
RNA. We selected 10 genes with differential expression of
homoeoloci and with three homoeolocus-specific tentative
contigs (TCs) identified from a BLAST search of the T.
aestivum TIGR database (Additional file 12: Table S4A).
The homoeologous sequences of each selected gene
were extended from the 3′end using the homoeologous
TC sequences to provide highly variable regions for
homoeolocus-specific primer design. All PCR primers
were designed using Primer 3 (http://frodo.wi.mit.edu/)
to amplify either all three homoeoloci (using common
forward and reverse primers) or specific homoeoloci (using
homoeolocus-specific reverse primers). Homoeolocus-
specificity was confirmed by RT-PCR with the complete
set of nullitetra lines. Each homoeolocus-specific PCR
amplified the homoeologous product (i.e., gel bands were
detected) in all samples except for the nullitetra lines
lacking the corresponding homoeologous chromosome.
Homoeologous sequences were confirmed by purification
of PCR products using the QIAquick PCR purification kit
(QIAGEN) and Sanger sequencing. We could amplify all
three homoeoloci for all 10 genes. For 6 of these, all three
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homoeolocus-specific primer sets amplified a single homo-
eolocus with 100% specificity. These genes were selected
for experimental verification of homoeolocus expression
patterns. For the remaining 4 genes, it was not possible to
design three completely specific primer sets, so their ex-
pression patterns were not verified. Details of primers and
their sequences are listed in Additional file 12: Table S4C.
Experimental validation of differential homoeologous
expression patterns
Two independent approaches were used to experimentally
validate differential homoeolocus expression patterns for 6
genes with all three homoeoloci amplified specifically (see
Additional file 12: Table S4B). First, homoeolocus-specific
PCR amplification products from the exponential amplifi-
cation phase were run on a 1% agarose gel stained with
ethidium bromide (Sigma) and band intensities were
compared to quantify the expression levels of each homo-
eolocus (A, B, D). Second, all three homoeoloci amplified
using common primers were purified by ethanol precipita-
tion, ligated into pGEM-T Easy Vector and transformed
into One Shot Top 10 competent cells (Invitrogen).
Transformed cells were spread onto Luria-Broth agar
plates containing ampicillin and incubated at 37°C over-
night. 96 colonies were picked and cultured in 2 ml
tubes at 37°C overnight. Plasmids were extracted using
the Wizard SV 96 Plasmid kit (Promega) and cloned
products were Sanger sequenced using T7 and SP6
primers. HSVs were used to assign the homoeolocus
identity (A, B or D) of each clone, reflecting the relative
proportions of the three homoeoloci in vivo.
Availability of supporting data
The Illumina RNA-Seq data supporting the results of
this article is available [62] in the NCBI sequence read
archive [SRA: SRP028357].
Additional files
Additional file 1: Table S1. Illumina RNA-sequencing data and read
mapping statistics. This table summarizes the RNA-Seq datasets obtained
from non-normalized cDNA libraries of wheat samples and the mapping
results for alignment of reads to a partial reference transcriptome
sequence using novoalign software.
Additional file 2: Table S2. Origin of EST sequences. This table shows
the species of origin for EST sequences used to construct the partial
wheat reference transcriptome.
Additional file 3: Figure S1. Length distribution for ESTs on wheat
chromosome groups 1 and 5. This figure shows the length distribution
for ESTs used to construct the partial wheat reference transcriptome.
Additional file 4: Figure S2. Schematic illustration of homoeologue-
specific variant (HSV) discovery in hexaploid bread wheat. This figure
shows the bioinformatics pipeline used to identify and quantify
homoeolocus-specific bread wheat sequences.
Additional file 5: Figure S3. Qualitative gene expression in bread
wheat. This figure shows the overlap between expressed genes in two
biological replicates for shoot and root tissues.
Additional file 6: Figure S4. Number of haplotypes observed within
RNA-Seq reads mapped to regions of the reference sequence covered by
homoeolocus-specific haplotypes. This figure shows the distribution of
the number of haplotypes in 26,498 regions from 872 genes expressed
from all three homoeologous chromosomes of group 1 (1A, 1B, 1D) or
group 5 (5A, 5B, 5D) in roots.
Additional file 7: Figure S5. Properties of transcript sequences for
wheat genes on chromosome groups 1 and 5 showing either equal
expression from all three homoeoloci (‘A=B=D’), differential expression of
homoeoloci (‘DE’) or an unknown pattern of expression bias (‘U’)in
shoots (white) or roots (blue). This figure shows how the total expression
level (RPKM) and HSV (homoeologue-specific variant) density varies
between genes with different patterns of expression from three
homoeoloci (A, B and D).
Additional file 8: Figure S6. Biased patterns of expression for
homoeoloci on wheat group 1 and 5 chromosomes. This figure shows
the contribution of transcripts from A, B and D homoeoloci for genes
showing each possible pattern of differential expression.
Additional file 9: Table S3. Significance of differential expression
patterns for genes expressed from all three group 1 or 5 homoeoloci in
shoots or roots. This table shows the significance level of differentially
expressed genes in two biological replicates of shoots and roots.
Additional file 10: Figure S7. Relationship between total gene
expression level (RPKM) and variation in expression among three
expressed homoeoloci. This figure shows the relationship between the
total expression level of genes expressed from all three homoeoloci and
the strength of differential expression bias.
Additional file 11: Figure S8. Gel images depicting amplification of A,
B and D homoeoloci of 6 genes by homoeologue-specific RT-PCR. This
figure shows experimental confirmation of the homoeologue-specificity
of A, B and D homoeoloci and the corresponding expression patterns of
6 group 1 or group 5 genes expressed from all three homoeoloci
(A, B and D).
Additional file 12: Table S4. Experimental validation of homoeolocus
expression patterns shown by RNA-Seq of euploid wheat roots. This table
details the experimental verification of homoeologue-specific variants
(HSVs) and expression patterns for 6 genes expressed from all three
homoeoloci (A, B and D).
Additional file 13: Figure S9. Total expression level of wheat genes on
group 1 and 5 chromosomes expressed from all three homoeoloci in
both shoots and roots. This figure shows the total expression level of
genes expressed from all three homoeoloci in both tissues is significantly
higher in roots compared with shoots.
Additional file 14: Table S5. Conflicting homoeolocus expression
patterns between shoot and root organs of bread wheat. This table
shows genes with different patterns of homoeolocus expression in shoot
versus root tissues.
Additional file 15: Table S6. Presence and expression of up to three
homoeoloci for wheat group 1 genes. This table shows the number of
genes physically present on and expressed from (in shoots or roots) each
combination of group 1 homoeologous chromosomes.
Additional file 16: Table S7. Dataset of assembled sequences for
wheat group 1 chromosome arms. This table shows the dataset of
assembled sequences for genes on wheat chromosomes 1A, 1B and 1D
from Wicker et al. [52].
Additional file 17: Table S8. Comparison of homoeolocus expression
between shoots and roots for genes on group 1 and 5 chromosomes.
This table shows that most genes are expressed from the same
homoeologous chromosomes in the two wheat tissues (shoots
and roots).
Abbreviations
HSV: Homoeologue-specific variant; Nullitetra: Nullisomic-tetrasomic.
Leach et al. BMC Genomics 2014, 15:276 Page 16 of 19
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Competing interests
The authors declare that they have no competing interests.
Authors’contributions
NPH conceived of and designed the study, and helped to write the
manuscript. LJL and EJB participated in the design and coordination of the
research. LJL performed the RNA-Seq data analysis, experimental verification
of genes with differential expression of homoeoloci by RT-PCR and PCR
product cloning and wrote the paper. CB grew the wheat plants and
collected samples for RNA-Sequencing. CJ performed some cloning
experiments. AM contributed to the data analysis. All authors read and
approved the final manuscript.
Acknowledgements
We thank Dr Nils Stein and Dr Thomas Wicker for kindly providing their
dataset of assembled chromosome 1 wheat genes. We also thank Steve
Reader (John Innes Centre, Norwich, UK) for provision of wheat materials and
David Buck and his team at the Wellcome Trust Centre for Human Genetics
(WTCHG, Oxford) for performing all of the RNA sequencing and providing
valuable support and assistance. This work was supported through funding
(of authors LJL, EJB, CJ, CB and AM) in a grant from King Abdullah University
of Science and Technology (KAUST) in Saudi Arabia to NPH and through
funding from the Biotechnology and Biological Sciences Research Council
(BB/F020759/1) to NPH.
Author details
1
Department of Plant Sciences, University of Oxford, Oxford, UK.
2
School of
Biosciences, University of Birmingham, Birmingham, UK.
3
Syed Babar Ali
School of Science and Engineering, Lahore University of Management
Sciences, Lahore, Pakistan.
Received: 7 November 2013 Accepted: 2 April 2014
Published: 11 April 2014
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doi:10.1186/1471-2164-15-276
Cite this article as: Leach et al.:Patterns of homoeologous gene
expression shown by RNA sequencing in hexaploid bread wheat. BMC
Genomics 2014 15:276.
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