BACKGROUND
allopolyploidization is an important driver of plant speciation  <cit> .
in the initial hybridization two  distinct genomes are combined within a single nucleus, with fertility subsequently being restored by a whole genome doubling   <cit> .
although a de novo polyploid carries a complete copy of each of its constituent genomes, the early post-allopolyploidization generations typically experience a spectrum of genomic changes , a response to “genome shock”  <cit> .
by definition, allopolyploids carry more than one copy of any given single copy gene, but these copies are not necessarily transcribed and expressed in an additive fashion .
the genomic perturbations and alterations in individual homoeologue transcription induced by the allopolyploidization process generate a level of genetic novelty which gives opportunities for selection  <cit> .
the independence of the hybridization and wgd events has allowed for an experimental demonstration that it is the former which induces most of the transcriptomic changes associated with alloployploidization, possibly as a result of its relaxation of transcriptional regulation .
however, in senecio sp., wgd was also found have an obvious effect, since the extent of the transcriptional changes appeared to be less marked in the allohexaploid setting than in its allohaploid progenitor  <cit> .
in a small number of cases, the opposite has been demonstrated, i.e., that allopolyploidization can further disrupt gene transcription  <cit> .
the most recent allopolyploidization event in the evolution of bread wheat  involved the formation of a hybrid between a cultivated form of the ab genome allotetraploid species t. turgidum and the d genome diploid goatgrass aegilops tauschii .
comparisons of transcription profiles between re-synthesized hexaploid wheat and its progenitors have suggested that additivity between homoeologues is commoner than non-additivity  <cit> .
however, the phenomenon of parental expression level dominance  has also been documented: this relates to the situation where, for a set of homoeologues transcribed at different levels in the parents, the total expression level of these homoeologues in the progeny is statistically similar to that of one parent  <cit> .
the suggestion has been made that in wheat, eld may underlie some of the vigour and adaptability of the species  <cit> .
the acquisition of draft genome sequences for bread wheat and its a and d genome donors  provides an unparalleled opportunity to track homoeologue transcript abundances during allohexaploidization .
here, the global gene transcription profiles in the leaf of allotriploid hybrids and their wgd-derived allohexaploid product have been compared.
the picture which emerges is one where both the hybridization and wgd events have an influence over the transcriptome.
RESULTS
the phenotype of de novo synthesized hexaploid wheat, its allotriploid form and its progenitors
the f <dig> plants  were generated from a wide cross between either t. turgidum  ssp.
turgidum accession as <dig> or ssp.
durum ldn  and a. tauschii accession as <dig> .
the chromosomes in the allotriploid metaphase i meiocytes typically formed  <dig> univalents , but a significant number of immature microspores formed a dyad, diagnostic of the presence of an unreduced gamete .
zygotes derived from the union of two unreduced gametes represent a wgd event, producing an allohexaploid  embryo .fig.
<dig> the morphology and cytology of t. turgidum as <dig> , a. tauschii as <dig> , the allotriploid as2255 × as <dig>  and the derived allohexaploid .
a fluorescent in situ hybridization  analysis of the  <dig> univalents presents at meiosis metaphase i in the meiocyte of an allotriploid plant.
the probe 6c6- <dig> hybridizing to the centromeres fluoresced green.
bar: 10 μm.
b allotriploid pollen mother cells comprise a mixture of dyads  and tetrads .
c multi-colour genomic in situ hybridization of a root tip mitotic cell from an allohexaploid plant, showing 2n = 6x =  <dig>  d sequential multi-colour fish of a root tip mitotic cell from an allohexaploid plant, showing that chromosomes of the a, b and d genome were all represented on basis of probes psc <dig>  , pas <dig> , and pta <dig> .
e morphology of 120 day old plants of as <dig>  as <dig> and their derived allotriploid  and allohexaploid .
f leaf width and length of the first four leaves of the plants.
whiskers indicate sd
the allotriploid plants were less vigorous than their t. turgidum progenitor .
their leaf length and width were intermediate between the two parents’ during the early stage of the plants’ development .
in contrast the leaf length of the allohexaploid plants in the first generation  was greater than their t. turgidum progenitor’s in both combinations .
as allotriploid and allohexaploid s <dig> plants were most distinguishable at this stage  and there were no noticeable differences in morphology among s1-s <dig> euploid plants , we collected leaves at this stage from s <dig> plants to isolate rna for sequencing.
for all s <dig> plants used, root tip cells were checked to make sure they contained a complete set of chromosomes without structure variation.
homoeologue discrimination and transcript abundance in the allotriploid, allohexaploid and parental plants
rna-seq analysis was applied to rna extracted from allotriploid, allohexaploid  and the progenitor plants .
the number of clean reads ranged from  <dig>  to  <dig>  million, and a mean of  <dig> % of these were mappable to the cv.
chinese spring  draft genome sequence.
after a stringent filtering step using the method by pfeifer et al.
<cit> , an average of 36% of the mapped reads could be allocated to one of the homoeologues.
the transcript abundance of each homoeologue was then quantified using htseq-count  <cit> , based on the high confidence gene  dataset  <cit> , comprising  <dig>  gene models.
counts were expressed as fragments per kilobase of exon model per million mapped base pairs   <cit> .
only homoeologues/genes showing an fpkm greater than unity in at least one sample within a lineage were retained.
abundances of biological duplicates proved to be strongly correlated .
a correlation dendrogram showed that the allotriploid was more closely related to the allohexaploid plant than to its tetraploid progenitor, with the diploid progenitors appearing as outliers , as would be expected from the various plants’ genome constitutions.
although the transcripts of the two t. turgidum accessions mapped for the most part to the a and b genome chromosomes, a small number were associated with a d genome location; similarly, a few of the a. tauschii transcripts apparently mapped to an a or b genome chromosome location .
these locations are likely artefacts, deriving from polymorphism between the parental genome sequences and corresponding subgenome sequences of cs.
these reads were discarded.
the median relative abundance of the ~ <dig>  genes for which a read of each homoeologue was obtained was close to unity in both hybridization lineages, while the peak value for fpkm was > <dig> .
applying a threshold of fpkm > <dig>   <dig>   and  <dig>   genes were identified for each species .
based on the criteria 90% sequence identity and 90% alignment, the cs gene models could be classified as  <dig>  triplets ,  <dig>  duplets  and  <dig>  singletons .
an average of 60% triplets, 50% duplets and 30% singletons were identified in the transcriptomes of hybridization combination .
for the most part the two hybrid lineages behaved similarly.
what follows relates mostly to the as2255 × as <dig> combination, unless indicated otherwise.
although the identity of the t. turgidum parent had little influence over the pattern of homoeologue transcription, there were some differences in the estimated overall gene number.
data relating to the ldn × as <dig> combination are given in additional file 3: dataset s <dig>  additional file 4: dataset s <dig>  additional file 5: dataset s <dig> and additional file 1: figures s3–s <dig> .
wgd restored homoeologue transcript abundances disturbed by hybridization
in all,  <dig> % of the set of d-subgenome homoeologues  in the allotriploid plants compared to diploid parents as <dig> of as2255 × as <dig> combination were down-regulated.
the equivalent frequency for the a and b genome homoeologues compared to tetraploid parents as <dig> was, respectively, only  <dig> % and  <dig> % .
the frequency of up-regulated homoeologues was small for all three genomes .
an even smaller proportion  was altered in the move from abd to aabbdd .
however,  <dig>  d-subgenome homoeologues appeared to be down-regulated when the contrast was made between the allohexaploid and as <dig>  a number which was ~25% less than in the contrast between the allotriploid and as <dig>  the proportion of a and b genome homoeologues down-regulated in the allohexaploid was also lower .
the implication is that wgd effectively reduced the number of differentially transcribed homoeologues, probably by minor changes but non-significant difference on statistical analyzing using fdr-adjusted p-value below  <dig>  that potentially cause inflated comparisons of expression level differences.
in order to confirm this suggestion we analyzed theses differential expressed genes only appeared between f <dig> and parents by boxplot analysis using r packages.
this corrective effect was supported by the contrasting effects of hybridization and wgd on homoeologue transcript abundances: for example,  <dig>   d-subgenome homoeologues were significantly down-regulated in the allotriploid plants , and their transcript abundances were marginally increased in allohexaploid , as demonstrated by the upward shift in the median abundance.
a similar contrasting effect was observed for homoeologues up-regulated in the allotriploid plants, even for the a and b genome homoeologues.
at the same time,  <dig>  d genome homoeologues remained down-regulated in the allohexaploid .
the large number of genes for which the level of transcription was corrected in this manner suggests that this is an important feature of the polyploid wheat transcriptome.fig.
<dig> variation in the transcription of homoelogues as a result of allotriploidization and wgd in the as2255 × as <dig> lineage.
a differentially transcribed homoeologues.
the number next to the symbol for the species represents the number of differentially up-regulated homoeologues vs. the neighboring species linked by a line.
a consistent colour has been used to refer to each genome .
numbers in the middle of each line represent the total numbers of differentially transcribed homoeologues .
b boxplots illustrating the effect of allotriploidization and wgd on transcript abundance: homoeologues from  the a genome,  the b genome,  the d genome.
differentially transcribed d genome homoeologues between the allotriploid and parent that were transmitted into allohexaploid are used as controls .
boxes span the data range between the first and third quartiles, and the median is represented as a horizontal line.
whiskers extend to the most extreme data point, which is no more than  <dig>  times the interquartile range away from the first and third quartiles.
the widths of the boxes are proportional to the gene numbers
a further effect of wgd on the transcription of the set of unequally transcribed homoeologues  was noted: this set represented 25%  of the genes defined in the t. turgidum parent as <dig> for which at least one of the homoeologues showed an fpkm greater than unity ; however this proportion rose to 38% in the allotriploid plants, falling to 22% in the allohexaploids.
the decrease effect on uth proportion from allotriploids to allohexaploids applied similarly to a genome vs d genome and b genome vs d genome genes in both lineages .table  <dig> changes in the transcript abundance of homoeologues induced by allotriploidization and wgd*
*values differ significantly  following the application of the benjamini-hochberg multiple test correction.
based on the cs gene models, the triplet genes were used in the comparison of homoeologues between a-, b, and d-subgenomes.
only those that at least one of three homoeologues were expressed with fpkm > <dig> in parents  were analyzed
wgd corrected the non-additive down-regulation of genes induced by hybridization
based on the cs gene models, it was possible to determine the transcript abundance of each gene in the parental lines and their allotriploid and allohexaploid derivatives .
when mid-parent values were compared with those recorded in the allotriploid and allohexaploid plants, non-additivity was assignable to  <dig> genes  of the genes in the allotriploids, the abundance of most of which  was below the mid-parent value .
go analysis showed that genes related to cellular component terms “plastid” and “thylakoid” were well represented .
the d-subgenome homoeologues were especially affected:  <dig>  were less abundant in the allotriploids than in as <dig> .
however, the transcript abundance of only  <dig> of these genes remained below the mid-parent value in the allohexaploid plants.fig.
<dig> non-additive transcription of genes in the allotriploid and allohexaploid in the lineage as2255 × as <dig>  a numbers of non-additively transcribed genes in the progeny compared to mid-parent value .
the red numbers shown refer to genes up-regulated  or down-regulated  in the allotriploid  and allohexaploid .
b the number of non-additive genes common to the allotriploid and allohexaploid.
go enrichment terms for the genes non-additively down-regulated in the allotriploid are shown below the figure.
c homoeologue expression patterns of non-additively expressed genes.
“up” and “down” refer to homoeologues differentially transcribed between the progeny and the parents, whereas “no change” implies that the transcription levels were statistically unchanged by either the allotriploidization or the wgd
eld was frequent in both the allotriploid and allohexaploid plants
there was a five fold greater number of eld-ab genes  than eld-d genes  .
in each case, the transcription level in the allotriploid was more similar to that of the parent showing the higher transcription level .
the pattern was largely retained at the allohexaploid: more than 72% of the eld-ab genes behaved equivalently in the allotriploid and allohexaploid.
of the set of  <dig>  eld-ab genes identified in the allohexaploid, only  <dig> were not classed as eld in the allotriploid .
a go analysis of the eld genes shared by f <dig> and s <dig> revealed an enrichment for genes assigned to the cellular component “plastid”.
genes homologous to components of the rna-dependent dna methylation  pathway, in particular those encoding argonaute  <dig> , defective in meristem silencing  <dig> , and the rna-binding protein involved in de novo <dig> , were among the eld-ab genes identified in both the allotriploid and allohexaploid .
meanwhile, almost all  of eld-ab genes identified in the allotriploid plant achieved this status thanks to the down-regulation of their d genome homoeologue .fig.
<dig> parental expression level dominance  genes in the allotriploid and allohexaploid.
a the number of genes with a transcription level similar to that in t. turgidum  or that in as <dig>  in both the as2255 × as <dig> and ldn × as <dig> lineages.
b the number of eld-ab genes common to the allotriploid and allohexaploidand the associated enriched go terms.
c genes encoding major components of the rna-dependent dna methylation pathway  were classified as eld-ab genes.
the histograms show the fpkms of the relevant homoeologues in as <dig> , as <dig> , allotriploid  and allohexaploid
complementation of genome-specific singletons in allotriploid and allohexaploid
as referred to earlier,  <dig> % of the  <dig>  annotated cs genes were classified as singletons .
of the  <dig>  singletons assigned to high confidence group  <dig>   <cit> , over a quarter were associated with an fpkm greater than unity, and most were consistently transcribed in the as2255 × as <dig> allotriploid and allohexaploid .
among the  <dig> d-subgenome singletons transcribed in the allotriploid,  <dig> were also transcribed in the allohexaploid.
a go analysis revealed some enrichment in the processes “cellular macromolecule biosynthesis” and “cellular biosynthesis” .
some additional processes were enriched for the b genome singletons, including “secondary metabolism” and “response to abiotic stimulus”; and similarly for the a genome singletons with respect to “cellular biosynthesis”.
a number of functions were represented among the set of singletons .
the singletons identified in the allotriploid and allohexaploid of the other lineage  covered a similar set of functions .
members of the set of d-subgenome singletons were more frequently down-regulated in both the allotriploid and allohexaploid .fig.
<dig> the transcription of singletons in the as2255 × as <dig> lineage.
a singletons classified according to genome origin; enriched go terms found in the shared singleton genes are shown below the venn diagram.
b the function of singletons derived from the mapman program
discussion
nascent allohexaploid wheat has been used to introduce the genetic divergence from the tetraploid and diploid progenitors into bread wheat.
the poor vigour displayed by the allotriploid is greatly enhanced by wgd, restoring much of the phenotype exhibited by its tetraploid aabb progenitor.
as a result, it has proved highly practical to exploit de novo synthetic hexaploid wheat as a bridge to enable the transfer of genetic material into the bread wheat genepool from its wild donors  <cit> .
understanding how homoeologues respond at the transcriptional level to both the allotriploidization event and then the subsequent wgd is of relevance if the maximum benefit is to be gained from synthetic hexaploids.
the capacity to capture the entire transcriptome allows a global picture of gene reprogamming to be acquired, in contrast to earlier studies which relied on relatively low numbers of expressed sequences.
up to now, the focus of experiments aimed at characterizing homoeologue transcript abundances in polyploid wheat has been on established amphiploids  <cit> , an approach which ignores the possibility that alloploidization and wgd may have different, even opposing, effects.
if genomic shock is a reality, it will be felt most intensely in the initial hybrid rather than in its subsequent derivatives, as was demonstrated here, where the extent of transcriptome reprogramming induced by the allotriploidization process was much greater than that induced by the subsequent wgd.
the re-programming of d genome homoeologue transcription is a prominent characteristic of the allotriploid transcriptome
hybridization appears to relax the regulation of transcription, thereby inducing differences between a hybrid and its progenitors with respect to the level of transcription of many genes.
in diploids such as arabidopsis thaliana  <cit>  and rice  <cit> , the sequence-based tagging of alleles is straightforward, but in polyploids such as brassica napus  <cit> , cotton  <cit>  and wheat  <cit> , the situation is complicated by the presence of homoeologues.
bread wheat has evolved from two independent and temporally well separated polyploidization events.
the first of these, which occurred ca.
<dig>  m years ago, involved the hybridization of t. urartu with an aegilops species belonging to the sitopsis section to form t. turgidum; the second was between a cultivated form of t. turgidum and a. tauschii and dates to some  <dig>  m years ago  <cit> .
both events are likely to have a profound effect on the transcriptome.
the asymmetry involved in combining a tetraploid with a diploid may account for the observation that it was the d genome homoeologues which suffered the greatest extent of down-regulation in the de novo allotriploid.
one possibility is that the silencing involved is activated by the small rna-mediated dna methylation system, given that there appears to be a concentration of 24 nt rnas around a number of genic loci in the d genome  <cit> .
here, the data imply that d genome homoeologue regulation was the major contributor to the altered transcription patterns exhibited by the allotriploid plants.
a major proportion of the non-additive transcribed genes and the eld-ab genes were also associated with the d-subgenome homoeologues.
curiously, non-additive transcription in the allotriploid plants was for the most part reversed in the allohexaploids, whereas a large proportion of eld genes maintained their status following the wgd.
it is conceivable therefore that the former genes are in some way responsible for the inferior growth of the allotriploid plants, while the latter help to restore vigour to the allohexaploids.
the restoration by wgd of parental homoeologue transcript abundance
wgd is required to restore fertility via the provision of pairs of homologous chromosomes.
it also has the effect, as shown here, of reversing some of the disturbance to transcription induced by the hybridization; a similar effect has been noted in senecio × baxteri when hybrids are created between tetraploid and diploid forms  <cit> .
in wheat, wgd appears to have a relatively small, non-genome specific effect on homoeologue transcription.
it reduced the number of uths present, an effect which may contribute to the observed reduction in non-additively transcribed genes and hence to the restoration of hybrid vigour.
this contrasts to the role of wgd in polyploid rice, in which wgd has been shown to disrupt transcription  <cit> .
the high rate of maintenance of eld status in the transition from allotriploid to allohexaploid  may go some way to explaining the morphological similarity between the allohexaploid and tetraploid plants.
several of the eld-ab genes were homologues of genes encoding rna-dependent dna methylation , which could imply that the maternal parent’s epigenetic modification system was maintained during the process of allohexaploidization.
singletons may represent a source of novel functionality
the bioinformatics-based analysis of the cs transcriptome suggested that over 50% of the gene content comprised singletons.
this number may well represent an overestimate, since the current wheat gene model set was derived from an assembly of rather short reads  <cit> .
a large number of the supposed singletons were found in the transcriptomes of both the allotriploid and allohexaploid, so they may well have a prominent effect on the phenotype of both hybrid forms.
the combination of singletons from the three genomes may provide extra functions in hexaploid wheat.
in maize, genes exhibiting so called “single parent transcription” - that is those which are silent in one of the parents of a hybrid, but transcribed both in the other parent and in the hybrid - have been shown to contribute materially to heterosis  <cit> .
in bread wheat, a number of genes underlying agronomic traits  have no known homoeologues  <cit> , which confirms the notion that singletons in allopolyploids can be very important determinants of biological function.
CONCLUSIONS
while wgd is believed to fix heterosis  <cit> , here many of the non-additively transcribed genes identified in allotriploid wheat did not behave in this fashion at the allohexaploid level; a plausible interpretation of this phenomenon is that the effect of wgd can be hybrid specific.
rather, the persistence of eld implies a role for this class of gene in determining the phenotype of hexaploid wheat.
the combination of some eld genes lends support to the “dominance model”, which proposes that the superior performance of a hybrid is due to the complementation of deleterious recessive alleles by dominant alleles at multiple genes  <cit> .

 allopolyploidization is an important driver of plant speciation. in the initial hybridization two distinct genomes are combined within a single nucleus, with fertility subsequently being restored by a whole genome doubling. although a de novo polyploid carries a complete copy of each of its constituent genomes, the early post-allopolyploidization generations typically experience a spectrum of genomic changes, a response to “genome shock”. by definition, allopolyploids carry more than one copy of any given single copy gene, but these copies are not necessarily transcribed and expressed in an additive fashion. the genomic perturbations and alterations in individual homoeologue transcription induced by the allopolyploidization process generate a level of genetic novelty which gives opportunities for selection. the independence of the hybridization and wgd events has allowed for an experimental demonstration that it is the former which induces most of the transcriptomic changes associated with alloployploidization, possibly as a result of its relaxation of transcriptional regulation. however, in senecio sp., wgd was also found have an obvious effect, since the extent of the transcriptional changes appeared to be less marked in the allohexaploid setting than in its allohaploid progenitor. in a small number of cases, the opposite has been demonstrated, i.e., that allopolyploidization can further disrupt gene transcription. the most recent allopolyploidization event in the evolution of bread wheat involved the formation of a hybrid between a cultivated form of the ab genome allotetraploid species t. turgidum and the d genome diploid goatgrass aegilops tauschii. comparisons of transcription profiles between re-synthesized hexaploid wheat and its progenitors have suggested that additivity between homoeologues is commoner than non-additivity. however, the phenomenon of parental expression level dominance has also been documented: this relates to the situation where, for a set of homoeologues transcribed at different levels in the parents, the total expression level of these homoeologues in the progeny is statistically similar to that of one parent. the suggestion has been made that in wheat, eld may underlie some of the vigour and adaptability of the species. the acquisition of draft genome sequences for bread wheat and its a and d genome donors provides an unparalleled opportunity to track homoeologue transcript abundances during allohexaploidization. here, the global gene transcription profiles in the leaf of allotriploid hybrids and their wgd-derived allohexaploid product have been compared. the picture which emerges is one where both the hybridization and wgd events have an influence over the transcriptome.  the phenotype of de novo synthesized hexaploid wheat, its allotriploid form and its progenitors the f plants were generated from a wide cross between either t. turgidum ssp. turgidum accession as or ssp. durum ldn and a. tauschii accession as. the chromosomes in the allotriploid metaphase i meiocytes typically formed univalents, but a significant number of immature microspores formed a dyad, diagnostic of the presence of an unreduced gamete. zygotes derived from the union of two unreduced gametes represent a wgd event, producing an allohexaploid embryo.fig. the morphology and cytology of t. turgidum as, a. tauschii as, the allotriploid as2255 × as and the derived allohexaploid. a fluorescent in situ hybridization analysis of the univalents presents at meiosis metaphase i in the meiocyte of an allotriploid plant. the probe 6c6- hybridizing to the centromeres fluoresced green. bar: 10 μm. b allotriploid pollen mother cells comprise a mixture of dyads and tetrads. c multi-colour genomic in situ hybridization of a root tip mitotic cell from an allohexaploid plant, showing 2n = 6x =  d sequential multi-colour fish of a root tip mitotic cell from an allohexaploid plant, showing that chromosomes of the a, b and d genome were all represented on basis of probes psc, pas, and pta. e morphology of 120 day old plants of as as and their derived allotriploid and allohexaploid. f leaf width and length of the first four leaves of the plants. whiskers indicate sd the allotriploid plants were less vigorous than their t. turgidum progenitor. their leaf length and width were intermediate between the two parents’ during the early stage of the plants’ development. in contrast the leaf length of the allohexaploid plants in the first generation was greater than their t. turgidum progenitor’s in both combinations. as allotriploid and allohexaploid s plants were most distinguishable at this stage and there were no noticeable differences in morphology among s1-s euploid plants, we collected leaves at this stage from s plants to isolate rna for sequencing. for all s plants used, root tip cells were checked to make sure they contained a complete set of chromosomes without structure variation. homoeologue discrimination and transcript abundance in the allotriploid, allohexaploid and parental plants rna-seq analysis was applied to rna extracted from allotriploid, allohexaploid and the progenitor plants. the number of clean reads ranged from to million, and a mean of % of these were mappable to the cv. chinese spring draft genome sequence. after a stringent filtering step using the method by pfeifer et al., an average of 36% of the mapped reads could be allocated to one of the homoeologues. the transcript abundance of each homoeologue was then quantified using htseq-count, based on the high confidence gene dataset, comprising gene models. counts were expressed as fragments per kilobase of exon model per million mapped base pairs. only homoeologues/genes showing an fpkm greater than unity in at least one sample within a lineage were retained. abundances of biological duplicates proved to be strongly correlated. a correlation dendrogram showed that the allotriploid was more closely related to the allohexaploid plant than to its tetraploid progenitor, with the diploid progenitors appearing as outliers, as would be expected from the various plants’ genome constitutions. although the transcripts of the two t. turgidum accessions mapped for the most part to the a and b genome chromosomes, a small number were associated with a d genome location; similarly, a few of the a. tauschii transcripts apparently mapped to an a or b genome chromosome location. these locations are likely artefacts, deriving from polymorphism between the parental genome sequences and corresponding subgenome sequences of cs. these reads were discarded. the median relative abundance of the ~ genes for which a read of each homoeologue was obtained was close to unity in both hybridization lineages, while the peak value for fpkm was >. applying a threshold of fpkm > and genes were identified for each species. based on the criteria 90% sequence identity and 90% alignment, the cs gene models could be classified as triplets, duplets and singletons. an average of 60% triplets, 50% duplets and 30% singletons were identified in the transcriptomes of hybridization combination. for the most part the two hybrid lineages behaved similarly. what follows relates mostly to the as2255 × as combination, unless indicated otherwise. although the identity of the t. turgidum parent had little influence over the pattern of homoeologue transcription, there were some differences in the estimated overall gene number. data relating to the ldn × as combination are given in additional file 3: dataset s additional file 4: dataset s additional file 5: dataset s and additional file 1: figures s3–s. wgd restored homoeologue transcript abundances disturbed by hybridization in all, % of the set of d-subgenome homoeologues in the allotriploid plants compared to diploid parents as of as2255 × as combination were down-regulated. the equivalent frequency for the a and b genome homoeologues compared to tetraploid parents as was, respectively, only % and %. the frequency of up-regulated homoeologues was small for all three genomes. an even smaller proportion was altered in the move from abd to aabbdd. however, d-subgenome homoeologues appeared to be down-regulated when the contrast was made between the allohexaploid and as a number which was ~25% less than in the contrast between the allotriploid and as the proportion of a and b genome homoeologues down-regulated in the allohexaploid was also lower. the implication is that wgd effectively reduced the number of differentially transcribed homoeologues, probably by minor changes but non-significant difference on statistical analyzing using fdr-adjusted p-value below that potentially cause inflated comparisons of expression level differences. in order to confirm this suggestion we analyzed theses differential expressed genes only appeared between f and parents by boxplot analysis using r packages. this corrective effect was supported by the contrasting effects of hybridization and wgd on homoeologue transcript abundances: for example, d-subgenome homoeologues were significantly down-regulated in the allotriploid plants, and their transcript abundances were marginally increased in allohexaploid, as demonstrated by the upward shift in the median abundance. a similar contrasting effect was observed for homoeologues up-regulated in the allotriploid plants, even for the a and b genome homoeologues. at the same time, d genome homoeologues remained down-regulated in the allohexaploid. the large number of genes for which the level of transcription was corrected in this manner suggests that this is an important feature of the polyploid wheat transcriptome.fig. variation in the transcription of homoelogues as a result of allotriploidization and wgd in the as2255 × as lineage. a differentially transcribed homoeologues. the number next to the symbol for the species represents the number of differentially up-regulated homoeologues vs. the neighboring species linked by a line. a consistent colour has been used to refer to each genome. numbers in the middle of each line represent the total numbers of differentially transcribed homoeologues. b boxplots illustrating the effect of allotriploidization and wgd on transcript abundance: homoeologues from the a genome, the b genome, the d genome. differentially transcribed d genome homoeologues between the allotriploid and parent that were transmitted into allohexaploid are used as controls. boxes span the data range between the first and third quartiles, and the median is represented as a horizontal line. whiskers extend to the most extreme data point, which is no more than times the interquartile range away from the first and third quartiles. the widths of the boxes are proportional to the gene numbers a further effect of wgd on the transcription of the set of unequally transcribed homoeologues was noted: this set represented 25% of the genes defined in the t. turgidum parent as for which at least one of the homoeologues showed an fpkm greater than unity; however this proportion rose to 38% in the allotriploid plants, falling to 22% in the allohexaploids. the decrease effect on uth proportion from allotriploids to allohexaploids applied similarly to a genome vs d genome and b genome vs d genome genes in both lineages.table changes in the transcript abundance of homoeologues induced by allotriploidization and wgd* *values differ significantly following the application of the benjamini-hochberg multiple test correction. based on the cs gene models, the triplet genes were used in the comparison of homoeologues between a-, b, and d-subgenomes. only those that at least one of three homoeologues were expressed with fpkm > in parents were analyzed wgd corrected the non-additive down-regulation of genes induced by hybridization based on the cs gene models, it was possible to determine the transcript abundance of each gene in the parental lines and their allotriploid and allohexaploid derivatives. when mid-parent values were compared with those recorded in the allotriploid and allohexaploid plants, non-additivity was assignable to genes of the genes in the allotriploids, the abundance of most of which was below the mid-parent value. go analysis showed that genes related to cellular component terms “plastid” and “thylakoid” were well represented. the d-subgenome homoeologues were especially affected: were less abundant in the allotriploids than in as. however, the transcript abundance of only of these genes remained below the mid-parent value in the allohexaploid plants.fig. non-additive transcription of genes in the allotriploid and allohexaploid in the lineage as2255 × as a numbers of non-additively transcribed genes in the progeny compared to mid-parent value. the red numbers shown refer to genes up-regulated or down-regulated in the allotriploid and allohexaploid. b the number of non-additive genes common to the allotriploid and allohexaploid. go enrichment terms for the genes non-additively down-regulated in the allotriploid are shown below the figure. c homoeologue expression patterns of non-additively expressed genes. “up” and “down” refer to homoeologues differentially transcribed between the progeny and the parents, whereas “no change” implies that the transcription levels were statistically unchanged by either the allotriploidization or the wgd eld was frequent in both the allotriploid and allohexaploid plants there was a five fold greater number of eld-ab genes than eld-d genes. in each case, the transcription level in the allotriploid was more similar to that of the parent showing the higher transcription level. the pattern was largely retained at the allohexaploid: more than 72% of the eld-ab genes behaved equivalently in the allotriploid and allohexaploid. of the set of eld-ab genes identified in the allohexaploid, only were not classed as eld in the allotriploid. a go analysis of the eld genes shared by f and s revealed an enrichment for genes assigned to the cellular component “plastid”. genes homologous to components of the rna-dependent dna methylation pathway, in particular those encoding argonaute, defective in meristem silencing, and the rna-binding protein involved in de novo, were among the eld-ab genes identified in both the allotriploid and allohexaploid. meanwhile, almost all of eld-ab genes identified in the allotriploid plant achieved this status thanks to the down-regulation of their d genome homoeologue.fig. parental expression level dominance genes in the allotriploid and allohexaploid. a the number of genes with a transcription level similar to that in t. turgidum or that in as in both the as2255 × as and ldn × as lineages. b the number of eld-ab genes common to the allotriploid and allohexaploidand the associated enriched go terms. c genes encoding major components of the rna-dependent dna methylation pathway were classified as eld-ab genes. the histograms show the fpkms of the relevant homoeologues in as, as, allotriploid and allohexaploid complementation of genome-specific singletons in allotriploid and allohexaploid as referred to earlier, % of the annotated cs genes were classified as singletons. of the singletons assigned to high confidence group, over a quarter were associated with an fpkm greater than unity, and most were consistently transcribed in the as2255 × as allotriploid and allohexaploid. among the d-subgenome singletons transcribed in the allotriploid, were also transcribed in the allohexaploid. a go analysis revealed some enrichment in the processes “cellular macromolecule biosynthesis” and “cellular biosynthesis”. some additional processes were enriched for the b genome singletons, including “secondary metabolism” and “response to abiotic stimulus”; and similarly for the a genome singletons with respect to “cellular biosynthesis”. a number of functions were represented among the set of singletons. the singletons identified in the allotriploid and allohexaploid of the other lineage covered a similar set of functions. members of the set of d-subgenome singletons were more frequently down-regulated in both the allotriploid and allohexaploid.fig. the transcription of singletons in the as2255 × as lineage. a singletons classified according to genome origin; enriched go terms found in the shared singleton genes are shown below the venn diagram. b the function of singletons derived from the mapman program discussion nascent allohexaploid wheat has been used to introduce the genetic divergence from the tetraploid and diploid progenitors into bread wheat. the poor vigour displayed by the allotriploid is greatly enhanced by wgd, restoring much of the phenotype exhibited by its tetraploid aabb progenitor. as a result, it has proved highly practical to exploit de novo synthetic hexaploid wheat as a bridge to enable the transfer of genetic material into the bread wheat genepool from its wild donors. understanding how homoeologues respond at the transcriptional level to both the allotriploidization event and then the subsequent wgd is of relevance if the maximum benefit is to be gained from synthetic hexaploids. the capacity to capture the entire transcriptome allows a global picture of gene reprogamming to be acquired, in contrast to earlier studies which relied on relatively low numbers of expressed sequences. up to now, the focus of experiments aimed at characterizing homoeologue transcript abundances in polyploid wheat has been on established amphiploids, an approach which ignores the possibility that alloploidization and wgd may have different, even opposing, effects. if genomic shock is a reality, it will be felt most intensely in the initial hybrid rather than in its subsequent derivatives, as was demonstrated here, where the extent of transcriptome reprogramming induced by the allotriploidization process was much greater than that induced by the subsequent wgd. the re-programming of d genome homoeologue transcription is a prominent characteristic of the allotriploid transcriptome hybridization appears to relax the regulation of transcription, thereby inducing differences between a hybrid and its progenitors with respect to the level of transcription of many genes. in diploids such as arabidopsis thaliana and rice, the sequence-based tagging of alleles is straightforward, but in polyploids such as brassica napus, cotton and wheat, the situation is complicated by the presence of homoeologues. bread wheat has evolved from two independent and temporally well separated polyploidization events. the first of these, which occurred ca.  m years ago, involved the hybridization of t. urartu with an aegilops species belonging to the sitopsis section to form t. turgidum; the second was between a cultivated form of t. turgidum and a. tauschii and dates to some  m years ago. both events are likely to have a profound effect on the transcriptome. the asymmetry involved in combining a tetraploid with a diploid may account for the observation that it was the d genome homoeologues which suffered the greatest extent of down-regulation in the de novo allotriploid. one possibility is that the silencing involved is activated by the small rna-mediated dna methylation system, given that there appears to be a concentration of 24 nt rnas around a number of genic loci in the d genome. here, the data imply that d genome homoeologue regulation was the major contributor to the altered transcription patterns exhibited by the allotriploid plants. a major proportion of the non-additive transcribed genes and the eld-ab genes were also associated with the d-subgenome homoeologues. curiously, non-additive transcription in the allotriploid plants was for the most part reversed in the allohexaploids, whereas a large proportion of eld genes maintained their status following the wgd. it is conceivable therefore that the former genes are in some way responsible for the inferior growth of the allotriploid plants, while the latter help to restore vigour to the allohexaploids. the restoration by wgd of parental homoeologue transcript abundance wgd is required to restore fertility via the provision of pairs of homologous chromosomes. it also has the effect, as shown here, of reversing some of the disturbance to transcription induced by the hybridization; a similar effect has been noted in senecio × baxteri when hybrids are created between tetraploid and diploid forms. in wheat, wgd appears to have a relatively small, non-genome specific effect on homoeologue transcription. it reduced the number of uths present, an effect which may contribute to the observed reduction in non-additively transcribed genes and hence to the restoration of hybrid vigour. this contrasts to the role of wgd in polyploid rice, in which wgd has been shown to disrupt transcription. the high rate of maintenance of eld status in the transition from allotriploid to allohexaploid may go some way to explaining the morphological similarity between the allohexaploid and tetraploid plants. several of the eld-ab genes were homologues of genes encoding rna-dependent dna methylation, which could imply that the maternal parent’s epigenetic modification system was maintained during the process of allohexaploidization. singletons may represent a source of novel functionality the bioinformatics-based analysis of the cs transcriptome suggested that over 50% of the gene content comprised singletons. this number may well represent an overestimate, since the current wheat gene model set was derived from an assembly of rather short reads. a large number of the supposed singletons were found in the transcriptomes of both the allotriploid and allohexaploid, so they may well have a prominent effect on the phenotype of both hybrid forms. the combination of singletons from the three genomes may provide extra functions in hexaploid wheat. in maize, genes exhibiting so called “single parent transcription” - that is those which are silent in one of the parents of a hybrid, but transcribed both in the other parent and in the hybrid - have been shown to contribute materially to heterosis. in bread wheat, a number of genes underlying agronomic traits have no known homoeologues, which confirms the notion that singletons in allopolyploids can be very important determinants of biological function.  while wgd is believed to fix heterosis, here many of the non-additively transcribed genes identified in allotriploid wheat did not behave in this fashion at the allohexaploid level; a plausible interpretation of this phenomenon is that the effect of wgd can be hybrid specific. rather, the persistence of eld implies a role for this class of gene in determining the phenotype of hexaploid wheat. the combination of some eld genes lends support to the “dominance model”, which proposes that the superior performance of a hybrid is due to the complementation of deleterious recessive alleles by dominant alleles at multiple genes. 
the hybridization event strongly down-regulated d-subgenome homoeologues, but this effect was in many cases reversed by whole genome doubling. the implication is that whole genome doubling helps to overcome the phenotypic weakness of the allotriploid, restoring a more favourable gene dosage in genes experiencing transcription level dominance in hexaploid wheat. here, rna-seq was used to obtain the genome-wide leaf transcriptome of two independent triticum turgidum × aegilops tauschii allotriploids, along with their spontaneous allohexaploids and their parental lines. singletons were frequently transcribed at both the allotriploid and allohexaploid plants. the formation of an allopolyploid is a two step process, comprising an initial wide hybridization event, which is later followed by a whole genome doubling.
 the formation of an allopolyploid is a two step process, comprising an initial wide hybridization event, which is later followed by a whole genome doubling. both processes can affect the transcription of homoeologues. here, rna-seq was used to obtain the genome-wide leaf transcriptome of two independent triticum turgidum × aegilops tauschii allotriploids, along with their spontaneous allohexaploids and their parental lines. the resulting sequence data were then used to characterize variation in homoeologue transcript abundance.  the hybridization event strongly down-regulated d-subgenome homoeologues, but this effect was in many cases reversed by whole genome doubling. the suppression of d-subgenome homoeologue transcription resulted in a marked frequency of parental transcription level dominance, especially with respect to genes encoding proteins involved in photosynthesis. singletons were frequently transcribed at both the allotriploid and allohexaploid plants.  the implication is that whole genome doubling helps to overcome the phenotypic weakness of the allotriploid, restoring a more favourable gene dosage in genes experiencing transcription level dominance in hexaploid wheat. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords genome duplicationinterspecific hybridizationpolyploidysynthetic wheattranscriptome evolutionhttp://dx.doi.org/ /501100001809national natural science foundation of china312717239133111731571665li aili mao long liu dengcai the hundred talent program of casb0217liu dengcai the caas knowledge innovation programissue-copyright-statement© the author 2017 