BACKGROUND
the widespread existence of allopolyploidy in the plant kingdom points to its important role in the evolution of many groups of plants  <cit> .
yet, reuniting divergent genomes from different species in one nucleus likely represents a traumatic experience that the newly formed allopolyploids must go through to survive and adapt.
conceivably, substantial reconciliation of incompatibility is required at the very early stages following allopolyploidization to enable the newly formed individuals to recover from the "genome shock"  <cit>  and establish as new species.
we are still largely ignorant about the means and their underlying mechanisms whereby compatibility is accomplished at the onset of allopolyploidy.
however, recent studies conducted over the last decade in diverse plant taxa have provided novel insights into the seemingly intangible enigma; the most striking being the documentation that allopolyploidization triggers instantaneous genetic and epigenetic changes that enable altered trajectories of gene regulation  <cit> .
these rapid and non-mendelian genetic, epigenetic and regulatory changes are thought as important in ameliorating the hurdles related to immediate accommodation of nascent allopolyploids, and may contribute to their establishment and evolution as competitive new species  <cit> .
paradoxically, initial genomic instability was not detected in all studied cases of successful speciation via allopolyploidy  <cit> .
it is, however, important to note that alteration in gene expression appears to represent a consensus feature of nascent plant allopolyploidy involving diverse taxa  <cit> .
moreover, the immediate alteration in gene expression may provide the basis for the evolution of homoeologue-specific tuning or partitioning -- a unique property of allopolyploid species  <cit> .
maintenance of homoeologue-specific tuning or partitioning in an allopolyploid species requires cytological diploidization, and, hence, disomic inheritance, which conceivably entails timely functional differentiation among homeologous chromosomes  <cit> .
common wheat  originated only about  <dig>  years ago  <cit>  from hybridization event -- most probably between a domesticated form of tetraploid wheat, t. turgidum  with a diploid goat grass species, aegilops tauschii   <cit> .
common wheat thus provides a classic example of formation of a new species in a single step.
deciphering the process and understanding its mechanistic underpinning is of great evolutionary interest.
exploring the pattern and spectrum of global gene expression changes associated with nascent wheat allohexaploidization represents an essential step towards this goal.
hitherto, there are three reports on global gene expression changes associated with nascent allopolyploidization in wheat, all employing genetically stable synthetic allohexaploid lines  <cit> .
in the study by pumphrey et al.
<cit> , it was found that as high as 16% of the  <dig> analyzed genes selected from a 70-mer customer wheat oligonucleotide microarray displayed non-additive expression in a first generation synthetic allohexaploid line.
in contrast, the study by chagué et al.
<cit>  documented that the great majority  of  <dig>  transcripts analyzed showed additive expression and, hence, leaving only about 7% of genes as non-additively expressed in two synthetic lines.
the expression patterns were highly stable across two generations and fairly consistent among the two synthetic lines, which shared the same tetraploid parental genotype  <cit> .
the third study focused only on transcripts that have detectable parent-specific features , and 19% of these genes showed non-additive expression  <cit> .
the striking discrepancy in the proportions of non-additively expressed genes associated with nascent allohexaploidization for the same species  by the three studies could be due to various factors, such as differences in the developmental stage of the leaf tissue investigated or the different organs studied .
most likely, the differences are due to the parental genotypes used  <cit> .
apparently, further independent investigations are needed to determine the molecular, cell biological and physiological opportunities provided by genome-wide combinations in novel syntheses of allohexaploid wheat.
the present study was aimed to address:  pattern and spectrum of changes in global gene expression at early generations of nascent allohexaploid wheat lines with chromosomal stability;  characteristics of the genes showing altered expression patterns, and their possible functional relevance; and  transgenerational heritability and inter-line conservation of the novel expression patterns.
RESULTS
cytological characterization of the two sets of newly formed allohexaploid wheat lines
we meticulously determined the chromosome configurations on root-tip mitosis of each individual plant for both nascent allohexaploid lines by the multicolor gish technique  <cit> .
we found that chromosomal numerical and structural variations, including aneuploidy and/or inter-genomic translocations, occurred in certain proportions of the plants in each line at both studied generations, s <dig> and s <dig> .
nonetheless, the two lines are considered as fairly stable at the chromosomal level compared with some other nascent allopolyploid plants , which is consistent with their tetraploid parental genotypes harboring the ph <dig> gene that suppresses meiotic pairing between homeologous chromosomes and, hence, ensuring diploid-like meiosis  <cit> .
inclusion of the tetraploid parents containing the functional ph <dig> gene in the synthesis of the allohexaploid wheat lines is important for evolutionary relevance as this is the case in the formation of natural common wheat  <cit> .
for this study, only those individual plants that showed complete transgenerational chromosomal integrity as judged by the multi-color gish were used as the rna sources.
these plants had a genome constitution consisting of three intact genomes corresponding to the b, a and d genomes of common wheat .
gene expression divergence between the parental plants of t. turgidum and ae.
tauschii, and conservation between the two subspecies of t. turgidum
the affymetrix genechip® wheat genome array  contained  <dig>  probe-sets representing  <dig>  different genes that mapped across the common wheat genome.
these genes are certainly not all expressed at a particular developmental stage of a given tissue, and we detected  <dig>  genes that showed reliable expression  between the two biological replications in the second-seedling-leaf tissue at the three-leaf-stage.
we compared the whole transcriptomic difference between each pair of the parental species, t. turgidum  vs. ae.
tauschii , and t. turgidum  vs. ae.
tauschii  .
because in both combinations, tq <dig> served as the paternal parent, the transcriptome of tq <dig> was used as a reference.
compared with tq <dig>   <dig>   and  <dig>   genes were up-regulated  in ttr <dig> and tth <dig>  respectively; whereas  <dig>   and  <dig>   genes were down-regulated in the two tetraploid wheat lines .
taken together, the percentages for the transcriptomic divergence between each pair of the parental species were calculated as  <dig> %  and  <dig> %  of all expressed genes, respectively .
as expected, a large number and proportion  of all the differentially expressed genes  between a given tetraploid and the diploid parental species were overlapping , and accordingly, the number of genes showing inter-subspecific difference between durum and carthlicum was relatively small  .
differential parental contribution and parental dominance gene expression in the two nascent allohexaploid wheat lines
we compared the expression difference of each of the two synthetic allohexaploid wheat lines across two selfed generations directly with their tetraploid and diploid parental species.
the number of differentially expressed genes between allo-at5s <dig> and its tetraploid parent t. turgidum ssp.
durum amounted to only  <dig> % and to its diploid parent ae.
tauschii to  <dig> %.
in the case of the allo-at9s <dig> the frequency of differentially expressed genes between the allohexaploid and its tetraploid t. turgidum ssp.
carthlicum was  <dig> % and with its diploid parent  <dig> % .
this indicates that the overall transcriptome of the nascent allohexaploid lines was more similar to that of t. turgidum than to ae.
tauschii, consistent with the two-third and one-third genomic contributions by the former and the later, respectively.
notably, the between-generation difference with respect to the number of differentially expressed genes for a given allohexaploid line vs. its parental species was evident .
notably, the two allohexaploid lines showed contrasting trends in the number of genes showing differential expression from their parental species across the two successive generations, s <dig> and s <dig> , underscoring genetic context-dependent differential expression dynamics in the two nascent allohexaploid lines.
for those genes that were expressed differentially between the parental species, their expression levels in the allohexaploid lines could be statistically equal to one of the parents but different from the other; that is, they display expression bias towards one of the two parents.
this phenomenon has been systematically investigated in allotetraploid cotton, and was termed "parental dominance gene expression"  <cit> .
since the parental species were of different ploidy-level, a conceivable contributing factor to the seemingly parental biased expression in the allohexaploid lines was differential hybridization affinity by the two parental species' cdnas on the same array  <cit> .
we used the experimentally measured mid-parent values  for each parental species combination which was generated by mixing the rnas of parental lines  at a ratio of 2: <dig> to exclude this confounding factor.
the rationale was: those genes that appeared as biased expression in synthetic allopolyploids but actually were due to differential hybridization-affinity would also show "biased expression" in mpvs, and were excluded .
thus, we confined our analysis to those genes that exhibited parental biased expression in the allohexaploid lines but not in the corresponding mpvs, that is, the bona fide parental expression dominance genes resulting from allopolyploidy-specific tuning.
broadly, two categories of parental expression dominance were recognized, that is, diploid parental dominance and tetraploid parental dominance, each category including two patterns.
for diploid parental dominance: pattern one was that expression levels in the diploid were greater than the tetraploid, and the allohexaploid values largely fell above the mpv, which were statistically equal to the higher expression levels of the diploid, but different from the lower expression levels of the tetraploid ; pattern two was that expression levels of the diploid were lower than the tetraploid, and the allohexaploid values largely fell below the mpv, which were statistically equal to the lower expression levels of the diploid but different from the higher expression levels of the tetraploid .
for tetraploid parental dominance: pattern one was that expression levels in the tetraploid were greater than the diploid, and the allohexaploid values largely fell above the mpv, which were statistically equal to the higher expression levels of the tetraploid, but different from the lower expression levels of the diploid ; pattern two was that expression levels of the tetraploid were lower than the diploid, and the allohexaploid values largely fell below the mpv, which were statistically equal to the lower expression levels of the tetraploid but different from the higher expression levels of the diploid .
in general, the number of genes showing diploid parental dominance was much fewer than those showing tetraploid parental dominance in each allohexaploid line at each generation .
the parental dominance expression genes in the allohexaploid lines showed either statistically additive or non-additive expression levels, with the former being the great majority while the later accounting for less than  <dig> % of these genes .
this accorded well with the earlier results in both cotton  <cit>  and wheat  <cit> .
it should be emphasized that this "phenotypic additivity" in expression pattern shown by the parental dominance expression genes may not solely result from the summing up of parental transcripts in which the occurrence of expression alterations did not transgress the expected parental additive expression range; rather, they could be caused by marked, yet compensatory, changes between the homeologous alleles.
we noted that the parental dominance expression genes showed low degrees of conservation.
that is, the proportions of these genes that were overlapping between the two generations of a given allohexaploid line and between the two lines at a given generation were small.
specifically, there were only  <dig> and  <dig> diploid parental dominance expression genes that were overlapped between the s <dig> and s <dig> generations in allo-at <dig> and allo-at <dig>  respectively, which accounted for  <dig> % and  <dig> % of the total diploid parental dominance expression genes in the two lines .
similarly, there were  <dig>  and  <dig> tetraploid parental dominance expression genes that were overlapped between the s <dig> and s <dig> generations in allo-at <dig> and allo-at <dig> and which accounted for  <dig> % and  <dig> % of the total tetraploid parental expression dominance genes in the two allohexaploid lines, respectively .
more limited conservation of dominance expression genes was observed between the two allohexaploid lines at a given generation, which applied particularly so for those of the diploid parent.
specifically, there were  <dig> %  and  <dig> %  diploid parental dominance expression genes being shared by the two lines at s <dig> and s <dig>  respectively, whereas the proportions were  <dig> %  and  <dig> %  for the tetraploid parental dominance expression genes at the two generations .
expectedly, there were only  <dig> and  <dig> genes with expression patterns that were consistently biased toward the diploid and tetraploid parental species, respectively, in both lines at both generations .
the foregoing results indicated that the parental dominance expression genes in the allohexaploid wheat lines could be classified into three distinct groups, "stochastic"  referring to those that were specific to one line  at one generation  only, "heritable"  referring to those that were specific to one line at both generations, and "persistent"  referring to those that were commonly detected in both lines at both generations.
consistent with the above analysis, the group ii and iii genes were of markedly greater proportions among the total tetraploid parental dominance expression genes than those of the diploid , suggesting that allopolyploidy-regulated gene expression is more stochastic in the transcriptome contributed by the diploid parent than in that by the tetraploid parent.
non-additive gene expression in the nascent allohexaploid wheat lines
another prominent pattern of altered gene expression commonly associated with nascent plant allopolyploidy is non-additivity.
by definition, if the expression level of a gene in an allopolyploid did not change from that of their parental lines, then its expression level would be statistically equal to that of the experimentally measured mid-parent values , described above; otherwise, it is defined as non-additive.
at this point, it appeared important to distinguish between the two expression patterns.
while the "parental dominance gene expression" refers to the statistically expression similarity in an allopolyploid to one of the parents but different from the other, the "non-additivity" refers to those genes the expression levels of which deviated from the empirically measured mid-parent values .
genes of these two expressing patterns were neither mutually exclusive nor inclusive.
for example, a gene may show expression deviation from that of the mpvs, that is, non-additivity, but at the same time its expression level may be statistically equal to one of the parents but different from the other, that is, expression dominance.
conversely, a gene could have shown parental dominance expression, but statistically did not deviate from the mpvs, and hence, was categorized as additive expression.
we quantitatively investigated the phenomenon of non-additivity by comparing the microarray-based gene expression levels of the two nascent allohexaploid wheat lines at two successive generations against the empirically measured mpvs .
we found that a great majority of the expressed genes showed additive expression compared with the corresponding mpvs, which if averaged across the two generations , accounted for  <dig> % and  <dig> % of the total expressed genes in allo-at <dig> and allo-at <dig>  respectively .
nonetheless, deviation from additivity in gene expression occurred in both lines, though with marked difference in regard to its expression pattern and preponderance of transgenerational inheritance .
for example, allo-at <dig> showed more inter-generational difference  than allo-at <dig>  .
also, higher proportions showed up- rather than down-regulation in allo-at <dig> , but the opposite was true in allo-at <dig>  .
a consensus feature for the non-additive genes across lines and generations was their over-representation by those showing parental differential or divergent expression , as also documented previously in various plant taxa  <cit> .
synthetic line and
total no.
and  of
up-
237
down:
246
up-
426
down:
629
up-
332
down:
438
up-
316
down:
425
up-
203
down:
410
up-
260
down:
418
no.
and  of high-diploid
no.
and  of high-tetraploid
no.
and  of low-diploid
no.
and  of low-tetraploid
no.
and  of over-dominance
no.
and  of under-
aof all expressed genes.
bof non-additively expressed genes.
the up- or down-regulated numbers of the nonadditively expressed genes.
the nonadditively expressed genes can be divided into six categories based on their expression: f represented the synthetic allohexaploid lines; p, paternal lines; and m, maternal lines.
high-paternal dominance, f = p, f > m, p > m; high-maternal dominance, f = m, f > p, m > p; low-paternal dominance, f = p, f < m, p < m; low-maternal dominance, f = m, f < p, m < p; over-dominance, f > p and f > m; under-dominance, f < p and f < m.
according to criteria specified previously  <cit> , the non-additive expressing genes could also be further classified into several patterns, including high- and low-diploid parental dominance, high- and low-tetraploid parental dominance, over-dominance and under-dominance .
the two lines manifested marked differences in the proportions of the non-additive genes showing the various patterns of expression.
for example, in allo-at <dig>  more tetraploid than diploid parental dominance expression genes were detected ; in allo-at <dig>  the number showing tetraploid parental dominance was the same as or similar to that showing diploid parental dominance .
the average numbers of non-additive genes showing over- and under-dominance in allo-at <dig>  were slightly lower than those in allo-at <dig> ; however, in both lines significantly more over- than under-dominance genes were detected .
the proportions of common non-additive genes in the two successive generations of each line  were low.
specifically, there were only  <dig> %  and  <dig> %  such genes being shared by the s <dig> and s <dig> generations in allo-at <dig> and allo-at <dig>  respectively .
this rendered the nonadditive genes that were specific to one allohexaploid line in only one generation  a high proportion of the total non-additive genes, which comprised  <dig> % and  <dig> % in allo-at <dig> and allo-at <dig>  respectively.
if comparing the extent of overlaps of the nonadditive genes between the two allohexaploid lines at a given generation, the proportions were even lower, with only  <dig> %  and  <dig> %  at generations s <dig> and s <dig>  respectively .
not shown in the figure was that there were only  <dig> non-additively expressed genes that were common to both lines at both generations, which were termed "persistent non-additive genes" .
validation of the microarray data by real-time quantitative -rt-pcr in the nascent allohexaploid wheat lines
to verify the microarray data, we selected nine genes that showed non-additive expression in one or both of the two allohexaploid lines at one or both generations, and subjected the genes to -rt-pcr analysis.
these nine genes included the three groups of non-additively expressed genes, that is, stochastic, heritable and persistent and, therefore,  <dig> comparisons for non-additivity were generated across the two lines and the two generations .
the expression level of each of these genes was compared with similarly constructed mpvs as used in the microarray experiments.
results indicated that of the  <dig> comparisons,  <dig> were confirmed as matching or highly similar to the microarray data for non-additive gene expression , thus confirming reliability of the microarray data and analysis.
enrichment for a specific category of genes involved in vesicle function by the non-additively expressed genes in the two nascent allohexaploid wheat lines
to investigate possible functional relevance of the two major groups of genes showing allohexaploid-specified regulation, that is, parental dominance and non-additivity, we conducted gene ontology  analysis for each group, both as an entirety and as further divided subgroups.
first, for the parental dominance expression genes, we found that  diverse go terms were involved, indicating that the genes showing this expression pattern had various molecular functions, participated in diverse biological processes, and localized to different cellular components ;  statistical tests for enrichment indicated that only those that exhibited the nonadditive expression pattern were enriched for a very specific go category of the cellular component involved in vesicle function , suggesting that this specific subgroup  of the parental dominance genes is probably functionally required by the nascent allohexaploid wheat lines;  if these nonadditive expression genes were analyzed as transgenerationally heritable and non-heritable subgroups , then only the heritable subgroup showed statistically significant enrichment for the vesicle-related genes ;  if the genes were analyzed as the three subgroups, stochastic, heritable and persistent , then none showed any enrichment .
taken together, this analysis led to an important conclusion that only those parental dominance genes that are non-additive as well as heritable are enriched for the vesicle-related genes.
this, however, becomes cryptic, if all the parental dominance genes were analyzed as a whole.
second, we carried out go analysis for the non-additively expressed genes, both as an entirety and as three distinct groups: stochastic , heritable , and consistent .
we found that:  similar to the situation of parental dominance expression genes, the non-additive genes as an entirety were also involved in diverse functional categories ;  statistical analysis for enrichment indicated that, remarkably, the same go terms of the cellular component, that is, those involved in vesicle function, showed significant enrichment, with those of group ii manifested a higher degree of enrichment than group i ;  the statistical insignificance of group iii for those same go-terms is apparently due to their too small number , and hence, lack of statistical power, rather than the absence of a real enrichment ; and  interestingly, the group iii genes also showed statistically meaningful homology to two dna polymerases .
taken the go analysis results together for the two sets of genes specifically tuned by allopolyploidy, it is clear that only the non-additively expressed ones  manifested strong and specific enrichment for the small category of go terms related to vesicle-function, with the subset of those exhibiting transgenerational inheritance being further enhanced for the enrichment.
discussion
in this study, we used the affymetrix genechip® wheat genome array platform as was done in two of the three studies  <cit> , as well as similar synthetic allohexaploid wheat lines as was used in one study  <cit> .
therefore, it is perhaps not surprising that our major results are more consistent with those of chagué et al.
<cit>  than to the other two studies.
however, the synthetic lines we used also differ from those of chagué et al.
in three important aspects:  the two lines we used share the same genotype of the diploid goat-grass species, ae.
tauschii, as the paternal parent, but with different subspecies durum and carthlicum of t. turgidum, as the maternal parent, whereas the two lines used by chagué et al.
are with the same genotype of the tetraploid maternal parent but different genotypes of the diploid paternal parent;  our lines and those of chagué et al.
are of different generations following initial allopolyploidization, with ours being at the fourth and fifth selfed generations and those of chagué et al.
being at the immediate and first selfed generations ;  different tissues are used as the rna source, that is, the second seedling-leaf  in our study and shoots  in the study of chagué et al.. therefore, being based on the same microarray platform and employing similar statistical methods, the results of our study have provided important comparisons as well as complementation to those of chagué et al.
<cit> .
for example, chagué et al.
<cit>  documented that the non-additively expressed genes between the s <dig> and s <dig> generations of one of the studied synthetic allohexaploid line were highly conserved.
in contrast, we showed here that it was the proportion of the genes with the altered expression pattern that was conserved between the two successive generations  of both allohexaploid lines studied, whereas identity of the genes per se were highly variable .
these differences could be due to one or more of the different factors involved or the combinatory effects thereof.
but it is clear, based on the results of chagué et al.
<cit>  and ours, that the early generations of allohexaploidy in wheat are extremely dynamic in gene expression, even in karyotypically normal and genetically stable plant individuals.
we believe this double-edged property  of nascent allopolyploidy is critical for bestowing stabilization on one hand and evolvability on the other of the newly formed allopolyploid plants towards establishment and speciation.
importance of chromosomal integrity in allohexaploid wheat evolution
it is well established by earlier cytological studies that, at the chromosomal level, the three constituent genomes of common wheat are largely intact.
we, thus, analyzed the chromosomal constitution of each individual plant by the multicolor gish technique  <cit> , and chose only those plants with transgenerational chromosomal integrity for the microarray analysis.
therefore, our study has paid attention to this critical point that was unheeded by most previous investigations, except the one by chagué et al.
<cit> .
conservation in expression pattern but stochasticity in gene identity characterize both parental dominance expression genes and non-additive expression genes
a major pattern of altered gene expression in the nascent allohexaploid wheat lines is parental dominance expression.
the experimental mpvs enabled unequivocal exclusion of those genes for which the detected biased expression was due to differential hybridization affinity by the parental species' cdnas to the same array, and therefore, allowed analysis of the bona fide parental expression dominance genes resulting from allohexaploidy-specified tuning.
similarly, comparisons with the mpvs allowed unambiguous identification of the genes showing non-additive expression in the nascent allohexaploid lines.
an important generalization that emerged from these analyses is that both the parental dominance expression genes and non-additive expression genes can be divided into three distinct groups depending on whether they occurred only in one allohexaploid line at only one generation, that is, stochastic , in one line at both generations, that is, heritable  or in both lines at both generations, that is, persistent .
although this classification is meaningful only in a relative sense, its mere occurrence implicates an important attribute; that is, if the tuned expression is important for the newly formed allohexaploid lines, then it is likely the pattern of expression rather than identity of the genes showing the pattern of expression bears the relevance.
this novel feature of allopolyploidy-specified gene regulation has not been reported previously, but it is reminiscent of the findings in allotetraploid cotton, in which the parental dominance expression was found to be even in opposite directions in different allotetraploid lines but with similar proportions of genes showing the pattern  <cit> .
this feature of altered genomewide expression patterns also bears remarkable resemblance to the phenomenon of nucleoli dominance dictated by localized epigenetic difference, that is, stochastic silencing of one of the parental rrna genes in an allopolyploid  <cit> , thus further suggesting possible commonality of the phenomenon in nascent plant allopolyploids.
it is important to note that the majority of the genes, albeit showing parental dominance expression in the allohexaploid wheat lines, did not transgress the parental additive expression-range relative to the corresponding mpvs, as were found in cotton  <cit> .
this is interesting given that the "appeared" expression additivity for this set of genes was actually achieved by allohexaploidy-specific tuning rather than merely the adding-up of parental transcripts, suggesting that a too high- or low-level of expression is likely detrimental and, hence, is being selected against.
non-additive gene expression in the newly formed allohexaploid wheat lines
deviation from additivity in expression pattern of genes was commonly observed in previous studies, albeit the spectrum of deviation can be dramatically variable.
although the functional relevance of non-additive gene expression remains largely unexplored  <cit> , it is suggested that the non-additivity-bestowed diverse novel gene expression patterns may provide for variations upon which selection can act  <cit> .
it is conceivable that for non-additive gene expression to play a biologically meaningful role, transgenerational heritability of the altered expression patterns and/or the genes is required.
indeed, several studies have indicated that the non-additive expression patterns can be established at the initial stages of allopolyploidization but with protracted effects over evolutionary timescales  <cit> .
results of this study suggest that it is the altered pattern of non-additive gene expression rather than the involved genes per se that showed a propensity of transgenerational inheritance, as evidenced by the results that the proportions of genes showing the altered expression patterns were highly similar between the two successive generations but the identities of the genes are highly variable.
this novel paradigm of allopolyploidy-associated gene expression has not been reported previously.
possible requirements for specific tuning of genes involved in vesicle function by the nascent allohexaploid wheat lines
gene ontology  analysis indicated that both groups of genes showing allopolyploidy-specific tuning, that is, parental dominance expression and non-additive expression, are involved in diverse functional go-categories.
nevertheless, detailed analysis revealed that only those genes that showed non-additive expression, regardless of whether they are exhibiting parental dominance expression or not, are enriched for a very specific go-category involved in vesicle function.
moreover, the enrichment was further enhanced for a subset of these genes showing transgenerational heritability.
this novel observation strongly implicates a functional relevance of non-additive gene expression being associated with nascent allopolyploidy, which makes sense given that an immediate outcome of allopolyploidization is enlarged cell volume but reduced surface-area to volume ratio  <cit> , and, therefore, tuned non-additive expression of vesicle-related genes may help circumvent this dilemma.
the molecular basis underlying the allopolyploidy-regulated gene expression remains to be fully elucidated  <cit> .
it has been proposed that both genetic regulatory mechanisms, such as cis- and trans-interactions  <cit> , and epigenetic alterations, such as altered dna methylation and small rna biogenesis  <cit> , likely play important roles.
further studies are needed to explore the exact roles played by these mechanisms in the allohexaploidization of wheat.
CONCLUSIONS
this study showed that the early stages of allohexaploidization, leading to the formation of allohexaploid wheat, exhibited two major patterns of global gene expression alteration: parental dominance expression and non-additive expression.
the mechanisms underpinning the expression changes associated with nascent allopolyploidy remain largely unknown  <cit> , but chromosomal instability is not a causal factor.
instead, molecular interactions, including altered cis-/trans-regulation  <cit> , dosage balance/compensation  <cit> , gene elimination and/or epigenetic remodeling  <cit> , brought about by the sudden merging of divergent parental species' genomes, are conceivably responsible  <cit> .
genes involved in each altered expression pattern could be classified into three distinct groups: stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation.
importantly, whereas both altered patterns of gene expression showed a propensity of transgenerational inheritance, identity of the involved genes is highly stochastic.
accordingly, diverse gene ontology  terms were implicated with both patterns of altered gene expression; however, those showing non-additive expression manifested a significant enrichment for a specific group of proteins associated with vesicle function.
our results suggest that global alteration in gene expression conditioned by nascent allopolyploidy is accomplished by means of both targeted regulation and stochastic changes, that likely play distinct functional roles in the stabilization and establishment of the newly formed allohexaploid plants as competitive populations and new species  <cit> .

 the widespread existence of allopolyploidy in the plant kingdom points to its important role in the evolution of many groups of plants. yet, reuniting divergent genomes from different species in one nucleus likely represents a traumatic experience that the newly formed allopolyploids must go through to survive and adapt. conceivably, substantial reconciliation of incompatibility is required at the very early stages following allopolyploidization to enable the newly formed individuals to recover from the "genome shock" and establish as new species. we are still largely ignorant about the means and their underlying mechanisms whereby compatibility is accomplished at the onset of allopolyploidy. however, recent studies conducted over the last decade in diverse plant taxa have provided novel insights into the seemingly intangible enigma; the most striking being the documentation that allopolyploidization triggers instantaneous genetic and epigenetic changes that enable altered trajectories of gene regulation. these rapid and non-mendelian genetic, epigenetic and regulatory changes are thought as important in ameliorating the hurdles related to immediate accommodation of nascent allopolyploids, and may contribute to their establishment and evolution as competitive new species. paradoxically, initial genomic instability was not detected in all studied cases of successful speciation via allopolyploidy. it is, however, important to note that alteration in gene expression appears to represent a consensus feature of nascent plant allopolyploidy involving diverse taxa. moreover, the immediate alteration in gene expression may provide the basis for the evolution of homoeologue-specific tuning or partitioning -- a unique property of allopolyploid species. maintenance of homoeologue-specific tuning or partitioning in an allopolyploid species requires cytological diploidization, and, hence, disomic inheritance, which conceivably entails timely functional differentiation among homeologous chromosomes. common wheat originated only about years ago from hybridization event -- most probably between a domesticated form of tetraploid wheat, t. turgidum with a diploid goat grass species, aegilops tauschii. common wheat thus provides a classic example of formation of a new species in a single step. deciphering the process and understanding its mechanistic underpinning is of great evolutionary interest. exploring the pattern and spectrum of global gene expression changes associated with nascent wheat allohexaploidization represents an essential step towards this goal. hitherto, there are three reports on global gene expression changes associated with nascent allopolyploidization in wheat, all employing genetically stable synthetic allohexaploid lines. in the study by pumphrey et al., it was found that as high as 16% of the analyzed genes selected from a 70-mer customer wheat oligonucleotide microarray displayed non-additive expression in a first generation synthetic allohexaploid line. in contrast, the study by chagué et al. documented that the great majority of transcripts analyzed showed additive expression and, hence, leaving only about 7% of genes as non-additively expressed in two synthetic lines. the expression patterns were highly stable across two generations and fairly consistent among the two synthetic lines, which shared the same tetraploid parental genotype. the third study focused only on transcripts that have detectable parent-specific features, and 19% of these genes showed non-additive expression. the striking discrepancy in the proportions of non-additively expressed genes associated with nascent allohexaploidization for the same species by the three studies could be due to various factors, such as differences in the developmental stage of the leaf tissue investigated or the different organs studied. most likely, the differences are due to the parental genotypes used. apparently, further independent investigations are needed to determine the molecular, cell biological and physiological opportunities provided by genome-wide combinations in novel syntheses of allohexaploid wheat. the present study was aimed to address: pattern and spectrum of changes in global gene expression at early generations of nascent allohexaploid wheat lines with chromosomal stability; characteristics of the genes showing altered expression patterns, and their possible functional relevance; and transgenerational heritability and inter-line conservation of the novel expression patterns.  cytological characterization of the two sets of newly formed allohexaploid wheat lines we meticulously determined the chromosome configurations on root-tip mitosis of each individual plant for both nascent allohexaploid lines by the multicolor gish technique. we found that chromosomal numerical and structural variations, including aneuploidy and/or inter-genomic translocations, occurred in certain proportions of the plants in each line at both studied generations, s and s. nonetheless, the two lines are considered as fairly stable at the chromosomal level compared with some other nascent allopolyploid plants, which is consistent with their tetraploid parental genotypes harboring the ph gene that suppresses meiotic pairing between homeologous chromosomes and, hence, ensuring diploid-like meiosis. inclusion of the tetraploid parents containing the functional ph gene in the synthesis of the allohexaploid wheat lines is important for evolutionary relevance as this is the case in the formation of natural common wheat. for this study, only those individual plants that showed complete transgenerational chromosomal integrity as judged by the multi-color gish were used as the rna sources. these plants had a genome constitution consisting of three intact genomes corresponding to the b, a and d genomes of common wheat. gene expression divergence between the parental plants of t. turgidum and ae. tauschii, and conservation between the two subspecies of t. turgidum the affymetrix genechip® wheat genome array contained probe-sets representing different genes that mapped across the common wheat genome. these genes are certainly not all expressed at a particular developmental stage of a given tissue, and we detected genes that showed reliable expression between the two biological replications in the second-seedling-leaf tissue at the three-leaf-stage. we compared the whole transcriptomic difference between each pair of the parental species, t. turgidum vs. ae. tauschii, and t. turgidum vs. ae. tauschii. because in both combinations, tq served as the paternal parent, the transcriptome of tq was used as a reference. compared with tq and genes were up-regulated in ttr and tth respectively; whereas and genes were down-regulated in the two tetraploid wheat lines. taken together, the percentages for the transcriptomic divergence between each pair of the parental species were calculated as % and % of all expressed genes, respectively. as expected, a large number and proportion of all the differentially expressed genes between a given tetraploid and the diploid parental species were overlapping, and accordingly, the number of genes showing inter-subspecific difference between durum and carthlicum was relatively small. differential parental contribution and parental dominance gene expression in the two nascent allohexaploid wheat lines we compared the expression difference of each of the two synthetic allohexaploid wheat lines across two selfed generations directly with their tetraploid and diploid parental species. the number of differentially expressed genes between allo-at5s and its tetraploid parent t. turgidum ssp. durum amounted to only % and to its diploid parent ae. tauschii to %. in the case of the allo-at9s the frequency of differentially expressed genes between the allohexaploid and its tetraploid t. turgidum ssp. carthlicum was % and with its diploid parent %. this indicates that the overall transcriptome of the nascent allohexaploid lines was more similar to that of t. turgidum than to ae. tauschii, consistent with the two-third and one-third genomic contributions by the former and the later, respectively. notably, the between-generation difference with respect to the number of differentially expressed genes for a given allohexaploid line vs. its parental species was evident. notably, the two allohexaploid lines showed contrasting trends in the number of genes showing differential expression from their parental species across the two successive generations, s and s, underscoring genetic context-dependent differential expression dynamics in the two nascent allohexaploid lines. for those genes that were expressed differentially between the parental species, their expression levels in the allohexaploid lines could be statistically equal to one of the parents but different from the other; that is, they display expression bias towards one of the two parents. this phenomenon has been systematically investigated in allotetraploid cotton, and was termed "parental dominance gene expression". since the parental species were of different ploidy-level, a conceivable contributing factor to the seemingly parental biased expression in the allohexaploid lines was differential hybridization affinity by the two parental species' cdnas on the same array. we used the experimentally measured mid-parent values for each parental species combination which was generated by mixing the rnas of parental lines at a ratio of 2: to exclude this confounding factor. the rationale was: those genes that appeared as biased expression in synthetic allopolyploids but actually were due to differential hybridization-affinity would also show "biased expression" in mpvs, and were excluded. thus, we confined our analysis to those genes that exhibited parental biased expression in the allohexaploid lines but not in the corresponding mpvs, that is, the bona fide parental expression dominance genes resulting from allopolyploidy-specific tuning. broadly, two categories of parental expression dominance were recognized, that is, diploid parental dominance and tetraploid parental dominance, each category including two patterns. for diploid parental dominance: pattern one was that expression levels in the diploid were greater than the tetraploid, and the allohexaploid values largely fell above the mpv, which were statistically equal to the higher expression levels of the diploid, but different from the lower expression levels of the tetraploid; pattern two was that expression levels of the diploid were lower than the tetraploid, and the allohexaploid values largely fell below the mpv, which were statistically equal to the lower expression levels of the diploid but different from the higher expression levels of the tetraploid. for tetraploid parental dominance: pattern one was that expression levels in the tetraploid were greater than the diploid, and the allohexaploid values largely fell above the mpv, which were statistically equal to the higher expression levels of the tetraploid, but different from the lower expression levels of the diploid; pattern two was that expression levels of the tetraploid were lower than the diploid, and the allohexaploid values largely fell below the mpv, which were statistically equal to the lower expression levels of the tetraploid but different from the higher expression levels of the diploid. in general, the number of genes showing diploid parental dominance was much fewer than those showing tetraploid parental dominance in each allohexaploid line at each generation. the parental dominance expression genes in the allohexaploid lines showed either statistically additive or non-additive expression levels, with the former being the great majority while the later accounting for less than % of these genes. this accorded well with the earlier results in both cotton and wheat. it should be emphasized that this "phenotypic additivity" in expression pattern shown by the parental dominance expression genes may not solely result from the summing up of parental transcripts in which the occurrence of expression alterations did not transgress the expected parental additive expression range; rather, they could be caused by marked, yet compensatory, changes between the homeologous alleles. we noted that the parental dominance expression genes showed low degrees of conservation. that is, the proportions of these genes that were overlapping between the two generations of a given allohexaploid line and between the two lines at a given generation were small. specifically, there were only and diploid parental dominance expression genes that were overlapped between the s and s generations in allo-at and allo-at respectively, which accounted for % and % of the total diploid parental dominance expression genes in the two lines. similarly, there were and tetraploid parental dominance expression genes that were overlapped between the s and s generations in allo-at and allo-at and which accounted for % and % of the total tetraploid parental expression dominance genes in the two allohexaploid lines, respectively. more limited conservation of dominance expression genes was observed between the two allohexaploid lines at a given generation, which applied particularly so for those of the diploid parent. specifically, there were % and % diploid parental dominance expression genes being shared by the two lines at s and s respectively, whereas the proportions were % and % for the tetraploid parental dominance expression genes at the two generations. expectedly, there were only and genes with expression patterns that were consistently biased toward the diploid and tetraploid parental species, respectively, in both lines at both generations. the foregoing results indicated that the parental dominance expression genes in the allohexaploid wheat lines could be classified into three distinct groups, "stochastic" referring to those that were specific to one line at one generation only, "heritable" referring to those that were specific to one line at both generations, and "persistent" referring to those that were commonly detected in both lines at both generations. consistent with the above analysis, the group ii and iii genes were of markedly greater proportions among the total tetraploid parental dominance expression genes than those of the diploid, suggesting that allopolyploidy-regulated gene expression is more stochastic in the transcriptome contributed by the diploid parent than in that by the tetraploid parent. non-additive gene expression in the nascent allohexaploid wheat lines another prominent pattern of altered gene expression commonly associated with nascent plant allopolyploidy is non-additivity. by definition, if the expression level of a gene in an allopolyploid did not change from that of their parental lines, then its expression level would be statistically equal to that of the experimentally measured mid-parent values, described above; otherwise, it is defined as non-additive. at this point, it appeared important to distinguish between the two expression patterns. while the "parental dominance gene expression" refers to the statistically expression similarity in an allopolyploid to one of the parents but different from the other, the "non-additivity" refers to those genes the expression levels of which deviated from the empirically measured mid-parent values. genes of these two expressing patterns were neither mutually exclusive nor inclusive. for example, a gene may show expression deviation from that of the mpvs, that is, non-additivity, but at the same time its expression level may be statistically equal to one of the parents but different from the other, that is, expression dominance. conversely, a gene could have shown parental dominance expression, but statistically did not deviate from the mpvs, and hence, was categorized as additive expression. we quantitatively investigated the phenomenon of non-additivity by comparing the microarray-based gene expression levels of the two nascent allohexaploid wheat lines at two successive generations against the empirically measured mpvs. we found that a great majority of the expressed genes showed additive expression compared with the corresponding mpvs, which if averaged across the two generations, accounted for % and % of the total expressed genes in allo-at and allo-at respectively. nonetheless, deviation from additivity in gene expression occurred in both lines, though with marked difference in regard to its expression pattern and preponderance of transgenerational inheritance. for example, allo-at showed more inter-generational difference than allo-at. also, higher proportions showed up- rather than down-regulation in allo-at, but the opposite was true in allo-at. a consensus feature for the non-additive genes across lines and generations was their over-representation by those showing parental differential or divergent expression, as also documented previously in various plant taxa. synthetic line and total no. and of up- 237 down: 246 up- 426 down: 629 up- 332 down: 438 up- 316 down: 425 up- 203 down: 410 up- 260 down: 418 no. and of high-diploid no. and of high-tetraploid no. and of low-diploid no. and of low-tetraploid no. and of over-dominance no. and of under- aof all expressed genes. bof non-additively expressed genes. the up- or down-regulated numbers of the nonadditively expressed genes. the nonadditively expressed genes can be divided into six categories based on their expression: f represented the synthetic allohexaploid lines; p, paternal lines; and m, maternal lines. high-paternal dominance, f = p, f > m, p > m; high-maternal dominance, f = m, f > p, m > p; low-paternal dominance, f = p, f < m, p < m; low-maternal dominance, f = m, f < p, m < p; over-dominance, f > p and f > m; under-dominance, f < p and f < m. according to criteria specified previously, the non-additive expressing genes could also be further classified into several patterns, including high- and low-diploid parental dominance, high- and low-tetraploid parental dominance, over-dominance and under-dominance. the two lines manifested marked differences in the proportions of the non-additive genes showing the various patterns of expression. for example, in allo-at more tetraploid than diploid parental dominance expression genes were detected; in allo-at the number showing tetraploid parental dominance was the same as or similar to that showing diploid parental dominance. the average numbers of non-additive genes showing over- and under-dominance in allo-at were slightly lower than those in allo-at; however, in both lines significantly more over- than under-dominance genes were detected. the proportions of common non-additive genes in the two successive generations of each line were low. specifically, there were only % and % such genes being shared by the s and s generations in allo-at and allo-at respectively. this rendered the nonadditive genes that were specific to one allohexaploid line in only one generation a high proportion of the total non-additive genes, which comprised % and % in allo-at and allo-at respectively. if comparing the extent of overlaps of the nonadditive genes between the two allohexaploid lines at a given generation, the proportions were even lower, with only % and % at generations s and s respectively. not shown in the figure was that there were only non-additively expressed genes that were common to both lines at both generations, which were termed "persistent non-additive genes". validation of the microarray data by real-time quantitative -rt-pcr in the nascent allohexaploid wheat lines to verify the microarray data, we selected nine genes that showed non-additive expression in one or both of the two allohexaploid lines at one or both generations, and subjected the genes to -rt-pcr analysis. these nine genes included the three groups of non-additively expressed genes, that is, stochastic, heritable and persistent and, therefore, comparisons for non-additivity were generated across the two lines and the two generations. the expression level of each of these genes was compared with similarly constructed mpvs as used in the microarray experiments. results indicated that of the comparisons, were confirmed as matching or highly similar to the microarray data for non-additive gene expression, thus confirming reliability of the microarray data and analysis. enrichment for a specific category of genes involved in vesicle function by the non-additively expressed genes in the two nascent allohexaploid wheat lines to investigate possible functional relevance of the two major groups of genes showing allohexaploid-specified regulation, that is, parental dominance and non-additivity, we conducted gene ontology analysis for each group, both as an entirety and as further divided subgroups. first, for the parental dominance expression genes, we found that diverse go terms were involved, indicating that the genes showing this expression pattern had various molecular functions, participated in diverse biological processes, and localized to different cellular components; statistical tests for enrichment indicated that only those that exhibited the nonadditive expression pattern were enriched for a very specific go category of the cellular component involved in vesicle function, suggesting that this specific subgroup of the parental dominance genes is probably functionally required by the nascent allohexaploid wheat lines; if these nonadditive expression genes were analyzed as transgenerationally heritable and non-heritable subgroups, then only the heritable subgroup showed statistically significant enrichment for the vesicle-related genes; if the genes were analyzed as the three subgroups, stochastic, heritable and persistent, then none showed any enrichment. taken together, this analysis led to an important conclusion that only those parental dominance genes that are non-additive as well as heritable are enriched for the vesicle-related genes. this, however, becomes cryptic, if all the parental dominance genes were analyzed as a whole. second, we carried out go analysis for the non-additively expressed genes, both as an entirety and as three distinct groups: stochastic, heritable, and consistent. we found that: similar to the situation of parental dominance expression genes, the non-additive genes as an entirety were also involved in diverse functional categories; statistical analysis for enrichment indicated that, remarkably, the same go terms of the cellular component, that is, those involved in vesicle function, showed significant enrichment, with those of group ii manifested a higher degree of enrichment than group i; the statistical insignificance of group iii for those same go-terms is apparently due to their too small number, and hence, lack of statistical power, rather than the absence of a real enrichment; and interestingly, the group iii genes also showed statistically meaningful homology to two dna polymerases. taken the go analysis results together for the two sets of genes specifically tuned by allopolyploidy, it is clear that only the non-additively expressed ones manifested strong and specific enrichment for the small category of go terms related to vesicle-function, with the subset of those exhibiting transgenerational inheritance being further enhanced for the enrichment. discussion in this study, we used the affymetrix genechip® wheat genome array platform as was done in two of the three studies, as well as similar synthetic allohexaploid wheat lines as was used in one study. therefore, it is perhaps not surprising that our major results are more consistent with those of chagué et al. than to the other two studies. however, the synthetic lines we used also differ from those of chagué et al. in three important aspects: the two lines we used share the same genotype of the diploid goat-grass species, ae. tauschii, as the paternal parent, but with different subspecies durum and carthlicum of t. turgidum, as the maternal parent, whereas the two lines used by chagué et al. are with the same genotype of the tetraploid maternal parent but different genotypes of the diploid paternal parent; our lines and those of chagué et al. are of different generations following initial allopolyploidization, with ours being at the fourth and fifth selfed generations and those of chagué et al. being at the immediate and first selfed generations; different tissues are used as the rna source, that is, the second seedling-leaf in our study and shoots in the study of chagué et al.. therefore, being based on the same microarray platform and employing similar statistical methods, the results of our study have provided important comparisons as well as complementation to those of chagué et al.. for example, chagué et al. documented that the non-additively expressed genes between the s and s generations of one of the studied synthetic allohexaploid line were highly conserved. in contrast, we showed here that it was the proportion of the genes with the altered expression pattern that was conserved between the two successive generations of both allohexaploid lines studied, whereas identity of the genes per se were highly variable. these differences could be due to one or more of the different factors involved or the combinatory effects thereof. but it is clear, based on the results of chagué et al. and ours, that the early generations of allohexaploidy in wheat are extremely dynamic in gene expression, even in karyotypically normal and genetically stable plant individuals. we believe this double-edged property of nascent allopolyploidy is critical for bestowing stabilization on one hand and evolvability on the other of the newly formed allopolyploid plants towards establishment and speciation. importance of chromosomal integrity in allohexaploid wheat evolution it is well established by earlier cytological studies that, at the chromosomal level, the three constituent genomes of common wheat are largely intact. we, thus, analyzed the chromosomal constitution of each individual plant by the multicolor gish technique, and chose only those plants with transgenerational chromosomal integrity for the microarray analysis. therefore, our study has paid attention to this critical point that was unheeded by most previous investigations, except the one by chagué et al.. conservation in expression pattern but stochasticity in gene identity characterize both parental dominance expression genes and non-additive expression genes a major pattern of altered gene expression in the nascent allohexaploid wheat lines is parental dominance expression. the experimental mpvs enabled unequivocal exclusion of those genes for which the detected biased expression was due to differential hybridization affinity by the parental species' cdnas to the same array, and therefore, allowed analysis of the bona fide parental expression dominance genes resulting from allohexaploidy-specified tuning. similarly, comparisons with the mpvs allowed unambiguous identification of the genes showing non-additive expression in the nascent allohexaploid lines. an important generalization that emerged from these analyses is that both the parental dominance expression genes and non-additive expression genes can be divided into three distinct groups depending on whether they occurred only in one allohexaploid line at only one generation, that is, stochastic, in one line at both generations, that is, heritable or in both lines at both generations, that is, persistent. although this classification is meaningful only in a relative sense, its mere occurrence implicates an important attribute; that is, if the tuned expression is important for the newly formed allohexaploid lines, then it is likely the pattern of expression rather than identity of the genes showing the pattern of expression bears the relevance. this novel feature of allopolyploidy-specified gene regulation has not been reported previously, but it is reminiscent of the findings in allotetraploid cotton, in which the parental dominance expression was found to be even in opposite directions in different allotetraploid lines but with similar proportions of genes showing the pattern. this feature of altered genomewide expression patterns also bears remarkable resemblance to the phenomenon of nucleoli dominance dictated by localized epigenetic difference, that is, stochastic silencing of one of the parental rrna genes in an allopolyploid, thus further suggesting possible commonality of the phenomenon in nascent plant allopolyploids. it is important to note that the majority of the genes, albeit showing parental dominance expression in the allohexaploid wheat lines, did not transgress the parental additive expression-range relative to the corresponding mpvs, as were found in cotton. this is interesting given that the "appeared" expression additivity for this set of genes was actually achieved by allohexaploidy-specific tuning rather than merely the adding-up of parental transcripts, suggesting that a too high- or low-level of expression is likely detrimental and, hence, is being selected against. non-additive gene expression in the newly formed allohexaploid wheat lines deviation from additivity in expression pattern of genes was commonly observed in previous studies, albeit the spectrum of deviation can be dramatically variable. although the functional relevance of non-additive gene expression remains largely unexplored, it is suggested that the non-additivity-bestowed diverse novel gene expression patterns may provide for variations upon which selection can act. it is conceivable that for non-additive gene expression to play a biologically meaningful role, transgenerational heritability of the altered expression patterns and/or the genes is required. indeed, several studies have indicated that the non-additive expression patterns can be established at the initial stages of allopolyploidization but with protracted effects over evolutionary timescales. results of this study suggest that it is the altered pattern of non-additive gene expression rather than the involved genes per se that showed a propensity of transgenerational inheritance, as evidenced by the results that the proportions of genes showing the altered expression patterns were highly similar between the two successive generations but the identities of the genes are highly variable. this novel paradigm of allopolyploidy-associated gene expression has not been reported previously. possible requirements for specific tuning of genes involved in vesicle function by the nascent allohexaploid wheat lines gene ontology analysis indicated that both groups of genes showing allopolyploidy-specific tuning, that is, parental dominance expression and non-additive expression, are involved in diverse functional go-categories. nevertheless, detailed analysis revealed that only those genes that showed non-additive expression, regardless of whether they are exhibiting parental dominance expression or not, are enriched for a very specific go-category involved in vesicle function. moreover, the enrichment was further enhanced for a subset of these genes showing transgenerational heritability. this novel observation strongly implicates a functional relevance of non-additive gene expression being associated with nascent allopolyploidy, which makes sense given that an immediate outcome of allopolyploidization is enlarged cell volume but reduced surface-area to volume ratio, and, therefore, tuned non-additive expression of vesicle-related genes may help circumvent this dilemma. the molecular basis underlying the allopolyploidy-regulated gene expression remains to be fully elucidated. it has been proposed that both genetic regulatory mechanisms, such as cis- and trans-interactions, and epigenetic alterations, such as altered dna methylation and small rna biogenesis, likely play important roles. further studies are needed to explore the exact roles played by these mechanisms in the allohexaploidization of wheat.  this study showed that the early stages of allohexaploidization, leading to the formation of allohexaploid wheat, exhibited two major patterns of global gene expression alteration: parental dominance expression and non-additive expression. the mechanisms underpinning the expression changes associated with nascent allopolyploidy remain largely unknown, but chromosomal instability is not a causal factor. instead, molecular interactions, including altered cis-/trans-regulation, dosage balance/compensation, gene elimination and/or epigenetic remodeling, brought about by the sudden merging of divergent parental species' genomes, are conceivably responsible. genes involved in each altered expression pattern could be classified into three distinct groups: stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. importantly, whereas both altered patterns of gene expression showed a propensity of transgenerational inheritance, identity of the involved genes is highly stochastic. accordingly, diverse gene ontology terms were implicated with both patterns of altered gene expression; however, those showing non-additive expression manifested a significant enrichment for a specific group of proteins associated with vesicle function. our results suggest that global alteration in gene expression conditioned by nascent allopolyploidy is accomplished by means of both targeted regulation and stochastic changes, that likely play distinct functional roles in the stabilization and establishment of the newly formed allohexaploid plants as competitive populations and new species. 
comparison with the parental plants coupled with inclusion of empirical mid-parent values revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of t. aestivum. multi-color gish was used to identify individual plants from two nascent allohexaploid wheat lines between triticum turgidum and aegilops tauschii, which had a stable chromosomal constitution analogous to that of common wheat. our results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. common wheat was formed via allohexaploidization about years ago, and became the most important crop plant. to gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat. nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function. genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from t. turgidum and ae. alteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse gene ontology terms.
 alteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. common wheat was formed via allohexaploidization about years ago, and became the most important crop plant. to gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat.  multi-color gish was used to identify individual plants from two nascent allohexaploid wheat lines between triticum turgidum and aegilops tauschii, which had a stable chromosomal constitution analogous to that of common wheat. genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from t. turgidum and ae. tauschii, using the affymetrix gene chip wheat genome-array. comparison with the parental plants coupled with inclusion of empirical mid-parent values revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse gene ontology terms. nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function.  our results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of t. aestivum. 