BACKGROUND
heterosis, or hybrid vigor, refers to the superior biological functions of f <dig> hybrids compared with their parental homozygous or inbred lines.
this phenomenon was first described by charles darwin and was later independently rediscovered by george h. shull and edward m. east in  <dig>  <cit> .
although it is not well understood at the molecular level, heterosis has been exploited over the past half-century in plant and animal breeding.
two classic hypotheses, “dominance” and “overdominance”, have been proposed to explain hybrid vigor.
the “dominance” hypothesis proposes that the detrimental allele from one parent is complemented by the superior allele from the other parent, and that the accumulated superior alleles in the f <dig> hybrids give rise to heterosis.
by comparison, the “overdominance” hypothesis signifies that hybrid vigor results from the interaction between alleles brought together in the hybrid  <cit> .
to attempt to discriminate between these hypotheses, extensive studies of gene effects and transcriptomics have been conducted  <cit> .
genetic analyses have revealed the genetic effects of additive, overdominance, dominance, and epistasis, and that interactions between different loci are associated with heterosis in different varieties  <cit> .
several studies analyzing transcription have indicated that degs commonly occur in inbred lines as well as between f <dig> hybrids and their parents.
for example, 4–18% of maize genes are differentially expressed in different tissues of the maize inbred lines b <dig> and mo <dig> according to microarray-based analyses  <cit> .
in arabidopsis seedlings, high-density single nucleotide polymorphism  analysis showed that 31% of all analyzed genes were differentially expressed between the parental inbred lines  <cit> , while, in another study,  <dig> % of genes were differentially expressed in different tissues of the hybrid rice lyp <dig> and its parents  <cit> .
recently, high-throughput rna-sequencing technology revealed that 4-week-old shoots of the 93– <dig> and nipponbare rice varieties had  <dig> % degs, as did their reciprocal hybrids  <cit> .
moreover, a newly published global survey based on rna sequencing technology found that approximately 70% of all expressed genes were differentially expressed between the two maize inbred parents b <dig> and mo <dig>  and that 42–55% were differentially expressed between the reciprocal f <dig> and its parents  <cit> .
although the molecular basis of heterosis has been attributed to the degs in the above hybrids, the underlying mechanism causing differential expression remain unknown.
several studies have shown that only one allele is expressed in heterozygotes  <cit> , and monoallelic expression or an imbalance in heterozygote allelic expression has been extensively studied in humans and other mammals  <cit> .
transcription profile analyses have indicated that monoallelic expression could be caused by x-chromosome silencing, autosomal imprinting, or random events.
some studies with vegetative tissues from maize f <dig> hybrids identified several genes exhibiting allele-specific expression   <cit> , which differed markedly between the different f <dig> hybrids and was altered in response to environmental stress.
this could contribute to heterosis.
the objectives of the present study were to explore global ase in hybrid rice and to reveal the mechanism of differential expression in f <dig> hybrids using rna sequencing.
three elite rice varieties were chosen that met the breeding objectives from different periods in china, guangluai # <dig> , teqing , and 93– <dig> , plus their f <dig> hybrids, which we show have different levels of heterosis.
two f <dig> hybrids, gl × tq and gl × 93- <dig>  exhibited high heterosis, and the third, 93-11 × tq, low heterosis.
to obtain sufficient snps to distinguish the maternal and paternal alleles in f <dig> hybrids, the genomes of the three parents were re-sequenced.
to identify more snps for further ase analysis, nuclear rna was extracted from leaves of the three f <dig> hybrids and their parents and subjected to illumina rna-seq technology.
we identify a global ase profile that reveals a potential mechanism for an increased biomass-based, grain-yield heterosis.
RESULTS
global ase analysis by rna sequencing
to obtain sufficient snps for ase analysis, we achieved a rice genome coverage of  <dig> – <dig>  fold to satisfy the minimum requirement of obtaining more than 90% snps  <cit> .
a total of  <dig> – <dig>  million reads  from three rice varieties serving as parents for the f1s were obtained using illumina dna-seq technology , and  <dig> – <dig>  snps were detected between each parent with  <dig> % accuracy .
a total of  <dig> – <dig>  million reads  were obtained from analogous tissue from the three f <dig> hybrids.
analysis disclosed that  <dig> – <dig>  genes at the secondary branch differentiation stage were expressed in the three f <dig> hybrids and their parents .
the expression levels of  <dig> genes in the f <dig> hybrids and their parents were also determined, and quantitative real time polymerase chain reaction  and rna-sequencing were shown to be highly correlated in terms of their analyses ).
we found that  <dig> – <dig>  of the snps located in gene bodies had an average read coverage of  <dig> – <dig>  in the f <dig> hybrids.
these snps were available for allelic expression analysis .
of these,  <dig> – <dig>  genes accounting for  <dig> – <dig> % of the total number of expressed genes containing snps could be used for distinguishing the alleles .
a total of  <dig> – <dig>  genes met the criterion of at least  <dig> snp reads, so could be used for further ase profile analyses.
through these ase profile analyses, the f <dig> hybrids were classified into three categories: monoallelic expression, in which only one allele from either the maternal or paternal parent is expressed; preferential allelic expression, in which expression levels differ by more than two-fold between alleles; and biallelic expression, in which expression levels vary by less than two-fold between alleles .
we found that  <dig> – <dig> % of the genes were monoallelic expression,  <dig> – <dig> % were preferential allelic expression, and  <dig> – <dig> % were biallelic expression .
further analysis of paternally and maternally derived read coverage in each heterozygote showed that the two parents contributed equally to the f <dig> hybrids.
our results are consistent with those from arabidopsis embryos  <cit> , and suggest that no obvious parent-of-origin effect occurred in the vegetative tissues of the rice hybrids .
a cis-regulation mechanism and genotype-dependent monoallelic expression
to understand the relationship between gene expression in the parents and ase in the f <dig> hybrids, the correlation between these parameters was determined for  <dig> – <dig>  genes, yielding correlation coefficients in the range of  <dig> – <dig>  .
a higher correlation coefficient was obtained from the same analysis with  <dig>  ase genes in the f <dig> hybrids .
these results suggest that a cis-regulatory mechanism is occurring, that is, if the allele is transcribed in the parent it is also transcribed in its f <dig> hybrids, and if not in the parent then not in its f <dig> hybrids.
our data also indicate that only  <dig> – <dig> % of gene expression in f <dig> hybrids is non-additive.
trans-acting regulation may thus also contribute to the regulation of gene expression in rice hybrids, but would not have a major effect.
we identified  <dig> monoallelic expression genes in the three f <dig> hybrids  .
of these,  <dig> were common to two f <dig> hybrids, but none were common to all three .
we randomly chose  <dig> monoallelic expression genes, accounting for  <dig> % of the total, from the three hybrids and used reverse-transcription -pcr sequencing to verify a reliability of  <dig> %  .
through further investigation of allelic expression patterns in three reciprocal crosses, gl × 93- <dig> and 93-11 × gl, gl × tq and tq × gl, and 9311 × tq and tq × 93- <dig>  we detected  <dig> monoallelic expression genes and  <dig> preferential allelic expression genes and found that all monoallelic expression and preferential allelic expression genes tested exhibited a genotype-dependent expression pattern, while  <dig> biallelic expression genes showed no difference between reciprocal crosses .
this shows that, regardless of the paternal or maternal origin in the reciprocal crossings, monoallelic expression and preferential allelic expression genes always express the allele from a given parent .
combining our results with previous observations from maize  <cit> , we suggest that a hitherto overlooked type of monoallelic expression occurs in eukaryotic organisms.
ase results in transcriptome divergence in the f <dig> hybrids
previous studies have demonstrated that higher levels of heterosis are associated with greater differences between the agronomic and/or metabolic traits of parents  <cit> , and that degs  < <dig> ) between f <dig> hybrids and their parents are among the major factors leading to heterosis  <cit> .
to ascertain the extent to which monoallelic expression and preferential allelic expression genes contribute to heterosis, we dissected the global degs between f <dig> hybrids and their parents.
the total number of degs in the three hybrids was correlated with heterosis level of both fresh and dry mass .
to explore which mechanism create degs in heterozygotes, we found that  <dig> % of monoallelic expression genes,  <dig> % of preferential allelic expression genes, and  <dig> % of biallelic expression genes are degs .
surprisingly, we discovered that the monoallelic expression and preferential allelic expression genes, comprising  <dig> % of the total analyzed genes, amounted to  <dig> % of the degs .
more specifically, the monoallelic expression genes, accounting for  <dig> % of the total analyzed genes, gave rise to  <dig> % of the degs, the preferential allelic expression genes  amounted to  <dig> %, and the biallelic expression genes  were composed of only  <dig> % of the degs .
by determining contributions to degs between f <dig> hybrids and their parents, we found that  <dig> % of degs from monoallelic expression,  <dig> % from preferential allelic expression, and  <dig> % from biallelic expression exhibited a considerably greater than 10-fold difference.
by contrast,  <dig> % of degs from biallelic expression,  <dig> % from preferential allelic expression, and  <dig> % from monoallelic expression genes displayed a less than four-fold difference between the f <dig> and their parents .
the  <dig> – <dig> % of the genes that were expressed in the f1s but silenced in one of the two parents showed monoallelic expression patterns .
these results demonstrate that the majority of degs  are attributable to ase, and that monoallelic expression genes in particular play an important role in gene expression divergence between f <dig> hybrids and their parents.
complementary effects are mainly contributed by ase genes
because the presence of a cis-regulatory mechanism of allelic expression would be in accordance with the “dominance” hypothesis, we analyzed the transcriptomic profiles of all ase genes in the three f <dig> hybrids and their parents.
a large number of genotype-dependent monoallelic expression genes in f <dig> hybrids would also be in accordance with the “dominance” hypothesis.
the results showed that an average of  <dig> %  of genes expressed in one parent and non-expressed in the other were categorized as monoallelic expression genes in f <dig> hybrids , whereas  <dig> %  of genes expressed at low levels in either parent, but enhanced in f <dig> hybrids, were categorized as monoallelic expression genes .
most of both types of monoallelic expression genes exhibited a mid-parent expression level .
therefore, the alleles only expressed in the f <dig> were those expressed in the inbred parent, while the alleles silenced in the inbred parent were also silenced in f <dig>  this means that the total expression level of the gene in the f <dig> is one half that in the parent, which is equivalent to a mid-parent expression level .
this dosage effect implies that a cis-regulatory mechanism is acting.
moreover, we found that more monoallelic expression genes were downregulated than upregulated in f <dig> , and that  <dig> % of preferential allelic expression genes exhibited the same patterns as monoallelic expression genes in the f <dig> hybrids .
the analysis of genes within the categories of activated and enhanced expression in f <dig> hybrids found complementary effects of superior alleles from both parents with average values of  <dig> % and  <dig> % for class iii and class iv genes, respectively.
by contrast, a biallelic expression pattern was exhibited by only  <dig> % and  <dig> % in class iii and class iv genes, respectively .
the proportion of genes with complementary effects in gl × tq and gl × 93- <dig> was higher than that in 93-11 × tq , which was consistent with the heterosis level of these f <dig> hybrids .
our data imply that ase genes, most notably monoallelic expression genes, are the main contributors to allelic complementary effects in hybrid rice.
ase genes have diverse biological functions in f <dig> hybrids
to ascertain the molecular and biological functions of monoallelic expression and preferential allelic expression genes, we performed gene ontology  analysis.
four go terms for molecular functions, nucleotide binding, receptor activity, protein binding, and kinase activity, were commonly found among both monoallelic expression and preferential allelic expression genes .
these functions indicate that monoallelic expression genes are mainly involved in protein modification, signal transduction, and response to endogenous stimulus pathways.
a wider diversity of functions, including biosynthesis, morphogenesis, and carbohydrate metabolism, was found for preferential allelic expression genes , suggesting that ase genes have important roles in biosynthesis, development, and their regulation.
to distinguish between the enrichment of monoallelic expression and preferential allelic expression gene functions and that of all genes for ase analysis, we performed go analysis of  <dig> – <dig>  genes in three f <dig> hybrids.
limited go terms were common to monoallelic expression and preferential allelic expression genes in different f <dig> hybrids , which might be a consequence of specific monoallelic expression and preferential allelic expression genes occurring in different f <dig> hybrids, suggesting that allele-specific expression genes have different roles in different genomic backgrounds.
discussion
previous studies of transcriptome analyses related to heterosis have mainly been conducted using microarray analyses, and rt-pcr and rna-sequencing technology  <cit> .
here, we combined rna-sequencing with dna re-sequencing technology to establish ase assays and achieved a 20-fold coverage of rice genome re-sequencing data to identify snps.
a strict statistical cut-off for snp calling enabled fully quantitative analyses of both overall and allele-specific gene expression profiles to be obtained for rice leaves at the stage of secondary branch differentiation.
these data were verified by pcr-sequencing and rt-pcr sequencing using analogous materials planted in the following year.
this developing stage is important as grain yield is directly correlated with the biomass established early in vegetative growth  <cit> .
snp accuracies of  <dig> % and  <dig> % were confirmed, indicating the reliability of both genome and transcriptome data, respectively.
monoallelic expression genes have been studied for nearly a half century in humans and other mammals  <cit> .
most cause x-chromosome silencing, autosomal imprinting, or random events  <cit> , and contribute to dose-dependent gene expression, immune responses, and disease biogenesis, including several types of cancers  <cit> .
ase and regulation mechanisms have also been studied in humans and other animals  <cit> ; however, by contrast, monoallelic expression is poorly understood in higher plants.
different studies have reported a high proportion of ase genes in maize hybrids  with cis-regulatory effects underlying the ase  <cit> , compared with a more limited number of monoallelic expression genes  <cit> .
other studies using f <dig> hybrids of japonica-indica, maize, and arabidopsis focused on endosperm-localized genes and identified more than  <dig> imprinted genes  <cit> .
however, no large-scale ase analysis has previously been carried out in rice hybrids.
we identified  <dig> monoallelic expression and  <dig>  preferential allelic expression genes in the three f <dig> hybrids via a global transcriptome analysis.
of the total genes analyzed,  <dig> – <dig> % exhibited monoallelic expression and  <dig> – <dig> % exhibited preferential allelic expression patterns.
the proportion of ase genes, moreover, did not differ significantly in the three f <dig> hybrids, which is similar to a previous report in maize hybrids  <cit> .
importantly, our results indicated that all monoallelic expression and preferential allelic expression genes tested exhibited genotype-dependent expression patterns in reciprocal crosses, which contrasts with the findings of monoallelic expression genes in humans and other mammals  <cit> .
the observed genotype-dependent ase in the vegetative tissue of hybrid rice could represent a common mechanism of allelic complementary effects in hybrids, and show the importance of parental genotype in both crossbreeding and hybrid breeding.
given the number of genes with genotype-dependent monoallelic expression involved in a wide range of go categories that play important biological functions, many potential opportunities exist for them to contribute to degs and to produce the diverse phenotypes of the f <dig> hybrids.
in the present study, the genotype-dependent ase genes might confer a fitness advantage to the heterozygous state relative to either of their homozygous parents.
as most monoallelic expression genes were silenced or suppressed in one of the parents, we speculate that genotype-dependent monoallelic expression might be the consequence of artificial parent selection by breeders.
to meet the breeding objectives, “superior alleles” are accumulated in the elite parent for long-term selection .
such alleles could maintain allelic diversity in different varieties and enlarge the allele difference in the rice germplasm.
therefore, our findings of allelic complementary effects in f <dig> hybrid rice could guide the selection of elite parents through complementing a variety of the superior alleles.
previous studies have indicated that differential gene expression is common between f <dig> and parents, and that it is a major contributor to heterosis at the transcription level  <cit> .
for example, hundreds of differentially expressed genes were detected at different developmental stages between the elite hybrid rice lyp <dig> and its parents  <cit> , and analogous results were obtained from studies with arabidopsis and maize  <cit> .
our study also found that the percentage of degs between f <dig> hybrids and parents exactly correlated with the heterosis level of each f <dig> hybrid .
such results do not divulge, nonetheless, how the alleles from inbred lines become the degs in heterozygotes, although the mechanism behind this is revealed to some extent by genome-wide, ase analysis using high-throughput rna sequencing.
moreover, in the present study, the most significant degs between the f <dig> and parent were expressed in the f <dig> but silenced in one of the parents, accounting for  <dig> % of the degs associated with ase.
of these,  <dig> % showed preferential allelic expression and  <dig> % were monoallelic expression.
altogether,  <dig> % of the ase genes contributed to  <dig> % of the total number of degs.
although the proportion of ase genes was similar in the three f <dig> hybrids, the total numbers of degs differed, with fewer detected in 93-11 × tq  with its lower heterosis level  and more found in gl × tq and gl × 93- <dig> with their higher heterosis levels.
the same results were also found between their parents, suggesting that transcriptome divergence in f <dig> hybrids is attributed to transcriptome divergence between parents.
crucially, increased parent allelic variation could be an important strategy for maintaining higher heterosis levels in hybrid rice breeding programs through enlarging the transcriptome diversity between parents, which have accumulated different sets of superior alleles.
a recent study has provided molecular evidence for a single gene model to support the “overdominance” hypothesis of heterosis in tomato hybrids  <cit> .
because of technological limitations, however, the maternal and paternal alleles in the f <dig> hybrids could not be distinguished effectively.
the recent development of high-throughput sequencing technology provides the opportunity to study ase in heterozygotes.
many genes have exhibited additive expression patterns in f <dig> hybrids in previous studies, and complementary effects at the gene expression level have been reported in hybrid maize  <cit> .
our data derived from global allelic expression profiles extend this result to hybrid rice, and also reveal the mechanism of deg formation in heterozygotes.
our observed high correlation  between allelic expression in f <dig> hybrids and gene expression in parental lines indicates that a cis-regulated mechanism plays an essential role in allelic expression.
allelic complementation effects, moreover, can be the outcome of a cis-regulatory mechanism mainly contributed to by ase genes.
the findings of the present study support the “dominance” hypothesis for indica hybrid rice varieties and reveal that the consequences of complementation, primarily by genotype-dependent monoallelic expression and preferential allelic expression alleles, are an accumulation of “superior” alleles that confer monoallelic expression and preferential allelic expression genes in f <dig> hybrids.
the expression of these “superior” alleles offers an opportunity to optimize the transcriptomes that give rise to heterosis in f <dig> hybrids.
although our results revealed that allelic complementary effects played a major contribution to gene expression in hybrid rice and support the “dominance” hypothesis, they do not exclude the contribution of different mechanisms to heterosis in hybrid rice and other crops.
CONCLUSIONS
allelic expression profiles in hybrid rice determined by rna-sequencing technology demonstrated a type of genotype-dependent monoallelic expression genes in plants.
degs between parents and f <dig> hybrids were mainly attributable to ase genes, which gave rise to the observed allelic complementary effects in f <dig> hybrids.

 heterosis, or hybrid vigor, refers to the superior biological functions of f hybrids compared with their parental homozygous or inbred lines. this phenomenon was first described by charles darwin and was later independently rediscovered by george h. shull and edward m. east in. although it is not well understood at the molecular level, heterosis has been exploited over the past half-century in plant and animal breeding. two classic hypotheses, “dominance” and “overdominance”, have been proposed to explain hybrid vigor. the “dominance” hypothesis proposes that the detrimental allele from one parent is complemented by the superior allele from the other parent, and that the accumulated superior alleles in the f hybrids give rise to heterosis. by comparison, the “overdominance” hypothesis signifies that hybrid vigor results from the interaction between alleles brought together in the hybrid. to attempt to discriminate between these hypotheses, extensive studies of gene effects and transcriptomics have been conducted. genetic analyses have revealed the genetic effects of additive, overdominance, dominance, and epistasis, and that interactions between different loci are associated with heterosis in different varieties. several studies analyzing transcription have indicated that degs commonly occur in inbred lines as well as between f hybrids and their parents. for example, 4–18% of maize genes are differentially expressed in different tissues of the maize inbred lines b and mo according to microarray-based analyses. in arabidopsis seedlings, high-density single nucleotide polymorphism analysis showed that 31% of all analyzed genes were differentially expressed between the parental inbred lines, while, in another study, % of genes were differentially expressed in different tissues of the hybrid rice lyp and its parents. recently, high-throughput rna-sequencing technology revealed that 4-week-old shoots of the 93– and nipponbare rice varieties had % degs, as did their reciprocal hybrids. moreover, a newly published global survey based on rna sequencing technology found that approximately 70% of all expressed genes were differentially expressed between the two maize inbred parents b and mo and that 42–55% were differentially expressed between the reciprocal f and its parents. although the molecular basis of heterosis has been attributed to the degs in the above hybrids, the underlying mechanism causing differential expression remain unknown. several studies have shown that only one allele is expressed in heterozygotes, and monoallelic expression or an imbalance in heterozygote allelic expression has been extensively studied in humans and other mammals. transcription profile analyses have indicated that monoallelic expression could be caused by x-chromosome silencing, autosomal imprinting, or random events. some studies with vegetative tissues from maize f hybrids identified several genes exhibiting allele-specific expression, which differed markedly between the different f hybrids and was altered in response to environmental stress. this could contribute to heterosis. the objectives of the present study were to explore global ase in hybrid rice and to reveal the mechanism of differential expression in f hybrids using rna sequencing. three elite rice varieties were chosen that met the breeding objectives from different periods in china, guangluai #, teqing, and 93–, plus their f hybrids, which we show have different levels of heterosis. two f hybrids, gl × tq and gl × 93- exhibited high heterosis, and the third, 93-11 × tq, low heterosis. to obtain sufficient snps to distinguish the maternal and paternal alleles in f hybrids, the genomes of the three parents were re-sequenced. to identify more snps for further ase analysis, nuclear rna was extracted from leaves of the three f hybrids and their parents and subjected to illumina rna-seq technology. we identify a global ase profile that reveals a potential mechanism for an increased biomass-based, grain-yield heterosis.  global ase analysis by rna sequencing to obtain sufficient snps for ase analysis, we achieved a rice genome coverage of – fold to satisfy the minimum requirement of obtaining more than 90% snps. a total of – million reads from three rice varieties serving as parents for the f1s were obtained using illumina dna-seq technology, and – snps were detected between each parent with % accuracy. a total of – million reads were obtained from analogous tissue from the three f hybrids. analysis disclosed that – genes at the secondary branch differentiation stage were expressed in the three f hybrids and their parents. the expression levels of genes in the f hybrids and their parents were also determined, and quantitative real time polymerase chain reaction and rna-sequencing were shown to be highly correlated in terms of their analyses ). we found that – of the snps located in gene bodies had an average read coverage of – in the f hybrids. these snps were available for allelic expression analysis. of these, – genes accounting for – % of the total number of expressed genes containing snps could be used for distinguishing the alleles. a total of – genes met the criterion of at least snp reads, so could be used for further ase profile analyses. through these ase profile analyses, the f hybrids were classified into three categories: monoallelic expression, in which only one allele from either the maternal or paternal parent is expressed; preferential allelic expression, in which expression levels differ by more than two-fold between alleles; and biallelic expression, in which expression levels vary by less than two-fold between alleles. we found that – % of the genes were monoallelic expression, – % were preferential allelic expression, and – % were biallelic expression. further analysis of paternally and maternally derived read coverage in each heterozygote showed that the two parents contributed equally to the f hybrids. our results are consistent with those from arabidopsis embryos, and suggest that no obvious parent-of-origin effect occurred in the vegetative tissues of the rice hybrids. a cis-regulation mechanism and genotype-dependent monoallelic expression to understand the relationship between gene expression in the parents and ase in the f hybrids, the correlation between these parameters was determined for – genes, yielding correlation coefficients in the range of –. a higher correlation coefficient was obtained from the same analysis with ase genes in the f hybrids. these results suggest that a cis-regulatory mechanism is occurring, that is, if the allele is transcribed in the parent it is also transcribed in its f hybrids, and if not in the parent then not in its f hybrids. our data also indicate that only – % of gene expression in f hybrids is non-additive. trans-acting regulation may thus also contribute to the regulation of gene expression in rice hybrids, but would not have a major effect. we identified monoallelic expression genes in the three f hybrids. of these, were common to two f hybrids, but none were common to all three. we randomly chose monoallelic expression genes, accounting for % of the total, from the three hybrids and used reverse-transcription -pcr sequencing to verify a reliability of %. through further investigation of allelic expression patterns in three reciprocal crosses, gl × 93- and 93-11 × gl, gl × tq and tq × gl, and 9311 × tq and tq × 93- we detected monoallelic expression genes and preferential allelic expression genes and found that all monoallelic expression and preferential allelic expression genes tested exhibited a genotype-dependent expression pattern, while biallelic expression genes showed no difference between reciprocal crosses. this shows that, regardless of the paternal or maternal origin in the reciprocal crossings, monoallelic expression and preferential allelic expression genes always express the allele from a given parent. combining our results with previous observations from maize, we suggest that a hitherto overlooked type of monoallelic expression occurs in eukaryotic organisms. ase results in transcriptome divergence in the f hybrids previous studies have demonstrated that higher levels of heterosis are associated with greater differences between the agronomic and/or metabolic traits of parents, and that degs < ) between f hybrids and their parents are among the major factors leading to heterosis. to ascertain the extent to which monoallelic expression and preferential allelic expression genes contribute to heterosis, we dissected the global degs between f hybrids and their parents. the total number of degs in the three hybrids was correlated with heterosis level of both fresh and dry mass. to explore which mechanism create degs in heterozygotes, we found that % of monoallelic expression genes, % of preferential allelic expression genes, and % of biallelic expression genes are degs. surprisingly, we discovered that the monoallelic expression and preferential allelic expression genes, comprising % of the total analyzed genes, amounted to % of the degs. more specifically, the monoallelic expression genes, accounting for % of the total analyzed genes, gave rise to % of the degs, the preferential allelic expression genes amounted to %, and the biallelic expression genes were composed of only % of the degs. by determining contributions to degs between f hybrids and their parents, we found that % of degs from monoallelic expression, % from preferential allelic expression, and % from biallelic expression exhibited a considerably greater than 10-fold difference. by contrast, % of degs from biallelic expression, % from preferential allelic expression, and % from monoallelic expression genes displayed a less than four-fold difference between the f and their parents. the – % of the genes that were expressed in the f1s but silenced in one of the two parents showed monoallelic expression patterns. these results demonstrate that the majority of degs are attributable to ase, and that monoallelic expression genes in particular play an important role in gene expression divergence between f hybrids and their parents. complementary effects are mainly contributed by ase genes because the presence of a cis-regulatory mechanism of allelic expression would be in accordance with the “dominance” hypothesis, we analyzed the transcriptomic profiles of all ase genes in the three f hybrids and their parents. a large number of genotype-dependent monoallelic expression genes in f hybrids would also be in accordance with the “dominance” hypothesis. the results showed that an average of % of genes expressed in one parent and non-expressed in the other were categorized as monoallelic expression genes in f hybrids, whereas % of genes expressed at low levels in either parent, but enhanced in f hybrids, were categorized as monoallelic expression genes. most of both types of monoallelic expression genes exhibited a mid-parent expression level. therefore, the alleles only expressed in the f were those expressed in the inbred parent, while the alleles silenced in the inbred parent were also silenced in f this means that the total expression level of the gene in the f is one half that in the parent, which is equivalent to a mid-parent expression level. this dosage effect implies that a cis-regulatory mechanism is acting. moreover, we found that more monoallelic expression genes were downregulated than upregulated in f, and that % of preferential allelic expression genes exhibited the same patterns as monoallelic expression genes in the f hybrids. the analysis of genes within the categories of activated and enhanced expression in f hybrids found complementary effects of superior alleles from both parents with average values of % and % for class iii and class iv genes, respectively. by contrast, a biallelic expression pattern was exhibited by only % and % in class iii and class iv genes, respectively. the proportion of genes with complementary effects in gl × tq and gl × 93- was higher than that in 93-11 × tq, which was consistent with the heterosis level of these f hybrids. our data imply that ase genes, most notably monoallelic expression genes, are the main contributors to allelic complementary effects in hybrid rice. ase genes have diverse biological functions in f hybrids to ascertain the molecular and biological functions of monoallelic expression and preferential allelic expression genes, we performed gene ontology analysis. four go terms for molecular functions, nucleotide binding, receptor activity, protein binding, and kinase activity, were commonly found among both monoallelic expression and preferential allelic expression genes. these functions indicate that monoallelic expression genes are mainly involved in protein modification, signal transduction, and response to endogenous stimulus pathways. a wider diversity of functions, including biosynthesis, morphogenesis, and carbohydrate metabolism, was found for preferential allelic expression genes, suggesting that ase genes have important roles in biosynthesis, development, and their regulation. to distinguish between the enrichment of monoallelic expression and preferential allelic expression gene functions and that of all genes for ase analysis, we performed go analysis of – genes in three f hybrids. limited go terms were common to monoallelic expression and preferential allelic expression genes in different f hybrids, which might be a consequence of specific monoallelic expression and preferential allelic expression genes occurring in different f hybrids, suggesting that allele-specific expression genes have different roles in different genomic backgrounds. discussion previous studies of transcriptome analyses related to heterosis have mainly been conducted using microarray analyses, and rt-pcr and rna-sequencing technology. here, we combined rna-sequencing with dna re-sequencing technology to establish ase assays and achieved a 20-fold coverage of rice genome re-sequencing data to identify snps. a strict statistical cut-off for snp calling enabled fully quantitative analyses of both overall and allele-specific gene expression profiles to be obtained for rice leaves at the stage of secondary branch differentiation. these data were verified by pcr-sequencing and rt-pcr sequencing using analogous materials planted in the following year. this developing stage is important as grain yield is directly correlated with the biomass established early in vegetative growth. snp accuracies of % and % were confirmed, indicating the reliability of both genome and transcriptome data, respectively. monoallelic expression genes have been studied for nearly a half century in humans and other mammals. most cause x-chromosome silencing, autosomal imprinting, or random events, and contribute to dose-dependent gene expression, immune responses, and disease biogenesis, including several types of cancers. ase and regulation mechanisms have also been studied in humans and other animals; however, by contrast, monoallelic expression is poorly understood in higher plants. different studies have reported a high proportion of ase genes in maize hybrids with cis-regulatory effects underlying the ase, compared with a more limited number of monoallelic expression genes. other studies using f hybrids of japonica-indica, maize, and arabidopsis focused on endosperm-localized genes and identified more than imprinted genes. however, no large-scale ase analysis has previously been carried out in rice hybrids. we identified monoallelic expression and preferential allelic expression genes in the three f hybrids via a global transcriptome analysis. of the total genes analyzed, – % exhibited monoallelic expression and – % exhibited preferential allelic expression patterns. the proportion of ase genes, moreover, did not differ significantly in the three f hybrids, which is similar to a previous report in maize hybrids. importantly, our results indicated that all monoallelic expression and preferential allelic expression genes tested exhibited genotype-dependent expression patterns in reciprocal crosses, which contrasts with the findings of monoallelic expression genes in humans and other mammals. the observed genotype-dependent ase in the vegetative tissue of hybrid rice could represent a common mechanism of allelic complementary effects in hybrids, and show the importance of parental genotype in both crossbreeding and hybrid breeding. given the number of genes with genotype-dependent monoallelic expression involved in a wide range of go categories that play important biological functions, many potential opportunities exist for them to contribute to degs and to produce the diverse phenotypes of the f hybrids. in the present study, the genotype-dependent ase genes might confer a fitness advantage to the heterozygous state relative to either of their homozygous parents. as most monoallelic expression genes were silenced or suppressed in one of the parents, we speculate that genotype-dependent monoallelic expression might be the consequence of artificial parent selection by breeders. to meet the breeding objectives, “superior alleles” are accumulated in the elite parent for long-term selection. such alleles could maintain allelic diversity in different varieties and enlarge the allele difference in the rice germplasm. therefore, our findings of allelic complementary effects in f hybrid rice could guide the selection of elite parents through complementing a variety of the superior alleles. previous studies have indicated that differential gene expression is common between f and parents, and that it is a major contributor to heterosis at the transcription level. for example, hundreds of differentially expressed genes were detected at different developmental stages between the elite hybrid rice lyp and its parents, and analogous results were obtained from studies with arabidopsis and maize. our study also found that the percentage of degs between f hybrids and parents exactly correlated with the heterosis level of each f hybrid. such results do not divulge, nonetheless, how the alleles from inbred lines become the degs in heterozygotes, although the mechanism behind this is revealed to some extent by genome-wide, ase analysis using high-throughput rna sequencing. moreover, in the present study, the most significant degs between the f and parent were expressed in the f but silenced in one of the parents, accounting for % of the degs associated with ase. of these, % showed preferential allelic expression and % were monoallelic expression. altogether, % of the ase genes contributed to % of the total number of degs. although the proportion of ase genes was similar in the three f hybrids, the total numbers of degs differed, with fewer detected in 93-11 × tq with its lower heterosis level and more found in gl × tq and gl × 93- with their higher heterosis levels. the same results were also found between their parents, suggesting that transcriptome divergence in f hybrids is attributed to transcriptome divergence between parents. crucially, increased parent allelic variation could be an important strategy for maintaining higher heterosis levels in hybrid rice breeding programs through enlarging the transcriptome diversity between parents, which have accumulated different sets of superior alleles. a recent study has provided molecular evidence for a single gene model to support the “overdominance” hypothesis of heterosis in tomato hybrids. because of technological limitations, however, the maternal and paternal alleles in the f hybrids could not be distinguished effectively. the recent development of high-throughput sequencing technology provides the opportunity to study ase in heterozygotes. many genes have exhibited additive expression patterns in f hybrids in previous studies, and complementary effects at the gene expression level have been reported in hybrid maize. our data derived from global allelic expression profiles extend this result to hybrid rice, and also reveal the mechanism of deg formation in heterozygotes. our observed high correlation between allelic expression in f hybrids and gene expression in parental lines indicates that a cis-regulated mechanism plays an essential role in allelic expression. allelic complementation effects, moreover, can be the outcome of a cis-regulatory mechanism mainly contributed to by ase genes. the findings of the present study support the “dominance” hypothesis for indica hybrid rice varieties and reveal that the consequences of complementation, primarily by genotype-dependent monoallelic expression and preferential allelic expression alleles, are an accumulation of “superior” alleles that confer monoallelic expression and preferential allelic expression genes in f hybrids. the expression of these “superior” alleles offers an opportunity to optimize the transcriptomes that give rise to heterosis in f hybrids. although our results revealed that allelic complementary effects played a major contribution to gene expression in hybrid rice and support the “dominance” hypothesis, they do not exclude the contribution of different mechanisms to heterosis in hybrid rice and other crops.  allelic expression profiles in hybrid rice determined by rna-sequencing technology demonstrated a type of genotype-dependent monoallelic expression genes in plants. degs between parents and f hybrids were mainly attributable to ase genes, which gave rise to the observed allelic complementary effects in f hybrids. 
a cis-regulatory mechanism was identified that contributes to allele-specific expression, leading to differential gene expression and allelic complementary effects in f hybrids. of the expressed genes, % exhibited an unexpected type of monoallelic expression and % showed preferential allelic expression that was genotype-dependent in reciprocal crosses. allele-specific expressioncomplementary effectsdifferentially expressed genesgenotype-dependent monoallelic expressionrice hybrids however, it is not clear why yield in heterozygotes is superior to that of the homozygous parents or how degs are produced. significant allelic complementary effects were detected in the f hybrids of rice. analysis of the allelic expression profiles of genes at the critical stage for highest biomass production from the leaves of three different rice f hybrids identified genotype-dependent allele-specific expression genes. global allele-specific expression analysis in hybrid rice has the potential to answer these questions. extensive studies on heterosis in plants using transcriptome analysis have identified differentially expressed genes in f hybrids.
 extensive studies on heterosis in plants using transcriptome analysis have identified differentially expressed genes in f hybrids. however, it is not clear why yield in heterozygotes is superior to that of the homozygous parents or how degs are produced. global allele-specific expression analysis in hybrid rice has the potential to answer these questions.  we report a genome-wide allele-specific expression analysis using rna-sequencing technology of – genes from three rice f hybrids. of the expressed genes, % exhibited an unexpected type of monoallelic expression and % showed preferential allelic expression that was genotype-dependent in reciprocal crosses. those genes exhibiting allele-specific expression comprised % of the genes differentially expressed between f hybrids and their parents. allele-specific expression accounted for % of the genes displaying more than a 10-fold expression level difference between an f and its parents, and almost all of the genes expressed in f but non-expressed in one parent. significant allelic complementary effects were detected in the f hybrids of rice.  analysis of the allelic expression profiles of genes at the critical stage for highest biomass production from the leaves of three different rice f hybrids identified genotype-dependent allele-specific expression genes. a cis-regulatory mechanism was identified that contributes to allele-specific expression, leading to differential gene expression and allelic complementary effects in f hybrids. allele-specific expressioncomplementary effectsdifferentially expressed genesgenotype-dependent monoallelic expressionrice hybrids 