BACKGROUND
heterosis is a phenomenon in which hybrids exhibit superior phenotypes, such as enhanced biomass production, development rate, grain yield, and stress tolerance, relative to their parents.
heterosis has been effectively utilized to increase crop production in the world.
it is estimated, for example, that hybrid rice, which occupies more than 50% of the total rice area in china, has a 10–20% yield advantage over inbred varieties  <cit> .
our knowledge of the genetic mechanisms of heterosis, however, has lagged behind its wide application.
two hypotheses-dominance  <cit>  and over-dominance  <cit> -were proposed in the early 20th century to interpret heterosis.
both describe nonadditive behavior as a consequence of genetic differences between distinct homozygous parental lines and their heterozygous hybrids  <cit> .
with the advent of molecular makers, quantitative trait locus  mapping has become a routine tool for studying the genetic basis of heterosis in crop plants; so far, however, the qtl analysis has not contributed much to the understanding of heterosis.
with the development of functional genomics, the technique of large-scale transcriptome analysis-based on cdna or expressed sequence tag  library sequencing, microarray hybridization, and serial analysis of gene expression -has been used to investigate heterosis in arabidopsis <cit> , maize  <cit> , and rice  <cit> .
such technologies offer the potential to unveil the molecular basis of heterosis at the transcriptional level  <cit> .
these technologies have drawbacks, however, such as low throughput, high cost, low sensitivity, cloning bias, high background signal, and pre-determined probes requirements  <cit> .
next-generation high-throughput rna sequencing technology  is a recently-developed method for discovering, profiling, and quantifying rna transcripts with several advantages over other expression profiling technologies including higher sensitivity and the ability to detect splicing isoforms and somatic mutations  <cit> .
using rna-seq, significant progress has been made in understanding the transcript expression of rice over the last two years  <cit> .
for example, transcriptome analysis of rice mature root tissue and root tips at two time points identified  <dig> root-enriched transcripts and  <dig> tip-enriched transcripts involved in different physiological processes  <cit> .
in addition, rna-seq has been applied to the identification of stress-inducible transcripts in rice  <cit> .
other than a transcriptome analysis of seedling shoots at the four-leaf stage  <cit> , however, little effort is being expended in attempts to investigate heterosis using rna-seq.
plant root systems serve a number of important functions, including anchoring the plant, absorbing water and nutrients, producing amino acids and hormones, and secreting organic acids, enzymes, and alkaloids.
in recent years, considerable research and interest has focused on root systems.
some of these studies have demonstrated that heterosis levels might be higher in root traits than in aboveground agronomic traits  <cit> , which suggests that roots might be an ideal organ for investigating the genetic basis of rice heterosis.
several attempts have been made to discover the molecular mechanism of root heterosis at the vegetative stage  <cit> , but little attention has been paid to heterosis in the root system during the late growth stage, when accumulation, transportation, and distribution of dry matter, and ultimately, yield potential, may be influenced.
there has currently been no systematic investigation into root heterosis at the two different developmental stages.
in this study, we focused our heterosis research on the late-stage high-vigor super-hybrid rice variety, xieyou  <dig>  which has a grain yield of up to  <dig>  ×  <dig> kg ⋅ hm- <dig> <cit>  and was designated as “super rice” by the chinese ministry of agriculture in  <dig>  xieyou  <dig> was bred by crossing the restorer line r <dig>  to the maternal line xieqingzao b .
we used rna-seq to investigate the global transcriptomes of roots from xieyou  <dig> and its parents at tillering and heading stages.
differentially expressed transcripts and their expression patterns were analyzed, and several potential candidate transcripts were found to be involved in carbohydrate metabolism and plant hormone signal transduction pathways.
we expect this genome-wide transcriptome comparison to provide a starting point to understand the causative mechanism of the altered gene expression in the hybrid and the molecular mechanism underlying rice root heterosis.
RESULTS
characterization of xieyou  <dig> and its two parental lines
we used rna-seq to investigate gene expression and function in a heterotic cross involving xieyou  <dig>  a super-hybrid rice variety with superior yield performance, its maternal line xieqingzao b, and its paternal line r <dig>  as suggested by pickett  <cit> , other yield-related traits might also show heterosis.
in this study, we found that the roots and aerial parts of xieyou  <dig> were more vigorous than those of either parent .
mid-parent heterosis  and best-parent heterosis  were calculated to measure the heterosis of aerial parts and roots .
the quantification of mph and hph and their statistical significance were shown in table  <dig>  we observed significant mph  for all traits at both stages except for shoot dry weight at the tillering stage.
furthermore, we also observed significant hph  for the root-shoot ratio at the tillering stage and root length, root dry weight, and shoot dry weight at the heading stage.
the mph and hph of root dry weight were greater than those of shoot dry weight at both stages, indicating that the level of heterosis was much higher in roots than in aerial parts.
at the tillering stage, the mph of root length, root dry weight, and root-shoot ratio varied from  <dig> % to  <dig> %.
by comparison, the degree of heterosis for these traits was larger at the heading stage, with the mph ranging from  <dig> % to  <dig> %.
**significant difference with p <  <dig> .
*significant difference with p <  <dig> .
mapping reads to the rice genome
to analyze the transcriptomes of the above three genotypes at tillering and heading stages, cdna libraries were prepared from rice roots and subjected to rna-seq analysis on the illumina hiseq  <dig> platform.
in total,  <dig> million short reads were generated from the two developmental stages, with  <dig> million high-quality 100-bp reads selected for further analysis .
the two biological replicates were in good agreement with respect to gene expression levels, with  <dig>  <r <dig> <  <dig>  .
we pooled the short reads and aligned them against the nipponbare reference genome ;  <dig> – <dig> % of reads were mapped to exonic regions,  <dig> – <dig> % to intronic regions, and  <dig> – <dig> % to intergenic regions .
we also found that  <dig>  of  <dig>   annotated transcripts in the nipponbare reference genome were detected by at least one sequence read.
r, x, and f denote r <dig>  xieqingzao b, and xieyou  <dig>  respectively.
numbers  <dig> and  <dig> denote samples from roots at tillering and heading stages, respectively.
transcriptome profiles of xieyou  <dig> and its parents
correlations between the hybrid and its parents at tillering and heading stages were investigated using cluster analysis with cluster  <dig>  software.
xieqingzao b had smaller differences in gene expression between the two stages than did r <dig> and xieyou  <dig> .
interestingly, with respect to root traits such as root dry weight, there were larger differences between the two stages in r <dig> and xieyou  <dig> than in xieqingzao b .
in addition, the transcriptome profile of xieyou  <dig> was similar to xieqingzao b  at the tillering stage, but it was closer to r <dig>  at the heading stage .
this is consistent with the results obtained from root dry weight at corresponding stages .
identification of differentially expressed genes  by rna-seq
gene expression levels were measured as reads per kilobase per million reads , with rpkm values ranging from  <dig> to over  <dig>  putative degs were identified using the following criteria:  false discovery rate  less than or equal to  <dig>  and  fold change  greater than or equal to  <dig>  using these criteria, we identified  <dig> transcripts as reliable degs at the tillering stage and  <dig> transcripts at the heading stage among the three genotypes.
for a detailed comparison, see additional file 3: table s <dig>  degs between the hybrid and its parents are designated as dghp, and those between the parental lines are designated as dgpp.
dghp may be relevant to heterosis because differences in expression between the hybrid and its parents should underlie their phenotypic differences, while dgpp only represents the differences between the two parental lines  <cit> .
in total,  <dig> and  <dig> dghp were identified at tillering and heading stages, respectively .
of the  <dig> dghp at the tillering stage,  <dig> transcripts showed differences between r <dig> and xieyou  <dig>  whereas only  <dig> exhibited differences between xieqingzao b and xieyou  <dig>  at the heading stage,  <dig> transcripts showed differences between xieqingzao b and xieyou  <dig>  whereas only  <dig> transcripts exhibited differences between r <dig> and xieyou  <dig> .
these results suggest that gene expression in xieyou  <dig> is more similar to that in xieqingzao b than to that in r <dig> at the tillering stage.
in contrast, at the heading stage, gene expression in xieyou  <dig> is more similar to that in r <dig> than to that in xieqingzao b. these observations are consistent with the results from the hierarchical cluster analysis described above.
r, x, and f refer to r <dig>  xieqingzao b, and xieyou  <dig>  respectively.
dgpp refers to degs between both parents, and dghp refers to degs between the hybrid and parents.
treating gene expression levels as quantitative traits, traditional quantitative genetic parameters, such as composite additive effect  and composite dominance effect , were estimated for our expression profile.
we classified dghp according to the dominance ratio hp , based on  <dig> % confidence intervals constructed for  -   and  +   .
depending on the sign of , hp was classified as either negative or positive .
dghp were then classified into four expression patterns: above high-parent  , high-parent level  , low-parent level  , and below low-parent  .
as shown in figure  <dig>  transcripts that were classified as lpl at the tillering stage and transcripts that were classified as hpl at the heading stage accounted for most of the dghp.
in addition, there were more down-regulated transcripts  than up-regulated transcripts  at the tillering stage.
at the heading stage, however, the results were completely reversed.
functional classification by gene ontology
go slim was used for the functional classification of dghp, and the annotations were plotted using web gene ontology annotation plot  software  <cit> .
in total,  <dig> of the  <dig> dghp at the tillering stage and  <dig> of the  <dig> dghp at the heading stage were assigned to at least one term in go molecular function, cellular component, and biological process categories.
transcripts from the two stages were further classified into  <dig> functional subcategories, providing an overview of ontology content .
in the biological process category, cellular process and metabolic process were the most highly represented groups, indicating that extensive metabolic activities were taking place in roots of hybrid plants during both stages.
in the molecular function category, binding and catalytic processes were prominently represented, while cell and cell parts dominated the cellular component category.
we further identified go terms in the biological process category that were over-represented  in dghp at tillering and heading stages .
these go terms served as indicators of significantly different biological processes underlying heterosis at tillering and heading stages.
go terms such as metabolic process, carbohydrate metabolic process, oxidation reduction, photosynthesis , photosynthesis, apoptosis, and response to oxidative stress were enriched in both sets of transcripts from the two stages, suggesting that the same biological processes were required to maintain root activities during both tillering and heading stages.
some striking differences were found, however, between the two sets of enriched go terms.
in particular, go terms related to protein phosphorylation, transport, cellulose biosynthetic process, and glycolysis were highly enriched in dghp at the heading stage .
kyoto encyclopedia of genes and genomes  pathway mapping
to identify metabolic pathways in which dghp were involved and enriched, pathway-based analysis was performed using the kegg pathway database.
as a result,  <dig> of  <dig> dghp at the tillering stage and  <dig> of  <dig> dghp at the heading stage were classified into  <dig> functional categories.
as shown in figure  <dig>  these transcripts belonged mainly to the following kegg pathways at both stages: carbohydrate metabolism, energy metabolism, amino acid metabolism, and lipid metabolism.
the above dghp were then classified into  <dig> subcategories that corresponded to their functions.
we further identified kegg orthology  terms that were over-represented in the dghp .
carbon fixation, photosynthesis, photosynthesis-antenna protein pathways, and fructose and mannose metabolism were over-represented at both stages.
in contrast, ko terms related to signal transduction, glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism were highly enriched in dghp and were exclusively expressed at the heading stage .
this suggests that there are considerable differences in root physiological processes between the tillering stage and the heading stage.
these annotations provide a valuable resource for investigating specific processes, functions, and pathways underlying heterosis.
validation by quantitative real-time pcr
a subset of  <dig> dghp was selected for qrt-pcr validation at tillering and heading stages.
qrt-pcr primers were designed based on rice annotation project database  annotations, and their sequences were listed in additional file 8: table s <dig>  we compared the results obtained from qrt-pcr with those generated from rna-seq analysis of these transcripts.
expression trends were consistent for all transcripts in both analyses, with a correlation coefficient of r <dig> =  <dig>  .
discussion
although heterosis has been widely exploited in plant breeding and plays an important role in agriculture, the molecular and genetic mechanisms underlying the phenomenon remain poorly understood.
differential gene expression between a hybrid and its parents may be associated with heterosis  <cit> .
in this study, we used rna-seq to investigate the relationship between transcriptional profiles and heterosis in a super-hybrid rice combination, xieyou  <dig>  in our rna-seq analysis,  <dig> million high-quality 100-bp paired-end reads were generated from the roots of xieyou  <dig> and its parental lines at tillering and heading stages, and  <dig>  annotated transcripts were identified.
on average,  <dig> reads were detected per identified annotated transcript, providing approximately 70-fold coverage of the transcriptome.
from the annotated transcripts,  <dig> dghp at the tillering stage and  <dig> dghp at the heading stage were identified.
these results suggest that the expression of dghp at the heading stage may play a more important role in root heterosis than that at the tillering stage.
additionally, only a small fraction of transcripts may be responsible for root heterosis at the transcriptional level in xieyou  <dig>
comparative analysis of annotated dghp
comparative transcriptome analysis revealed a subset of transcripts that were differently expressed between the hybrid and its parents at tillering and heading stages.
some potential regulators for heterosis in root development were uncovered.
at the tillering stage, a large number of dghp related to carbon fixation in photosynthetic organisms, photosynthesis, and photosynthesis-antenna proteins were found.
transcripts involved in plant hormone signal transduction, carbon fixation in photosynthetic organisms, photosynthesis, photosynthesis-antenna proteins, and carbohydrate metabolism  were highly expressed in roots at the heading stage.
we therefore conclude that carbohydrate metabolism and plant hormone signal transduction pathways may contribute significantly to root development.
another result of interest is the differential expression of photosynthetic transcripts at both stages.
the observed gene expression may be related to culture effects because the expression of these transcripts is not generally observed in roots of soil-grown plants  <cit> .
in our study, plants were cultured under hydroponic conditions; the roots may have thus been passively exposed to light, which could strongly activate photosynthesis in root tissues.
a similar result was observed in another recent study, along with the finding that a large number of degs were involved in photosynthesis in roots  <cit> .
in this study, we found that most of the dghp were down-regulated at the tillering stage and up-regulated at the heading stage.
because xieyou  <dig> is a late-stage high-vigor super-hybrid rice variety, root heterosis might be expected to be stronger at the heading stage than at the tillering stage.
for these reasons, subsequent analyses that focus on the expression of dghp at the heading stage are warranted.
the role of carbohydrate metabolism in heterosis
carbohydrate metabolism is an essential process in plants that produces both energy sources and structural components of cells and cell walls  <cit> .
it also generates compatible solutes for osmotic adjustment in roots  <cit> .
in our study, significant differences in carbohydrate metabolism, including glycolysis and metabolism of starch/sucrose, fructose/mannose, glucose/galactose, and pyruvate, were detected in the root tissues of the hybrid and its parents.
this result is consistent with the fact that a variety of carbohydrates are present in root tissues and exudates  <cit> .
many of the dghp-encoded enzymes involved in carbohydrate metabolism belong to a complex network that regulates carbohydrate synthesis/turnover in roots .
the starting point for the carbohydrate synthetic pathway in root tissues is sucrose, which is the main form of photosynthate that is transported from shoots to roots.
ogawa et al.
<cit>  determined that sucrose is transported to the root elongation zone and the surrounding tissue of the lateral root primordia, where it is converted to hexose by invertase or sucrose synthase .
hexose serves as an energy source, a compatible solute for root system formation, and a substance for cell wall synthesis.
in this regard, transcripts encoding sus  were up-regulated in xieyou  <dig> compared with the parental lines.
as reported previously, ossus <dig> is highly expressed in roots  <cit> , and sus <dig> is predominantly expressed in elongating tissues, including roots where rapid formation of secondary wall takes place just after cell elongation  <cit> .
the high level of sus expression in xieyou  <dig> may play a major role in sink organs by providing carbon sources for sink metabolism and sus expression may promote root elongation.
some enzymes involved in glycolysis pathways, such as phosphofructose kinase , fructose-bisphosphate aldolase , glyceraldehyde-3-phosphate dehydrogenase , phosphoglycerate kinase , pyruvate kinase , alcohol dehydrogenase , pyruvate decarboxylase , and lactate dehydrogenase , were up-regulated in xieyou  <dig>  the reaction catalyzed by pfk is the rate-limiting step of glycolysis; up-regulated pfk  may therefore reduce the limitation.
in addition, three dghp  that encode pk were up-regulated in xieyou  <dig>  interestingly, the expression of ospk <dig>  is stronger in adventitious roots than in the primary root, both of which serve as execution sites of absorption  <cit> .
this suggests that xieyou  <dig> may have a stronger absorption capacity than its parental lines.
transcriptional expression levels of fba , which plays an important physiological role in accelerating cell growth and promoting root elongation  <cit> , were up-regulated.
in addition, transcriptional expression levels of ldh , pdc , and adh , which are involved in glycolysis pathways, were up-regulated.
it has been suggested that the lactate initially produced by ldh lowers the ph, which in turn activates pdc and adh  <cit> .
ldh, pdc, and adh transcripts may be involved in inducing the hypoxia pathway; their up-regulation in this study may be due to the anaerobic stress that the roots might have experienced in the hydroponic solution  <cit> .
in contrast to animal cells, plant cells are enclosed by cell walls, which not only determine cell shape and provide structural support but also protect the plant against environmental stresses and regulate plant growth  <cit> .
cellulose is the most abundant biopolymer and the main structural component of plant cell walls.
our transcriptional profile analysis identified up-regulated transcripts that were related to cellulose synthesis, including cellulose synthase   and β- <dig>  4-endoglucanase  .
cesa up-regulation can promote root hair elongation, thus improving the absorption of water and nutrients by roots  <cit> .
interestingly, a previous study found that high expression of osglu <dig>  can affect root cell wall cellulose synthesis and thus modulate root elongation and protect roots from environmental stresses  <cit> .
complex regulation of plant hormone signal transduction
in multicellular organisms, cellular communication is important for coordinating the growth and differentiation of cells into new tissues and organs.
as is well known, hormones act as signaling molecules in plants by mediating physiological responses.
similar to the results in studies by kyndt et al.
<cit>  and wang et al.
<cit> , our transcriptome analysis uncovered many dghp that were involved in the phytohormone response in root tissues.
to illustrate, the abscisic acid  pathway is involved in the repression of lateral root development and adaption to environmental stresses  <cit> .
in our study, many aba-responsive transcripts exhibited different expression levels between the hybrid and its parents.
for example, mrna levels of four transcripts  encoding snf1-related protein kinase  <dig> , whose autophosphorylation is required for kinase activity towards downstream targets, were significantly more highly expressed in xieyou  <dig> than in its parents.
in addition, similar to previous studies by cohen et al.
<cit>  and santiago et al.
<cit> , pyr/pyl aba receptors  were down-regulated, and type-2c protein phosphatase   was up-regulated.
these results fit into the negative-feedback regulatory mechanism.
such re-setting of the aba signaling pathway provides xieyou  <dig> with a dynamic mechanism for monitoring aba levels and modulating aba response  <cit> .
transcripts involved in the cytokinin  signaling pathway were also differentially expressed between the hybrid and its two parents in our study.
the ctk pathway is involved in two aspects of root growth inhibition: the impedance of primary root elongation and the regulation of lateral root initiation  <cit> .
a subset of ctk-responsive transcripts such as type-a response regulators   and type-b response regulators  , showed significantly different expression patterns in root tissues.
rrs-b are positive transcriptional regulators in the ctk signaling pathway, whereas rrs-a act as negative regulators  <cit> .
the most likely model for this interaction is one in which rrs-a inhibit the activation of rrs-b by competing for phosphotransfer from upstream histidine phosphotransfer proteins; this model has been demonstrated in a few bacterial two-component systems  <cit> .
furthermore, previous studies have demonstrated that root elongation and lateral root formation in type-a mutants is more sensitive to ctk inhibition, and type-b mutants exhibit the opposite behavior  <cit> .
the up-regulated rrs-a and down-regulated rrs-b observed in our study may thus work together to lessen the sensitivity of xieyou  <dig> root tissues to ctk inhibition.
comparison of significant dghp with qtls for yield and root traits
in previous studies involving recombinant inbred lines and backcross populations derived from a cross between r <dig> and xieqingzao b, we identified a large number of qtls for root traits, including root dry weight , root length , root-shoot ratio , total root length , root volume , root surface area , root tip number , and root average diameter   <cit> .
the comparison of dghp with qtls that were mapped consistently across years or environments revealed that  <dig> dghp in the carbohydrate metabolism pathway, encoding pfk, sus, pdc, adh, egase, and cesa, and  <dig> dghp in the plant hormone signal transduction pathway, encoding rr-b and snrk <dig>  were located in root-related qtl regions .
as reported by liang et al.
<cit> , root traits, including rl, trl, rsa, rv, rtn, and rdw, are positively correlated with yield-related traits such as grain yield per plant , number of panicles , number of spikelets per panicle , and heading date .
we therefore investigated the potential association among the dghp, qtls for yield-related traits, and heterosis within many qtl regions, including associations between cesa  and rm180–rm <dig> for gyd, np, nsp, and hd; cesa  and rm148–rm <dig> for hd; and pfk  and rm6972–rm <dig> for gyd.
interestingly, pfk, which is involved in glycolysis, was located in the interval rm6972–rm <dig> not only for rdw but also for gyd.
in addition, three dghp encoding cesa were detected in the interval rm180–rm <dig> on chromosome  <dig> for three different yield-related traits and six root-related traits .
in hybrids, diversity in gene expression can be the result of variation in cis-regulatory elements  or trans-regulatory elements   <cit> .
if differential expression of a gene is cis-induced, the gene may be located in a qtl region, whereas if it is trans-induced, a qtl may correspond with the trans-regulatory elements and be located distantly from the gene locus.
in our dghp collections, we indeed found that some dghp encoded transcription factors, which may be involved in trans-regulation, in the qtl regions .
the expression of candidate transcripts in these qtl regions may serve as a starting point to understand the molecular mechanisms underlying heterosis.
the application of these results is expected to provide an opportunity to breed elite rice varieties that process both yield and root heterosis.
CONCLUSIONS
in this study, we used rna-seq to systematically investigate the global transcriptomes of roots from the super-hybrid rice xieyou  <dig> and its parents at tillering and heading stages, generating a useful resource for the rice community.
we analyzed dghp and compared them with qtls for yield and root traits, providing clues for candidate transcripts that may significantly contribute to root development and yield production.
the changes in the expression of candidate transcripts may provide valuable information for future studies on molecular mechanisms underlying root heterosis.

 heterosis is a phenomenon in which hybrids exhibit superior phenotypes, such as enhanced biomass production, development rate, grain yield, and stress tolerance, relative to their parents. heterosis has been effectively utilized to increase crop production in the world. it is estimated, for example, that hybrid rice, which occupies more than 50% of the total rice area in china, has a 10–20% yield advantage over inbred varieties. our knowledge of the genetic mechanisms of heterosis, however, has lagged behind its wide application. two hypotheses-dominance and over-dominance -were proposed in the early 20th century to interpret heterosis. both describe nonadditive behavior as a consequence of genetic differences between distinct homozygous parental lines and their heterozygous hybrids. with the advent of molecular makers, quantitative trait locus mapping has become a routine tool for studying the genetic basis of heterosis in crop plants; so far, however, the qtl analysis has not contributed much to the understanding of heterosis. with the development of functional genomics, the technique of large-scale transcriptome analysis-based on cdna or expressed sequence tag library sequencing, microarray hybridization, and serial analysis of gene expression -has been used to investigate heterosis in arabidopsis, maize, and rice. such technologies offer the potential to unveil the molecular basis of heterosis at the transcriptional level. these technologies have drawbacks, however, such as low throughput, high cost, low sensitivity, cloning bias, high background signal, and pre-determined probes requirements. next-generation high-throughput rna sequencing technology is a recently-developed method for discovering, profiling, and quantifying rna transcripts with several advantages over other expression profiling technologies including higher sensitivity and the ability to detect splicing isoforms and somatic mutations. using rna-seq, significant progress has been made in understanding the transcript expression of rice over the last two years. for example, transcriptome analysis of rice mature root tissue and root tips at two time points identified root-enriched transcripts and tip-enriched transcripts involved in different physiological processes. in addition, rna-seq has been applied to the identification of stress-inducible transcripts in rice. other than a transcriptome analysis of seedling shoots at the four-leaf stage, however, little effort is being expended in attempts to investigate heterosis using rna-seq. plant root systems serve a number of important functions, including anchoring the plant, absorbing water and nutrients, producing amino acids and hormones, and secreting organic acids, enzymes, and alkaloids. in recent years, considerable research and interest has focused on root systems. some of these studies have demonstrated that heterosis levels might be higher in root traits than in aboveground agronomic traits, which suggests that roots might be an ideal organ for investigating the genetic basis of rice heterosis. several attempts have been made to discover the molecular mechanism of root heterosis at the vegetative stage, but little attention has been paid to heterosis in the root system during the late growth stage, when accumulation, transportation, and distribution of dry matter, and ultimately, yield potential, may be influenced. there has currently been no systematic investigation into root heterosis at the two different developmental stages. in this study, we focused our heterosis research on the late-stage high-vigor super-hybrid rice variety, xieyou which has a grain yield of up to × kg ⋅ hm- and was designated as “super rice” by the chinese ministry of agriculture in xieyou was bred by crossing the restorer line r to the maternal line xieqingzao b. we used rna-seq to investigate the global transcriptomes of roots from xieyou and its parents at tillering and heading stages. differentially expressed transcripts and their expression patterns were analyzed, and several potential candidate transcripts were found to be involved in carbohydrate metabolism and plant hormone signal transduction pathways. we expect this genome-wide transcriptome comparison to provide a starting point to understand the causative mechanism of the altered gene expression in the hybrid and the molecular mechanism underlying rice root heterosis.  characterization of xieyou and its two parental lines we used rna-seq to investigate gene expression and function in a heterotic cross involving xieyou a super-hybrid rice variety with superior yield performance, its maternal line xieqingzao b, and its paternal line r as suggested by pickett, other yield-related traits might also show heterosis. in this study, we found that the roots and aerial parts of xieyou were more vigorous than those of either parent. mid-parent heterosis and best-parent heterosis were calculated to measure the heterosis of aerial parts and roots. the quantification of mph and hph and their statistical significance were shown in table we observed significant mph for all traits at both stages except for shoot dry weight at the tillering stage. furthermore, we also observed significant hph for the root-shoot ratio at the tillering stage and root length, root dry weight, and shoot dry weight at the heading stage. the mph and hph of root dry weight were greater than those of shoot dry weight at both stages, indicating that the level of heterosis was much higher in roots than in aerial parts. at the tillering stage, the mph of root length, root dry weight, and root-shoot ratio varied from % to %. by comparison, the degree of heterosis for these traits was larger at the heading stage, with the mph ranging from % to %. **significant difference with p <. *significant difference with p <. mapping reads to the rice genome to analyze the transcriptomes of the above three genotypes at tillering and heading stages, cdna libraries were prepared from rice roots and subjected to rna-seq analysis on the illumina hiseq platform. in total, million short reads were generated from the two developmental stages, with million high-quality 100-bp reads selected for further analysis. the two biological replicates were in good agreement with respect to gene expression levels, with <r <. we pooled the short reads and aligned them against the nipponbare reference genome; – % of reads were mapped to exonic regions, – % to intronic regions, and – % to intergenic regions. we also found that of annotated transcripts in the nipponbare reference genome were detected by at least one sequence read. r, x, and f denote r xieqingzao b, and xieyou respectively. numbers and denote samples from roots at tillering and heading stages, respectively. transcriptome profiles of xieyou and its parents correlations between the hybrid and its parents at tillering and heading stages were investigated using cluster analysis with cluster software. xieqingzao b had smaller differences in gene expression between the two stages than did r and xieyou. interestingly, with respect to root traits such as root dry weight, there were larger differences between the two stages in r and xieyou than in xieqingzao b. in addition, the transcriptome profile of xieyou was similar to xieqingzao b at the tillering stage, but it was closer to r at the heading stage. this is consistent with the results obtained from root dry weight at corresponding stages. identification of differentially expressed genes by rna-seq gene expression levels were measured as reads per kilobase per million reads, with rpkm values ranging from to over putative degs were identified using the following criteria: false discovery rate less than or equal to and fold change greater than or equal to using these criteria, we identified transcripts as reliable degs at the tillering stage and transcripts at the heading stage among the three genotypes. for a detailed comparison, see additional file 3: table s degs between the hybrid and its parents are designated as dghp, and those between the parental lines are designated as dgpp. dghp may be relevant to heterosis because differences in expression between the hybrid and its parents should underlie their phenotypic differences, while dgpp only represents the differences between the two parental lines. in total, and dghp were identified at tillering and heading stages, respectively. of the dghp at the tillering stage, transcripts showed differences between r and xieyou whereas only exhibited differences between xieqingzao b and xieyou at the heading stage, transcripts showed differences between xieqingzao b and xieyou whereas only transcripts exhibited differences between r and xieyou. these results suggest that gene expression in xieyou is more similar to that in xieqingzao b than to that in r at the tillering stage. in contrast, at the heading stage, gene expression in xieyou is more similar to that in r than to that in xieqingzao b. these observations are consistent with the results from the hierarchical cluster analysis described above. r, x, and f refer to r xieqingzao b, and xieyou respectively. dgpp refers to degs between both parents, and dghp refers to degs between the hybrid and parents. treating gene expression levels as quantitative traits, traditional quantitative genetic parameters, such as composite additive effect and composite dominance effect, were estimated for our expression profile. we classified dghp according to the dominance ratio hp, based on % confidence intervals constructed for - and +. depending on the sign of, hp was classified as either negative or positive. dghp were then classified into four expression patterns: above high-parent, high-parent level, low-parent level, and below low-parent. as shown in figure transcripts that were classified as lpl at the tillering stage and transcripts that were classified as hpl at the heading stage accounted for most of the dghp. in addition, there were more down-regulated transcripts than up-regulated transcripts at the tillering stage. at the heading stage, however, the results were completely reversed. functional classification by gene ontology go slim was used for the functional classification of dghp, and the annotations were plotted using web gene ontology annotation plot software. in total, of the dghp at the tillering stage and of the dghp at the heading stage were assigned to at least one term in go molecular function, cellular component, and biological process categories. transcripts from the two stages were further classified into functional subcategories, providing an overview of ontology content. in the biological process category, cellular process and metabolic process were the most highly represented groups, indicating that extensive metabolic activities were taking place in roots of hybrid plants during both stages. in the molecular function category, binding and catalytic processes were prominently represented, while cell and cell parts dominated the cellular component category. we further identified go terms in the biological process category that were over-represented in dghp at tillering and heading stages. these go terms served as indicators of significantly different biological processes underlying heterosis at tillering and heading stages. go terms such as metabolic process, carbohydrate metabolic process, oxidation reduction, photosynthesis, photosynthesis, apoptosis, and response to oxidative stress were enriched in both sets of transcripts from the two stages, suggesting that the same biological processes were required to maintain root activities during both tillering and heading stages. some striking differences were found, however, between the two sets of enriched go terms. in particular, go terms related to protein phosphorylation, transport, cellulose biosynthetic process, and glycolysis were highly enriched in dghp at the heading stage. kyoto encyclopedia of genes and genomes pathway mapping to identify metabolic pathways in which dghp were involved and enriched, pathway-based analysis was performed using the kegg pathway database. as a result, of dghp at the tillering stage and of dghp at the heading stage were classified into functional categories. as shown in figure these transcripts belonged mainly to the following kegg pathways at both stages: carbohydrate metabolism, energy metabolism, amino acid metabolism, and lipid metabolism. the above dghp were then classified into subcategories that corresponded to their functions. we further identified kegg orthology terms that were over-represented in the dghp. carbon fixation, photosynthesis, photosynthesis-antenna protein pathways, and fructose and mannose metabolism were over-represented at both stages. in contrast, ko terms related to signal transduction, glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism were highly enriched in dghp and were exclusively expressed at the heading stage. this suggests that there are considerable differences in root physiological processes between the tillering stage and the heading stage. these annotations provide a valuable resource for investigating specific processes, functions, and pathways underlying heterosis. validation by quantitative real-time pcr a subset of dghp was selected for qrt-pcr validation at tillering and heading stages. qrt-pcr primers were designed based on rice annotation project database annotations, and their sequences were listed in additional file 8: table s we compared the results obtained from qrt-pcr with those generated from rna-seq analysis of these transcripts. expression trends were consistent for all transcripts in both analyses, with a correlation coefficient of r =. discussion although heterosis has been widely exploited in plant breeding and plays an important role in agriculture, the molecular and genetic mechanisms underlying the phenomenon remain poorly understood. differential gene expression between a hybrid and its parents may be associated with heterosis. in this study, we used rna-seq to investigate the relationship between transcriptional profiles and heterosis in a super-hybrid rice combination, xieyou in our rna-seq analysis, million high-quality 100-bp paired-end reads were generated from the roots of xieyou and its parental lines at tillering and heading stages, and annotated transcripts were identified. on average, reads were detected per identified annotated transcript, providing approximately 70-fold coverage of the transcriptome. from the annotated transcripts, dghp at the tillering stage and dghp at the heading stage were identified. these results suggest that the expression of dghp at the heading stage may play a more important role in root heterosis than that at the tillering stage. additionally, only a small fraction of transcripts may be responsible for root heterosis at the transcriptional level in xieyou comparative analysis of annotated dghp comparative transcriptome analysis revealed a subset of transcripts that were differently expressed between the hybrid and its parents at tillering and heading stages. some potential regulators for heterosis in root development were uncovered. at the tillering stage, a large number of dghp related to carbon fixation in photosynthetic organisms, photosynthesis, and photosynthesis-antenna proteins were found. transcripts involved in plant hormone signal transduction, carbon fixation in photosynthetic organisms, photosynthesis, photosynthesis-antenna proteins, and carbohydrate metabolism were highly expressed in roots at the heading stage. we therefore conclude that carbohydrate metabolism and plant hormone signal transduction pathways may contribute significantly to root development. another result of interest is the differential expression of photosynthetic transcripts at both stages. the observed gene expression may be related to culture effects because the expression of these transcripts is not generally observed in roots of soil-grown plants. in our study, plants were cultured under hydroponic conditions; the roots may have thus been passively exposed to light, which could strongly activate photosynthesis in root tissues. a similar result was observed in another recent study, along with the finding that a large number of degs were involved in photosynthesis in roots. in this study, we found that most of the dghp were down-regulated at the tillering stage and up-regulated at the heading stage. because xieyou is a late-stage high-vigor super-hybrid rice variety, root heterosis might be expected to be stronger at the heading stage than at the tillering stage. for these reasons, subsequent analyses that focus on the expression of dghp at the heading stage are warranted. the role of carbohydrate metabolism in heterosis carbohydrate metabolism is an essential process in plants that produces both energy sources and structural components of cells and cell walls. it also generates compatible solutes for osmotic adjustment in roots. in our study, significant differences in carbohydrate metabolism, including glycolysis and metabolism of starch/sucrose, fructose/mannose, glucose/galactose, and pyruvate, were detected in the root tissues of the hybrid and its parents. this result is consistent with the fact that a variety of carbohydrates are present in root tissues and exudates. many of the dghp-encoded enzymes involved in carbohydrate metabolism belong to a complex network that regulates carbohydrate synthesis/turnover in roots. the starting point for the carbohydrate synthetic pathway in root tissues is sucrose, which is the main form of photosynthate that is transported from shoots to roots. ogawa et al. determined that sucrose is transported to the root elongation zone and the surrounding tissue of the lateral root primordia, where it is converted to hexose by invertase or sucrose synthase. hexose serves as an energy source, a compatible solute for root system formation, and a substance for cell wall synthesis. in this regard, transcripts encoding sus were up-regulated in xieyou compared with the parental lines. as reported previously, ossus is highly expressed in roots, and sus is predominantly expressed in elongating tissues, including roots where rapid formation of secondary wall takes place just after cell elongation. the high level of sus expression in xieyou may play a major role in sink organs by providing carbon sources for sink metabolism and sus expression may promote root elongation. some enzymes involved in glycolysis pathways, such as phosphofructose kinase, fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, alcohol dehydrogenase, pyruvate decarboxylase, and lactate dehydrogenase, were up-regulated in xieyou the reaction catalyzed by pfk is the rate-limiting step of glycolysis; up-regulated pfk may therefore reduce the limitation. in addition, three dghp that encode pk were up-regulated in xieyou interestingly, the expression of ospk is stronger in adventitious roots than in the primary root, both of which serve as execution sites of absorption. this suggests that xieyou may have a stronger absorption capacity than its parental lines. transcriptional expression levels of fba, which plays an important physiological role in accelerating cell growth and promoting root elongation, were up-regulated. in addition, transcriptional expression levels of ldh, pdc, and adh, which are involved in glycolysis pathways, were up-regulated. it has been suggested that the lactate initially produced by ldh lowers the ph, which in turn activates pdc and adh. ldh, pdc, and adh transcripts may be involved in inducing the hypoxia pathway; their up-regulation in this study may be due to the anaerobic stress that the roots might have experienced in the hydroponic solution. in contrast to animal cells, plant cells are enclosed by cell walls, which not only determine cell shape and provide structural support but also protect the plant against environmental stresses and regulate plant growth. cellulose is the most abundant biopolymer and the main structural component of plant cell walls. our transcriptional profile analysis identified up-regulated transcripts that were related to cellulose synthesis, including cellulose synthase and β- 4-endoglucanase. cesa up-regulation can promote root hair elongation, thus improving the absorption of water and nutrients by roots. interestingly, a previous study found that high expression of osglu can affect root cell wall cellulose synthesis and thus modulate root elongation and protect roots from environmental stresses. complex regulation of plant hormone signal transduction in multicellular organisms, cellular communication is important for coordinating the growth and differentiation of cells into new tissues and organs. as is well known, hormones act as signaling molecules in plants by mediating physiological responses. similar to the results in studies by kyndt et al. and wang et al., our transcriptome analysis uncovered many dghp that were involved in the phytohormone response in root tissues. to illustrate, the abscisic acid pathway is involved in the repression of lateral root development and adaption to environmental stresses. in our study, many aba-responsive transcripts exhibited different expression levels between the hybrid and its parents. for example, mrna levels of four transcripts encoding snf1-related protein kinase, whose autophosphorylation is required for kinase activity towards downstream targets, were significantly more highly expressed in xieyou than in its parents. in addition, similar to previous studies by cohen et al. and santiago et al., pyr/pyl aba receptors were down-regulated, and type-2c protein phosphatase was up-regulated. these results fit into the negative-feedback regulatory mechanism. such re-setting of the aba signaling pathway provides xieyou with a dynamic mechanism for monitoring aba levels and modulating aba response. transcripts involved in the cytokinin signaling pathway were also differentially expressed between the hybrid and its two parents in our study. the ctk pathway is involved in two aspects of root growth inhibition: the impedance of primary root elongation and the regulation of lateral root initiation. a subset of ctk-responsive transcripts such as type-a response regulators and type-b response regulators, showed significantly different expression patterns in root tissues. rrs-b are positive transcriptional regulators in the ctk signaling pathway, whereas rrs-a act as negative regulators. the most likely model for this interaction is one in which rrs-a inhibit the activation of rrs-b by competing for phosphotransfer from upstream histidine phosphotransfer proteins; this model has been demonstrated in a few bacterial two-component systems. furthermore, previous studies have demonstrated that root elongation and lateral root formation in type-a mutants is more sensitive to ctk inhibition, and type-b mutants exhibit the opposite behavior. the up-regulated rrs-a and down-regulated rrs-b observed in our study may thus work together to lessen the sensitivity of xieyou root tissues to ctk inhibition. comparison of significant dghp with qtls for yield and root traits in previous studies involving recombinant inbred lines and backcross populations derived from a cross between r and xieqingzao b, we identified a large number of qtls for root traits, including root dry weight, root length, root-shoot ratio, total root length, root volume, root surface area, root tip number, and root average diameter. the comparison of dghp with qtls that were mapped consistently across years or environments revealed that dghp in the carbohydrate metabolism pathway, encoding pfk, sus, pdc, adh, egase, and cesa, and dghp in the plant hormone signal transduction pathway, encoding rr-b and snrk were located in root-related qtl regions. as reported by liang et al., root traits, including rl, trl, rsa, rv, rtn, and rdw, are positively correlated with yield-related traits such as grain yield per plant, number of panicles, number of spikelets per panicle, and heading date. we therefore investigated the potential association among the dghp, qtls for yield-related traits, and heterosis within many qtl regions, including associations between cesa and rm180–rm for gyd, np, nsp, and hd; cesa and rm148–rm for hd; and pfk and rm6972–rm for gyd. interestingly, pfk, which is involved in glycolysis, was located in the interval rm6972–rm not only for rdw but also for gyd. in addition, three dghp encoding cesa were detected in the interval rm180–rm on chromosome for three different yield-related traits and six root-related traits. in hybrids, diversity in gene expression can be the result of variation in cis-regulatory elements or trans-regulatory elements. if differential expression of a gene is cis-induced, the gene may be located in a qtl region, whereas if it is trans-induced, a qtl may correspond with the trans-regulatory elements and be located distantly from the gene locus. in our dghp collections, we indeed found that some dghp encoded transcription factors, which may be involved in trans-regulation, in the qtl regions. the expression of candidate transcripts in these qtl regions may serve as a starting point to understand the molecular mechanisms underlying heterosis. the application of these results is expected to provide an opportunity to breed elite rice varieties that process both yield and root heterosis.  in this study, we used rna-seq to systematically investigate the global transcriptomes of roots from the super-hybrid rice xieyou and its parents at tillering and heading stages, generating a useful resource for the rice community. we analyzed dghp and compared them with qtls for yield and root traits, providing clues for candidate transcripts that may significantly contribute to root development and yield production. the changes in the expression of candidate transcripts may provide valuable information for future studies on molecular mechanisms underlying root heterosis. 
an extensive transcriptome dataset was obtained by rna-seq, giving a comprehensive overview of the root transcriptomes at tillering and heading stages in a heterotic rice cross and providing a useful resource for the rice research community. using comparative transcriptome analysis, we detected dghp and identified a group of potential candidate transcripts. several significant dghp that could be mapped to quantitative trait loci for yield and root traits are also involved in carbohydrate metabolism and plant hormone signal transduction pathways. the changes in the expression of the candidate transcripts may lay a foundation for future studies on molecular mechanisms underlying root heterosis. heterosis is a phenomenon in which hybrids exhibit superior performance relative to parental phenotypes. approximately million high-quality paired-end reads were generated and aligned against the nipponbare reference genome. in addition to the heterosis of above-ground agronomic traits on which most existing studies have focused, root heterosis is also an indispensable component of heterosis in the entire plant and of major importance to plant breeding. consequently, systematic investigations of root heterosis, particularly in reproductive-stage rice, are needed. at the heading stage, the dghp were significantly enriched in pathways related to processes such as carbohydrate metabolism and plant hormone signal transduction, with most of the key genes that are involved in the two pathways being up-regulated in the hybrid.
 heterosis is a phenomenon in which hybrids exhibit superior performance relative to parental phenotypes. in addition to the heterosis of above-ground agronomic traits on which most existing studies have focused, root heterosis is also an indispensable component of heterosis in the entire plant and of major importance to plant breeding. consequently, systematic investigations of root heterosis, particularly in reproductive-stage rice, are needed. the recent advent of rna sequencing technology provides an opportunity to conduct in-depth transcript profiling for heterosis studies.  using the illumina hiseq platform, the root transcriptomes of the super-hybrid rice variety xieyou and its parents were analyzed at tillering and heading stages. approximately million high-quality paired-end reads were generated and aligned against the nipponbare reference genome. we found that of annotated transcripts were represented by at least one sequence read. a total of and transcripts that were differentially expressed between the hybrid and its parents were identified at tillering and heading stages, respectively. out of the dghp, % were down-regulated at the tillering stage and % were up-regulated at the heading stage. at the heading stage, the dghp were significantly enriched in pathways related to processes such as carbohydrate metabolism and plant hormone signal transduction, with most of the key genes that are involved in the two pathways being up-regulated in the hybrid. several significant dghp that could be mapped to quantitative trait loci for yield and root traits are also involved in carbohydrate metabolism and plant hormone signal transduction pathways.  an extensive transcriptome dataset was obtained by rna-seq, giving a comprehensive overview of the root transcriptomes at tillering and heading stages in a heterotic rice cross and providing a useful resource for the rice research community. using comparative transcriptome analysis, we detected dghp and identified a group of potential candidate transcripts. the changes in the expression of the candidate transcripts may lay a foundation for future studies on molecular mechanisms underlying root heterosis. heterosisroottranscriptomerna-seqhybrid rice 