BACKGROUND
rapeseed  is one of the primary sources of vegetable oil for human nutrition; it occupies a pivotal position on oil supply in china.
rapeseed production has remarkably increased as a result of the popularization of elite cultivars with high yield and good quality, and molecular design breeding has become one of the most available breeding methods.
dissection of the genetic architecture underlying the important agronomic traits will greatly facilitate the genetic improvement of rapeseed.
brassica napus is an allopolyploid species derived from the natural interspecies hybridization between brassica rapa and brassica oleracea, the former contributing the a genome and the latter contributing the c genome.
the seed oil content is a very important economic character in rapeseed production.
and its genetic architecture has been investigated by quantitative trait locus  mapping based on appropriate mapping populations.
several qtls controlling seed oil content have been identified  <cit> .
qiu et al.
<cit>  analyzed the qtls of seed oil and erucic acid content using a comparative linkage map of oilseed rape.
recently, zhao et al.
<cit>  detected eight qtls with additive effects and nine pairs of loci with additive-additive epistasis.
however, the resolution is still not enough to pinpoint the candidate genes for the surveyed trait because of too many genes in the identified qtl regions.
systematic integration of genetic mapping with transcription and genome annotations is considered as a promising avenue to identify causal genes  <cit> .
the differentially expressed genes are potential contributors to the difference between individuals.
with rapid advance in biotechnology, the subtractive suppression hybridization  has been used to investigate differentially expressed genes underlying oil content of rapeseed  <cit> .
some important genes, such as yeast glycerol-3-phosphate dehydrogenase, arabidopsis fae <dig> and yeast slc1- <dig> genes, have been suggested for use in the genetic improvement of the oil content of rapeseed  <cit> .
however, this technology has a few drawbacks, such as low resolution and low dynamic range of expression  <cit>  and the relevant studies cannot explore integrated molecular mechanism underlying seed oil content.
rna sequencing  that uses deep-sequencing technologies, as an approach to transcriptome profiling, possess several advantages over the other expression profiling technologies, such as higher sensitivity and the ability to detect splicing isoforms and somatic mutations  <cit> .
transcriptome sequencing can provide massive sequence data for analysis of gene expression.
transcriptome sequencing has been applied in studies on brassica napus, such as investigating single nucleotide polymorphism   <cit> , and measuring the contributions of homologues to gene expression  <cit> .
however, little effort is being expended in attempts to investigate oil-related biological processes using rna-seq.
given that we have been able to dissect genetic architecture of oil content into several chromosomal loci by qtl mapping, the combined use of the qtl mapping with transcriptome profiling represents a practical solution to further refine the mapping resolution and identify potential candidate genes  <cit> .
here, we integrated qtl mapping and brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed.
as rapeseed oil is accumulated during the development of pod and the differential expression in the parents offers a snapshot on that of the mapping population, in this study, we focused our research on the german variety sollux and chinese variety gaoyou, which have been used as the parents to develop the qtl mapping population for oil content  <cit> .
we applied the rna-seq technique to characterize gene expression of brassica napu pod at the growth stages of 5– <dig>  15– <dig> and 25– <dig> days after flowering .
bioinformatics analysis was performed to investigate candidate differentially expressed genes  and their expression patterns.
intervarietal degs of lipid pathways were found to help us understand oil content difference between the two varieties.
more importantly, degs involved in qtl regions are important candidate genes for breeding program especially those degs associated with lipid metabolism.
our study provides information on the growth of pod at the molecular level, identifies candidate genes responsible for oil content, and is also an application case of quantitative genetics integrated with next-generation sequencing  analysis and comparative genomics that have broad application prospects.
RESULTS
gene differential expression analysis
after the quality control, approximately  <dig>  million pair-end reads  and  <dig>  million single-end reads  were generated.
on average of  <dig>  % of reads were mapped to the reference genome by the tophat .
after the annotation merging by cuffmerge,  <dig>  genes were annotated, within which,  <dig>  genes have been annotated with the publishing of the reference genome.
meanwhile,  <dig>  different transcripts were identified in the merged annotation.
based on the gene expression levels calculated by fpkm , we found a close overlapping between stages and varieties.
on one hand,  <dig> ,  <dig>  and  <dig>  co-expressed genes of the varieties were observed in three different stages, s1-g <dig>  s2-g <dig> and s3-g <dig> respectively .
on the other hand, in all three stages,  <dig>  genes expressed in the gaoyou and  <dig>  genes expressed in the sollux.fig.
<dig> venn diagram for the genes expressed in each of the three stages of brassica napus pod in sollux and gaoyou.
a  <dig>  genes are co-expressed at s <dig> and g <dig> while  <dig>   and  <dig>   are variety-specifically expressed genes.
b  <dig>  genes are co-expressed at s <dig> and g <dig> while  <dig>   and  <dig>   are variety-specifically expressed genes.
c  <dig>  genes are co-expressioned at s <dig> and g <dig> while  <dig>   and  <dig>   are variety-specifically expressed genes.
d among all genes,  <dig>  are co-expressed at all the stages in the gaoyou,  <dig>  are co-expressed in g <dig> and g <dig>   <dig>  are co-expressed in g <dig> and g <dig>  and  <dig>  are co-expressed in g <dig> and g <dig>  the numbers of stage-specifically expressed genes are  <dig>  ,  <dig>  , and  <dig>  , respectively.
e among all the genes,  <dig>  are co-expressed at all the stages in the sollux,  <dig>  are co-expressed in s <dig> and s <dig>   <dig>  are co-expressed in s <dig> and s <dig>  and  <dig>  are co-expressed in s <dig> and s <dig>  the numbers of stage-specifically expressed genes are  <dig>  ,  <dig>  , and  <dig>  , respectively
according to the criteria to determine differential expression of gene   <cit> ,  <dig> candidate degs between gaoyou and sollux at the three stages were identified .
there was some difference in percentage of up-regulated and down-regulated degs between the two varieties.
at 5– <dig> daf,  <dig> degs were identified.
the number of lowly expressed genes in gaoyou  was larger than that in sollux.
at 15– <dig> daf, the number of degs was  <dig>  and  <dig>  % of them exhibited lower expression in gaoyou.
the percentage of higher expression genes was  <dig>  % in gaoyou at 25– <dig> daf.
the numbers of up-regulated and down-regulated genes across different stages in sollux and gaoyou were presented in table  <dig>  more genes differentially expressed were up-regulated in the early growth stage than in the late stage for the gaoyou, but it was diametrically opposite for the sollux, indicating that the gaoyou had more active gene expression in the early stage, while the sollux had more in the late stage.
heat map from the hierarchical clustering of degs is shown in fig.
<dig>  the aforementioned genes up-regulated and down-regulated between varieties and/or stages were indicated by hierarchical clustering analysis.table  <dig> number and classification of degs between the two varieties at the three stages
s and g denote the sollux and the gaoyou, respectively.
the numbers 1– <dig> denote the three stages, respectively.
up-regulated and down-regulated mean that the expression level in the gaoyou is higher and lower than that in sollux, respectively.
the degs were defined by the criteria of fdr <  <dig> , and the p value <  <dig>
up-regulated and down-regulated, respectively, mean the expression level of the later stage is higher and lower than that of the former stage
fig.
<dig> hierarchical clustering analysis of gene expression based on rpkm data.
s <dig>  s <dig> and s <dig> are stages  <dig>   <dig> and  <dig>  respectively for sollux, and g <dig>  g <dig> and g <dig> are stages  <dig>   <dig> and  <dig>  respectively, for gaoyou.
the color key represents rpkm  normalized log <dig> transformed counts.
red represents high expression and green represents low expression.
each row represents a gene
functional classification by gene ontology
the gene ontology  project provides an ontology of defined terms concerning gene product properties, which can promote our understanding on the gene function at the molecular level.
the expressed genes could be classified into  <dig> go subcategories.
as shown in fig.
3a, the go category had three major terms: cellular component, molecular function and biological process.
the proportions of genes assigned in the three terms were  <dig>  %,  <dig>  % and  <dig>  %, respectively.fig.
<dig> go classifications of genes.
genes are divided into three main categories: biological process, molecular function and cellular component by go analysis.
a go classification of all genes.
b go classification of degs among the two varieties at three stages.
c go classification among three contrasts  of sollux.
d go classification among three contrasts  of gaoyou
go analysis of the degs between the three stages of the sollux and of the gaoyou showed  <dig> and  <dig> go sub-categories for the two varieties , respectively.
different go patterns were observed between the two varieties, for instance, the go: <dig>  only occurred in the comparison of s1-s <dig> for the sollux, but in the comparison of g1-g <dig>  g2-g <dig> and g1-g <dig> for the gaoyou.
the candidate degs of s1-g <dig>  s2-g <dig> and s3-g <dig> were grouped into  <dig> go sub-categories  and  <dig> go terms.
go analysis provided much insightful information regarding the go terms.
there was one go sub-categories showed significant difference in the three comparisons, it’s go: <dig> .
the p value based on gene percentage was below  <dig> .
to know the significant terms of the deggss , enrichment analysis was conducted against the background of go term distribution of all expression genes.
the significantly enriched go terms of these deggss were listed in additional file 1: table s <dig>  additional file 2: table s <dig> and additional file 3: table s <dig>  terms with a variety of different functions enriched, in particular, go terms such as lipid x metabolic process  , lipid storage   and sphingolipid metabolic process   showed lipid-related function.
these results represent an overall condition of the deggss function; in particular, these lipid-related terms provide information to understand intervarietal oil content differences at gene function level.
metabolic pathway analysis
the information about metabolic pathways of the transcriptome is very valuable for understanding the physiological process of the pod after flowering.
the predicted gene sequences were mapped to  <dig>  kegg orthology  terms through the kaas.
to learn more about the differences between the two varieties, the metabolic pathways of degs were analyzed by classification.
as a result, the degs at the three stages were grouped into  <dig> ko terms.
these ko terms were classified into  <dig> functional categories  and  <dig> branch pathways in total.
the degs between the two varieties at 5– <dig> daf, 15– <dig> daf and 25– <dig> daf were classified into  <dig>   <dig> and  <dig> branch pathways, respectively.
as shown in fig.
<dig>  xenobiotics biodegradation and metabolism, and carbohydrate metabolism pathways account for large proportion of the deggss.
they were remarkable by enrichment test with p values of  <dig>  × 10− <dig> and  <dig> , respectively.
carbohydrate metabolism is important for the pod development, and then may affect the accumulation of other organics.
maybe lipid accumulation was also affected by it.fig.
<dig> kegg pathway categories of differentially expressed genes at 5– <dig> daf, 15– <dig> daf and 25– <dig> daf
differentially expressed and lipid-related genes in the qtl regions
nine significant qtls of oil content were reported in the previous studies  <cit> , named as oila <dig>  oila <dig>  oila <dig>  oila <dig>  oilc <dig>  oilc <dig>  oilc <dig>  oilc08- <dig> and oilc08- <dig>  which were located in linkage groups a <dig>  a <dig>  a <dig>  a <dig>  c <dig>  c <dig>  c <dig> and c <dig>  respectively.
the physical information of these qtls is presented in additional file 4: table s <dig>  deggss and homologous genes to those involved in lipid metabolism were identified in the qtl regions by sequence alignment.
our analysis revealed a total of  <dig> deggss in the qtl regions; the numbers of the deggss located in the six qtls  were  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  respectively .
<dig> lipid-related homologous genes were discovered in the qtl regions.
there were  <dig> and  <dig> lipid-related genes in oilc <dig> and oilc <dig> regions.
<dig> lipid-related genes resided in the oilc08- <dig>  in which, there was one deggs.
this lipid-related deg is sac-pip , which participates in phospholipid signaling pathway.
in the oilc <dig> region, no deggs was overlapped with the  <dig> lipid-related genes.
<dig> lipid-related homologous genes were located in the oil qtls of a genome but did not overlap with deggss.
further investigation of these genes is warranted.table  <dig> number of genes, deggs, and lipid-related homologous genes in the qtl region
deggs means differentially expressed genes between gaoyou and sollux.
homologous denotes genes homologous to lipid-related genes in arabidopsis thaliana
identification of the degs or the lipid-related homologous genes in the qtls can substantially narrow down the number of candidate genes.
the annotated functions of all these genes will provide better information.
so, we listed all of the lipid-related genes  with their homologous gene names and the degs  with the gi numbers of their homologous genes in nr.
discussion
comparative analysis of gene expression between different stages and varieties
pods of the brassica napus play a key role in rapeseed growth.
in addition to the protective function of encapsulating from pest and pathogens, the photosynthetically active pod wall contributes nutrients to fuel seed growth  <cit> .
thus, we used the pod to investigate the molecular mechanism of oil-related biological processes in three specific growth stages.
to reduce the sequencing cost, we used the pooled samples to quantify the expression levels of the two varieties at the three growth stages.
this cost-effective strategy would offer an affordable way to identify potential differentially expressed genes between the two varieties, although it could not provide a valid estimation of sampling and measurement errors as in individual sequencing.
however, as in many other rna-seq studies that have no or very few replicates due to the still high cost of sequencing  <cit> , our study did not have biological replicates.
therefore, it is still necessary to note that all the identified degs or deggss are essentially candidates because of lack of biological replicates, although the fdr  could be alleviated or reduced by our pooled samples sequencing strategy to a certain extent.
the differential expression of a subset of genes can be identified through the comparative analysis of their transcriptomes.
at the first stage , the least number of differentially expressed genes were detected and less than half of these degs had a higher expression in gaoyou.
many of them are involved in the energy metabolism and carbohydrate metabolism.
at the stage of 15– <dig> daf, some of deggss were found to be involved in the pathways of carbohydrate metabolism, amino acid metabolism, transportation and catabolism, and signal transduction, especially in the carbohydrate metabolism where  <dig> deggss were involved.
among the deggss, there is one related with lipid metabolism which was up-regulated in gaoyou.
at 25– <dig> daf, abundant deggss were involved in some pathways, such as xenobiotics biodegradation and metabolism, carbohydrate metabolism, biosynthesis of other secondary metabolites and metabolism of terpenoids and polyketides.
most of the xenobiotics biodegradation and metabolism-related deggss exhibited higher expression in the sollux than in the gaoyou.
in different growth periods of rapeseed, compared with the number of degs involved in different pathways, more degs participated in the pathway of ribosome .
out of these degs involved in ribosome, 83 % in the gaoyou and 58 % in the sollux were up-regulated during three growth stages .
in cells, ribosome serves as the primary site of biological protein synthesis .
the expression pattern of the degs involved in ribosome indicates that protein synthesis increases with pod development.
some degs involved in carbon fixation in photosynthetic organisms exhibited different expression patterns in the sollux and in the gaoyou.
these genes were down-regulated from  <dig> to  <dig> daf to 15– <dig> daf and up-regulated from  <dig> to  <dig> daf to 25– <dig> daf in sollux.
in gaoyou, these genes were down-regulated from  <dig> to  <dig> daf to 15– <dig> daf, and also down-regulated in expression from  <dig> to  <dig> daf to 25– <dig> daf.
it indicates that the gaoyou and the sollux have different active periods of carbon fixation in pod.
lipid-related genes in pod development after flowering
rapeseed contains various components and many genes are involved in different metabolic pathways to participate in the formation of oil content.
genes homologous to acyl lipids-related genes from arabidopsis acyl-lipid metabolism database were analyzed.
in the sollux, there was only one lipid-related deg between s <dig> and s <dig>  while  <dig> and  <dig> were detected between s <dig> and s <dig>  and between s <dig> and s <dig>  respectively, most of which were up-regulated at the later stage.
six lipid-related degs with high expression values were pi5p-ii, cdp-dags, lpla, kdta, pi3p and pipk-ia.
apart from pi5p-ii, the others were up-regulated.
the pi5p-ii, lpla, pi3p and pipk-ia are involved in the phospholipid signaling pathway, and the cdp-dags and kdta are involved in the mitochondrial phospholipid synthesis and mitochondrial lipopolysaccharide synthesis.
during the growth stage of the gaoyou,  <dig> out of  <dig> differentially expressed lipid-related genes were up-regulated during the g1-g <dig>  the numbers of lipid-related degs were  <dig> and  <dig> during the g2-g <dig> and g1-g <dig>  respectively, and  <dig> and  <dig> degs were down-regulated, respectively .table  <dig> the differentially expressed oil-related homologous genes in the two varieties
s and g denote the sollux and the gaoyou, respectively, and the numbers 1– <dig> denote the three stages, respectively; the information of degs and corresponding oil-related homologous genes in arabidopsis are listed in additional file 7: table s7
the gaoyou performs better than the sollux for the oil content in hangzhou, china, which can be partly explained by the transcriptional level in our study.
the result indicated that vast majority of the lipid-related genes exhibited no difference in expression level between the two varieties.
at the stages of 5– <dig> daf, there was no significant lipid-related deg between the gaoyou and the sollux.
eight lipid-related degs were detected at 15– <dig> daf and 25– <dig> af.
out of the eight degs, three genes  exhibited a higher expression level in the gaoyou.
the cellular function of obo is related to oil bodies, which plays an important role in the tag synthesis  <cit> .
therefore, the expression level of obo could affect the final size of oil body.
mcmt  plays a key role in chain elongation in fatty acid biosynthesis and polyketide biosynthesis  <cit> , as it provides 2-carbon units to fatty acids and commits them to fatty acid chain synthesis.
sac-pip  participates in phospholipid signaling pathway.
other five degs  had higher expression level in sollux.
both pi5p-ii and gpi-plc are related to phospholipid signaling which is crucial for plant growth and development  <cit> .
at3g <dig> is a putative phospholipid-transporting atpase that cannot be classified into a certain pathway.
the aforementioned three genes that had a higher expression in sollux had no a direct correlation between gene function and the lipid synthesis/degradation.
mfp, a multifunctional protein, can catalyze four separate reactions, two of which play an important role in the catabolism of all fatty acids  <cit> .
on the basis of the above results, we conclude that the expression pattern of lipid-related genes may result in better performance in character of oil content for the gaoyou under the cultivation condition in hangzhou, china.
combining significant degs and qtls for oil content
the arabidopsis thaliana lipid-related homologous genes were mapped to the brassica napus, of which some lipid-related candidate loci reside in the regions of these qtls.
benefiting from identification of more genes involved in acyl-lipid metabolism in arabidopsis, more lipid-related candidate genes could be identified in the qtl regions by sequence alignment.
according to the genome annotation of the chinese cabbage , a total of  <dig>  genes were found in the regions of the nine qtls, whereas it is still a challenge to screen lipid-related candidate genes.
through the comparative analysis with arabidopsis lipid-related genes, candidate lipid-related genes in the region of qtls of the brassica napus can be efficiently distinguished.
integration of the homologous, qtl and expression information is the directly and efficiently way to identify the candidate genes which are quite significant for our study.
most of the arabidopsis lipid-related homologous genes are not associated with the qtls; it is probably due to the complexity in the genetic architecture of seed oil content and/or the limited knowledge on gene annotation.
another potential reason is that only limited qtls harboring major-effect genes are detected in qtl mapping because of insufficient power.
most deggss in the qtl regions are not homologous to the lipid-related genes of arabidopsis.
the annotation of these genes was achieved by sequence alignment between the predicted protein sequence and the nr database.
a subset of the genes are functionally related to the oil content, whereas the others have no clear relationship with the lipid-related pathways.
in oila <dig>  there is one deggs homologous to the gdsl esterase/lipase with function in hydrolase activity.
it is located in the endomembrane system and involved in lipid metabolic process  <cit> .
further study on the other deggss in these qtl regions that have no straightforward relationship with the seed oil content are needed for understanding their potential roles and importance in the oil content related metabolic pathways.
these continuing works will provide valuable resources for breeding program of the brassica napus.
methods
plant materials and rna extraction
the two parents of the sollux/gaoyou doubled haploid  population , sollux and gaoyou, were used for transcriptome profiling in this study.
the sollux is a typical german winter rapeseed cultivar and the gaoyou is an inbred line from chinese variety gaoyou  <dig> with no vernalization requirement.
while both parents have high oil content in seeds and do not show large difference in oil content, there are large inherited differences in many morphological traits.
in the six years mapping experiments in hangzhou, china  <cit> , the gaoyou had higher oil content and better agronomic performance than the sollux.
there was a transgressive segregation phenomenon where lower or higher oil content beyond the parents could be observed in the dh population, potentially ascribable to recombination of functional genes underlying oil content , suggesting a genetic difference in oil content between the parents.
the differences between the lines with the highest and lowest oil contents were averagely up to  <dig>  % over the six years mapping experiments.table  <dig> phenotypic variation of seed oil content  in the sollux/gaoyou dh population and the parents
<dig>   <dig>   <dig>   <dig>   <dig> and  <dig> indicate the years to conduct experiments in hangzhou, china; min and max indicate the minimum and the maximum of observation values in the dh population derived from the crossing between the sollux and the gaoyou
sollux and gaoyou were planted in the experimental farm of zhejiang academy of agricultural sciences for this study in  <dig>  gaoyou plants were first grown in growth chamber  during seedling stage and moved into greenhouse when getting the same bolting stage as sollux.
total rna of two cultivars were extracted from pods using trizol reagent  at the three different stages: 5– <dig> daf, 15– <dig> daf and 25– <dig> daf, respectively, and were stored in liquid nitrogen at the temperature of −80 °c.
the rna was purified using rneasy plant mini kit .
the quality was verified using a  <dig> bioanalyzer rna nanochip , and the rna integrity number  of all the samples were more than  <dig> .
the rna samples from six plants of each variety at each stage were pooled for rna-seq.
cdna library construction and sequencing
sequence libraries were prepared according to the manufacturer’s instructions .
poly-a-containing mrna was isolated from the total rna, subjected to two purification rounds using poly-t oligo-attached magnetic beads, and fragmented using divalent cations at 94 °c.
taking these fragments as templates, the first-strand cdna was synthesized using random hexamer primers and super-script tm iii .
following the second strand cdna synthesis and adaptor ligation, 200-bp and 50-bp cdna fragments were isolated using gel electrophoresis and amplified by 18 cycles of pcr.
after the amplification, the libraries were sequenced using illumina hiseq  <dig>  the adaptor sequences, empty reads and low quality sequences were filtered from the raw reads.
gene annotation and bioinformatics analysis
bowtie indexes the reference sequences with a burrows-wheeler index which can keep its memory footprint small  <cit> .
tophat is a fast mapping tool for rna-seq reads that can identify splice junction between exons  <cit> .
cufflinks can assembly the reads into transcripts based on the mapping results  <cit> .
tophat v <dig> .
<dig> and bowtie v <dig> .
<dig> were used to align the high quality reads of six samples  to the brassica napus genome sequences  <cit> .
then cufflinks was used to assembly the transcripts of each sample.
in addition to cufflinks, there are several other softwares such as isoem that can be used to infer isoform and gene expression levels from high-throughput transcriptome sequencing  <cit> , and malta  <cit>  that can be used to transcriptome assembly and quantification from ion torrent rna-seq data.
the brassica napus genome annotation downloaded from the ncbi database was merged with the annotation obtained from cufflinks by cuffmerge.
the predicted genes from the merged genome annotation were alignment with the plant part of nr database by blastx  <dig> .26+ with evalue less than 1e- <dig>  so the gene functions can be obtained from homologous genes in the nr database.
go annotation were performed by alignment between genes and go database.
website tool wego   <cit>  was used to produce go functional classification of all the expressed genes and to interpret the distribution of gene functions at the macro level.
the go enrichment analysis of the deggss  was performed by hypergeometric test in r. r function dhyper was used to calculate p-values, the go terms with p-value less than  <dig>  were considered to be enrichment terms.
at last, the predicted genes were submitted to the kaas   <cit>  website to obtain kegg annotations.
as same as the go enrichment analysis, the kegg enrichment analysis was also performed.
cuffdiff was used to identify degs .
the degs were filtered using a p value cut off of  <dig>  and a false discovery rate below  <dig>   <cit> .
it is also helpful for us to measure gene expression level by calculating fpkm, similar to rpkm  which is used earlier  <cit> .
as genes with similar expression patterns are often functionally correlated, to identify such clusters, the hierarchical clustering of degs between the two varieties at the three stages was performed using r package gplots .
analysis of genes involved in qtl regions
nine significant qtls for oil content in brassica napus have been detected in the previous studies, four of which are located in a genome, and the other five are located in c genome.
the parents of the mapping population in that study are the same as used in the present study.
the genome sequence of brassica napus were downloaded from the brassica napus database .
in order to distinguish genes homologous to the genes involved in lipid metabolism collected in arabidopsis acyl-lipid metabolism , sequence alignment was performed between genes in the qtl regions and the lipid-related genes were identified by blastn with evalue less than 10− <dig>
CONCLUSIONS
this study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages.
integrated analysis of transcriptome profiling, qtl mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of brassica napus.
additional files
additional file 1: table s1: the top  <dig> most represented go terms of deg in the biological process category at the 5– <dig> daf .
additional file 2: table s2: the top  <dig> most represented go terms of deg in the biological process category at the 15– <dig> daf .
additional file 3: table s3: the top  <dig> most represented go terms of deg in the biological process category at the 25– <dig> daf .
additional file 4: table s4: the physical location of the oil related qtls.
additional file 5: table s5: the lipid-related homologous genes in arabidopsis for the genes in qtl regions.
additional file 6: table s6: the gi number of genes in nr database homologous with degs between varieties in qtl regions.
additional file 7: table s7: lipid-related degs in brassica napus and their lipid-related homologous genes in arabidopsis.
competing interests
the authors declare that they have no competing financial interests.
authors’ contributions
xyl, jyz and hmx conceived and designed the experiments.
xdk contributed to the analysis of the data and drafted the manuscript.
xyl and hmx helped to revise the manuscript.
fc and jxh collected samples and prepared rna.
all the authors read and approved the final manuscript.
this research was financially supported by national basic research programs , national natural science foundations of china , zhejiang provincial natural science foundation of china , science and technology department of zhejiang province, china , and the national institutes of health grant da <dig>

 rapeseed is one of the primary sources of vegetable oil for human nutrition; it occupies a pivotal position on oil supply in china. rapeseed production has remarkably increased as a result of the popularization of elite cultivars with high yield and good quality, and molecular design breeding has become one of the most available breeding methods. dissection of the genetic architecture underlying the important agronomic traits will greatly facilitate the genetic improvement of rapeseed. brassica napus is an allopolyploid species derived from the natural interspecies hybridization between brassica rapa and brassica oleracea, the former contributing the a genome and the latter contributing the c genome. the seed oil content is a very important economic character in rapeseed production. and its genetic architecture has been investigated by quantitative trait locus mapping based on appropriate mapping populations. several qtls controlling seed oil content have been identified. qiu et al. analyzed the qtls of seed oil and erucic acid content using a comparative linkage map of oilseed rape. recently, zhao et al. detected eight qtls with additive effects and nine pairs of loci with additive-additive epistasis. however, the resolution is still not enough to pinpoint the candidate genes for the surveyed trait because of too many genes in the identified qtl regions. systematic integration of genetic mapping with transcription and genome annotations is considered as a promising avenue to identify causal genes. the differentially expressed genes are potential contributors to the difference between individuals. with rapid advance in biotechnology, the subtractive suppression hybridization has been used to investigate differentially expressed genes underlying oil content of rapeseed. some important genes, such as yeast glycerol-3-phosphate dehydrogenase, arabidopsis fae and yeast slc1- genes, have been suggested for use in the genetic improvement of the oil content of rapeseed. however, this technology has a few drawbacks, such as low resolution and low dynamic range of expression and the relevant studies cannot explore integrated molecular mechanism underlying seed oil content. rna sequencing that uses deep-sequencing technologies, as an approach to transcriptome profiling, possess several advantages over the other expression profiling technologies, such as higher sensitivity and the ability to detect splicing isoforms and somatic mutations. transcriptome sequencing can provide massive sequence data for analysis of gene expression. transcriptome sequencing has been applied in studies on brassica napus, such as investigating single nucleotide polymorphism, and measuring the contributions of homologues to gene expression. however, little effort is being expended in attempts to investigate oil-related biological processes using rna-seq. given that we have been able to dissect genetic architecture of oil content into several chromosomal loci by qtl mapping, the combined use of the qtl mapping with transcriptome profiling represents a practical solution to further refine the mapping resolution and identify potential candidate genes. here, we integrated qtl mapping and brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed. as rapeseed oil is accumulated during the development of pod and the differential expression in the parents offers a snapshot on that of the mapping population, in this study, we focused our research on the german variety sollux and chinese variety gaoyou, which have been used as the parents to develop the qtl mapping population for oil content. we applied the rna-seq technique to characterize gene expression of brassica napu pod at the growth stages of 5– 15– and 25– days after flowering. bioinformatics analysis was performed to investigate candidate differentially expressed genes and their expression patterns. intervarietal degs of lipid pathways were found to help us understand oil content difference between the two varieties. more importantly, degs involved in qtl regions are important candidate genes for breeding program especially those degs associated with lipid metabolism. our study provides information on the growth of pod at the molecular level, identifies candidate genes responsible for oil content, and is also an application case of quantitative genetics integrated with next-generation sequencing analysis and comparative genomics that have broad application prospects.  gene differential expression analysis after the quality control, approximately million pair-end reads and million single-end reads were generated. on average of  % of reads were mapped to the reference genome by the tophat. after the annotation merging by cuffmerge, genes were annotated, within which, genes have been annotated with the publishing of the reference genome. meanwhile, different transcripts were identified in the merged annotation. based on the gene expression levels calculated by fpkm, we found a close overlapping between stages and varieties. on one hand,, and co-expressed genes of the varieties were observed in three different stages, s1-g s2-g and s3-g respectively. on the other hand, in all three stages, genes expressed in the gaoyou and genes expressed in the sollux.fig. venn diagram for the genes expressed in each of the three stages of brassica napus pod in sollux and gaoyou. a genes are co-expressed at s and g while and are variety-specifically expressed genes. b genes are co-expressed at s and g while and are variety-specifically expressed genes. c genes are co-expressioned at s and g while and are variety-specifically expressed genes. d among all genes, are co-expressed at all the stages in the gaoyou, are co-expressed in g and g are co-expressed in g and g and are co-expressed in g and g the numbers of stage-specifically expressed genes are,, and, respectively. e among all the genes, are co-expressed at all the stages in the sollux, are co-expressed in s and s are co-expressed in s and s and are co-expressed in s and s the numbers of stage-specifically expressed genes are,, and, respectively according to the criteria to determine differential expression of gene, candidate degs between gaoyou and sollux at the three stages were identified. there was some difference in percentage of up-regulated and down-regulated degs between the two varieties. at 5– daf, degs were identified. the number of lowly expressed genes in gaoyou was larger than that in sollux. at 15– daf, the number of degs was and  % of them exhibited lower expression in gaoyou. the percentage of higher expression genes was  % in gaoyou at 25– daf. the numbers of up-regulated and down-regulated genes across different stages in sollux and gaoyou were presented in table  more genes differentially expressed were up-regulated in the early growth stage than in the late stage for the gaoyou, but it was diametrically opposite for the sollux, indicating that the gaoyou had more active gene expression in the early stage, while the sollux had more in the late stage. heat map from the hierarchical clustering of degs is shown in fig.  the aforementioned genes up-regulated and down-regulated between varieties and/or stages were indicated by hierarchical clustering analysis.table number and classification of degs between the two varieties at the three stages s and g denote the sollux and the gaoyou, respectively. the numbers 1– denote the three stages, respectively. up-regulated and down-regulated mean that the expression level in the gaoyou is higher and lower than that in sollux, respectively. the degs were defined by the criteria of fdr < , and the p value <  up-regulated and down-regulated, respectively, mean the expression level of the later stage is higher and lower than that of the former stage fig. hierarchical clustering analysis of gene expression based on rpkm data. s s and s are stages and respectively for sollux, and g g and g are stages and respectively, for gaoyou. the color key represents rpkm normalized log transformed counts. red represents high expression and green represents low expression. each row represents a gene functional classification by gene ontology the gene ontology project provides an ontology of defined terms concerning gene product properties, which can promote our understanding on the gene function at the molecular level. the expressed genes could be classified into go subcategories. as shown in fig. 3a, the go category had three major terms: cellular component, molecular function and biological process. the proportions of genes assigned in the three terms were  %,  % and  %, respectively.fig. go classifications of genes. genes are divided into three main categories: biological process, molecular function and cellular component by go analysis. a go classification of all genes. b go classification of degs among the two varieties at three stages. c go classification among three contrasts of sollux. d go classification among three contrasts of gaoyou go analysis of the degs between the three stages of the sollux and of the gaoyou showed and go sub-categories for the two varieties, respectively. different go patterns were observed between the two varieties, for instance, the go: only occurred in the comparison of s1-s for the sollux, but in the comparison of g1-g g2-g and g1-g for the gaoyou. the candidate degs of s1-g s2-g and s3-g were grouped into go sub-categories and go terms. go analysis provided much insightful information regarding the go terms. there was one go sub-categories showed significant difference in the three comparisons, it’s go:. the p value based on gene percentage was below. to know the significant terms of the deggss, enrichment analysis was conducted against the background of go term distribution of all expression genes. the significantly enriched go terms of these deggss were listed in additional file 1: table s additional file 2: table s and additional file 3: table s terms with a variety of different functions enriched, in particular, go terms such as lipid x metabolic process, lipid storage and sphingolipid metabolic process showed lipid-related function. these results represent an overall condition of the deggss function; in particular, these lipid-related terms provide information to understand intervarietal oil content differences at gene function level. metabolic pathway analysis the information about metabolic pathways of the transcriptome is very valuable for understanding the physiological process of the pod after flowering. the predicted gene sequences were mapped to kegg orthology terms through the kaas. to learn more about the differences between the two varieties, the metabolic pathways of degs were analyzed by classification. as a result, the degs at the three stages were grouped into ko terms. these ko terms were classified into functional categories and branch pathways in total. the degs between the two varieties at 5– daf, 15– daf and 25– daf were classified into and branch pathways, respectively. as shown in fig.  xenobiotics biodegradation and metabolism, and carbohydrate metabolism pathways account for large proportion of the deggss. they were remarkable by enrichment test with p values of  × 10− and, respectively. carbohydrate metabolism is important for the pod development, and then may affect the accumulation of other organics. maybe lipid accumulation was also affected by it.fig. kegg pathway categories of differentially expressed genes at 5– daf, 15– daf and 25– daf differentially expressed and lipid-related genes in the qtl regions nine significant qtls of oil content were reported in the previous studies, named as oila oila oila oila oilc oilc oilc oilc08- and oilc08- which were located in linkage groups a a a a c c c and c respectively. the physical information of these qtls is presented in additional file 4: table s deggss and homologous genes to those involved in lipid metabolism were identified in the qtl regions by sequence alignment. our analysis revealed a total of deggss in the qtl regions; the numbers of the deggss located in the six qtls were and respectively. lipid-related homologous genes were discovered in the qtl regions. there were and lipid-related genes in oilc and oilc regions. lipid-related genes resided in the oilc08- in which, there was one deggs. this lipid-related deg is sac-pip, which participates in phospholipid signaling pathway. in the oilc region, no deggs was overlapped with the lipid-related genes. lipid-related homologous genes were located in the oil qtls of a genome but did not overlap with deggss. further investigation of these genes is warranted.table number of genes, deggs, and lipid-related homologous genes in the qtl region deggs means differentially expressed genes between gaoyou and sollux. homologous denotes genes homologous to lipid-related genes in arabidopsis thaliana identification of the degs or the lipid-related homologous genes in the qtls can substantially narrow down the number of candidate genes. the annotated functions of all these genes will provide better information. so, we listed all of the lipid-related genes with their homologous gene names and the degs with the gi numbers of their homologous genes in nr. discussion comparative analysis of gene expression between different stages and varieties pods of the brassica napus play a key role in rapeseed growth. in addition to the protective function of encapsulating from pest and pathogens, the photosynthetically active pod wall contributes nutrients to fuel seed growth. thus, we used the pod to investigate the molecular mechanism of oil-related biological processes in three specific growth stages. to reduce the sequencing cost, we used the pooled samples to quantify the expression levels of the two varieties at the three growth stages. this cost-effective strategy would offer an affordable way to identify potential differentially expressed genes between the two varieties, although it could not provide a valid estimation of sampling and measurement errors as in individual sequencing. however, as in many other rna-seq studies that have no or very few replicates due to the still high cost of sequencing, our study did not have biological replicates. therefore, it is still necessary to note that all the identified degs or deggss are essentially candidates because of lack of biological replicates, although the fdr could be alleviated or reduced by our pooled samples sequencing strategy to a certain extent. the differential expression of a subset of genes can be identified through the comparative analysis of their transcriptomes. at the first stage, the least number of differentially expressed genes were detected and less than half of these degs had a higher expression in gaoyou. many of them are involved in the energy metabolism and carbohydrate metabolism. at the stage of 15– daf, some of deggss were found to be involved in the pathways of carbohydrate metabolism, amino acid metabolism, transportation and catabolism, and signal transduction, especially in the carbohydrate metabolism where deggss were involved. among the deggss, there is one related with lipid metabolism which was up-regulated in gaoyou. at 25– daf, abundant deggss were involved in some pathways, such as xenobiotics biodegradation and metabolism, carbohydrate metabolism, biosynthesis of other secondary metabolites and metabolism of terpenoids and polyketides. most of the xenobiotics biodegradation and metabolism-related deggss exhibited higher expression in the sollux than in the gaoyou. in different growth periods of rapeseed, compared with the number of degs involved in different pathways, more degs participated in the pathway of ribosome. out of these degs involved in ribosome, 83 % in the gaoyou and 58 % in the sollux were up-regulated during three growth stages. in cells, ribosome serves as the primary site of biological protein synthesis. the expression pattern of the degs involved in ribosome indicates that protein synthesis increases with pod development. some degs involved in carbon fixation in photosynthetic organisms exhibited different expression patterns in the sollux and in the gaoyou. these genes were down-regulated from to daf to 15– daf and up-regulated from to daf to 25– daf in sollux. in gaoyou, these genes were down-regulated from to daf to 15– daf, and also down-regulated in expression from to daf to 25– daf. it indicates that the gaoyou and the sollux have different active periods of carbon fixation in pod. lipid-related genes in pod development after flowering rapeseed contains various components and many genes are involved in different metabolic pathways to participate in the formation of oil content. genes homologous to acyl lipids-related genes from arabidopsis acyl-lipid metabolism database were analyzed. in the sollux, there was only one lipid-related deg between s and s while and were detected between s and s and between s and s respectively, most of which were up-regulated at the later stage. six lipid-related degs with high expression values were pi5p-ii, cdp-dags, lpla, kdta, pi3p and pipk-ia. apart from pi5p-ii, the others were up-regulated. the pi5p-ii, lpla, pi3p and pipk-ia are involved in the phospholipid signaling pathway, and the cdp-dags and kdta are involved in the mitochondrial phospholipid synthesis and mitochondrial lipopolysaccharide synthesis. during the growth stage of the gaoyou, out of differentially expressed lipid-related genes were up-regulated during the g1-g the numbers of lipid-related degs were and during the g2-g and g1-g respectively, and and degs were down-regulated, respectively.table the differentially expressed oil-related homologous genes in the two varieties s and g denote the sollux and the gaoyou, respectively, and the numbers 1– denote the three stages, respectively; the information of degs and corresponding oil-related homologous genes in arabidopsis are listed in additional file 7: table s7 the gaoyou performs better than the sollux for the oil content in hangzhou, china, which can be partly explained by the transcriptional level in our study. the result indicated that vast majority of the lipid-related genes exhibited no difference in expression level between the two varieties. at the stages of 5– daf, there was no significant lipid-related deg between the gaoyou and the sollux. eight lipid-related degs were detected at 15– daf and 25– af. out of the eight degs, three genes exhibited a higher expression level in the gaoyou. the cellular function of obo is related to oil bodies, which plays an important role in the tag synthesis. therefore, the expression level of obo could affect the final size of oil body. mcmt plays a key role in chain elongation in fatty acid biosynthesis and polyketide biosynthesis, as it provides 2-carbon units to fatty acids and commits them to fatty acid chain synthesis. sac-pip participates in phospholipid signaling pathway. other five degs had higher expression level in sollux. both pi5p-ii and gpi-plc are related to phospholipid signaling which is crucial for plant growth and development. at3g is a putative phospholipid-transporting atpase that cannot be classified into a certain pathway. the aforementioned three genes that had a higher expression in sollux had no a direct correlation between gene function and the lipid synthesis/degradation. mfp, a multifunctional protein, can catalyze four separate reactions, two of which play an important role in the catabolism of all fatty acids. on the basis of the above results, we conclude that the expression pattern of lipid-related genes may result in better performance in character of oil content for the gaoyou under the cultivation condition in hangzhou, china. combining significant degs and qtls for oil content the arabidopsis thaliana lipid-related homologous genes were mapped to the brassica napus, of which some lipid-related candidate loci reside in the regions of these qtls. benefiting from identification of more genes involved in acyl-lipid metabolism in arabidopsis, more lipid-related candidate genes could be identified in the qtl regions by sequence alignment. according to the genome annotation of the chinese cabbage, a total of genes were found in the regions of the nine qtls, whereas it is still a challenge to screen lipid-related candidate genes. through the comparative analysis with arabidopsis lipid-related genes, candidate lipid-related genes in the region of qtls of the brassica napus can be efficiently distinguished. integration of the homologous, qtl and expression information is the directly and efficiently way to identify the candidate genes which are quite significant for our study. most of the arabidopsis lipid-related homologous genes are not associated with the qtls; it is probably due to the complexity in the genetic architecture of seed oil content and/or the limited knowledge on gene annotation. another potential reason is that only limited qtls harboring major-effect genes are detected in qtl mapping because of insufficient power. most deggss in the qtl regions are not homologous to the lipid-related genes of arabidopsis. the annotation of these genes was achieved by sequence alignment between the predicted protein sequence and the nr database. a subset of the genes are functionally related to the oil content, whereas the others have no clear relationship with the lipid-related pathways. in oila there is one deggs homologous to the gdsl esterase/lipase with function in hydrolase activity. it is located in the endomembrane system and involved in lipid metabolic process. further study on the other deggss in these qtl regions that have no straightforward relationship with the seed oil content are needed for understanding their potential roles and importance in the oil content related metabolic pathways. these continuing works will provide valuable resources for breeding program of the brassica napus. methods plant materials and rna extraction the two parents of the sollux/gaoyou doubled haploid population, sollux and gaoyou, were used for transcriptome profiling in this study. the sollux is a typical german winter rapeseed cultivar and the gaoyou is an inbred line from chinese variety gaoyou with no vernalization requirement. while both parents have high oil content in seeds and do not show large difference in oil content, there are large inherited differences in many morphological traits. in the six years mapping experiments in hangzhou, china, the gaoyou had higher oil content and better agronomic performance than the sollux. there was a transgressive segregation phenomenon where lower or higher oil content beyond the parents could be observed in the dh population, potentially ascribable to recombination of functional genes underlying oil content, suggesting a genetic difference in oil content between the parents. the differences between the lines with the highest and lowest oil contents were averagely up to  % over the six years mapping experiments.table phenotypic variation of seed oil content in the sollux/gaoyou dh population and the parents and indicate the years to conduct experiments in hangzhou, china; min and max indicate the minimum and the maximum of observation values in the dh population derived from the crossing between the sollux and the gaoyou sollux and gaoyou were planted in the experimental farm of zhejiang academy of agricultural sciences for this study in gaoyou plants were first grown in growth chamber during seedling stage and moved into greenhouse when getting the same bolting stage as sollux. total rna of two cultivars were extracted from pods using trizol reagent at the three different stages: 5– daf, 15– daf and 25– daf, respectively, and were stored in liquid nitrogen at the temperature of −80 °c. the rna was purified using rneasy plant mini kit. the quality was verified using a bioanalyzer rna nanochip, and the rna integrity number of all the samples were more than. the rna samples from six plants of each variety at each stage were pooled for rna-seq. cdna library construction and sequencing sequence libraries were prepared according to the manufacturer’s instructions. poly-a-containing mrna was isolated from the total rna, subjected to two purification rounds using poly-t oligo-attached magnetic beads, and fragmented using divalent cations at 94 °c. taking these fragments as templates, the first-strand cdna was synthesized using random hexamer primers and super-script tm iii. following the second strand cdna synthesis and adaptor ligation, 200-bp and 50-bp cdna fragments were isolated using gel electrophoresis and amplified by 18 cycles of pcr. after the amplification, the libraries were sequenced using illumina hiseq the adaptor sequences, empty reads and low quality sequences were filtered from the raw reads. gene annotation and bioinformatics analysis bowtie indexes the reference sequences with a burrows-wheeler index which can keep its memory footprint small. tophat is a fast mapping tool for rna-seq reads that can identify splice junction between exons. cufflinks can assembly the reads into transcripts based on the mapping results. tophat v. and bowtie v. were used to align the high quality reads of six samples to the brassica napus genome sequences. then cufflinks was used to assembly the transcripts of each sample. in addition to cufflinks, there are several other softwares such as isoem that can be used to infer isoform and gene expression levels from high-throughput transcriptome sequencing, and malta that can be used to transcriptome assembly and quantification from ion torrent rna-seq data. the brassica napus genome annotation downloaded from the ncbi database was merged with the annotation obtained from cufflinks by cuffmerge. the predicted genes from the merged genome annotation were alignment with the plant part of nr database by blastx.26+ with evalue less than 1e- so the gene functions can be obtained from homologous genes in the nr database. go annotation were performed by alignment between genes and go database. website tool wego was used to produce go functional classification of all the expressed genes and to interpret the distribution of gene functions at the macro level. the go enrichment analysis of the deggss was performed by hypergeometric test in r. r function dhyper was used to calculate p-values, the go terms with p-value less than were considered to be enrichment terms. at last, the predicted genes were submitted to the kaas website to obtain kegg annotations. as same as the go enrichment analysis, the kegg enrichment analysis was also performed. cuffdiff was used to identify degs. the degs were filtered using a p value cut off of and a false discovery rate below. it is also helpful for us to measure gene expression level by calculating fpkm, similar to rpkm which is used earlier. as genes with similar expression patterns are often functionally correlated, to identify such clusters, the hierarchical clustering of degs between the two varieties at the three stages was performed using r package gplots. analysis of genes involved in qtl regions nine significant qtls for oil content in brassica napus have been detected in the previous studies, four of which are located in a genome, and the other five are located in c genome. the parents of the mapping population in that study are the same as used in the present study. the genome sequence of brassica napus were downloaded from the brassica napus database. in order to distinguish genes homologous to the genes involved in lipid metabolism collected in arabidopsis acyl-lipid metabolism, sequence alignment was performed between genes in the qtl regions and the lipid-related genes were identified by blastn with evalue less than 10−  this study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages. integrated analysis of transcriptome profiling, qtl mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of brassica napus. additional files additional file 1: table s1: the top most represented go terms of deg in the biological process category at the 5– daf. additional file 2: table s2: the top most represented go terms of deg in the biological process category at the 15– daf. additional file 3: table s3: the top most represented go terms of deg in the biological process category at the 25– daf. additional file 4: table s4: the physical location of the oil related qtls. additional file 5: table s5: the lipid-related homologous genes in arabidopsis for the genes in qtl regions. additional file 6: table s6: the gi number of genes in nr database homologous with degs between varieties in qtl regions. additional file 7: table s7: lipid-related degs in brassica napus and their lipid-related homologous genes in arabidopsis. competing interests the authors declare that they have no competing financial interests. authors’ contributions xyl, jyz and hmx conceived and designed the experiments. xdk contributed to the analysis of the data and drafted the manuscript. xyl and hmx helped to revise the manuscript. fc and jxh collected samples and prepared rna. all the authors read and approved the final manuscript. this research was financially supported by national basic research programs, national natural science foundations of china, zhejiang provincial natural science foundation of china, science and technology department of zhejiang province, china, and the national institutes of health grant da 
dissection of the genetic architecture underlying oil-related biological processes will greatly facilitates the genetic improvement of rapeseed. gene annotation analysis was integrated with qtl mapping and brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed. then, degs between sollux and gaoyou were identified as the candidate genes affecting seed oil content by combining those degs with the quantitative trait locus mapping results, of which, one was found to be homologous to arabidopsis thaliana lipid-related genes. bioinformatics analysis was performed to investigate differentially expressed genes. this study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages. the differential gene expression during pod development offers a snapshot on the genes responsible for oil accumulation in. integrated analysis of transcriptome profiling, qtl mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of brassica napus. transcriptomerna-seqqtl regionscomparative genomicslipid-related candidate genesbrassica napusissue-copyright-statement© the author 2015 intervarietal degs of lipid pathways in qtl regions represent important candidate genes for oil-related traits. the rna samples were collected for rna-seq at 5- 15- and 25- days after flowering. to identify candidate genes in the linkage peaks reported previously, we used rna sequencing technology to analyze the pod transcriptomes of german cultivar sollux and chinese inbred line gaoyou. brassica napus is an important oilseed crop. four hundred sixty five and two thousand, one hundred fourteen candidate degs were identified, respectively, between two varieties at the same stages and across different periods of each variety.
 brassica napus is an important oilseed crop. dissection of the genetic architecture underlying oil-related biological processes will greatly facilitates the genetic improvement of rapeseed. the differential gene expression during pod development offers a snapshot on the genes responsible for oil accumulation in. to identify candidate genes in the linkage peaks reported previously, we used rna sequencing technology to analyze the pod transcriptomes of german cultivar sollux and chinese inbred line gaoyou. methods the rna samples were collected for rna-seq at 5- 15- and 25- days after flowering. bioinformatics analysis was performed to investigate differentially expressed genes. gene annotation analysis was integrated with qtl mapping and brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed.  four hundred sixty five and two thousand, one hundred fourteen candidate degs were identified, respectively, between two varieties at the same stages and across different periods of each variety. then, degs between sollux and gaoyou were identified as the candidate genes affecting seed oil content by combining those degs with the quantitative trait locus mapping results, of which, one was found to be homologous to arabidopsis thaliana lipid-related genes. discussion intervarietal degs of lipid pathways in qtl regions represent important candidate genes for oil-related traits. integrated analysis of transcriptome profiling, qtl mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of brassica napus.  this study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transcriptomerna-seqqtl regionscomparative genomicslipid-related candidate genesbrassica napusissue-copyright-statement© the author 2015 