Transcriptome analysis of Brassica napus pod using RNA-Seq and identification of lipid-related candidate genes

Abstract

Background: 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 (RNA-Seq) 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 5-7, 15-17 and 25-27 days after flowering (DAF). Bioinformatics analysis was performed to investigate differentially expressed genes (DEGs). Gene annotation analysis was integrated with QTL mapping and Brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed.

Results: 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, 33 DEGs between Sollux and Gaoyou were identified as the candidate genes affecting seed oil content by combining those DEGs with the quantitative trait locus (QTL) 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.

Conclusions: This study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages.

Fig. 1

Venn diagram for the genes expressed in each of the three stages of Brassica napus pod in Sollux and Gaoyou. a 98,804 genes are co-expressed at S1 and G1 while 10,477 (S1) and 7,633 (G1) are variety-specifically expressed genes. b 64,272 genes are co-expressed at S2 and G2 while 22,334 (S2) and 5,848 (G2) are variety-specifically expressed genes. c 57,127 genes are co-expressioned at S3 and G3 while 15,294 (S3) and 8,613 (G3) are variety-specifically expressed genes. d Among all genes, 51,538 are co-expressed at all the stages in the Gaoyou, 11,407 are co-expressed in G1 and G2. 2,806 are co-expressed in G3 and G2, and 9,733 are co-expressed in G1 and G3. The numbers of stage-specifically expressed genes are 33,759 (G1), 4,396 (G2), and 1,663 (G3), respectively. e Among all the genes, 60,202 are co-expressed at all the stages in the Sollux, 18,648 are co-expressed in S1 and S2, 3,444 are co-expressed in S3 and S2, and 5,624 are co-expressed in S1 and S3. The numbers of stage-specifically expressed genes are 24,807 (S1), 4,312 (S2), and 3,151 (S3), respectively

Fig. 2

Hierarchical clustering analysis of gene expression based on RPKM data. S1, S2 and S3 are stages 1, 2 and 3, respectively for Sollux, and G1, G2 and G3 are stages 1, 2 and 3, respectively, for Gaoyou. The color key represents RPKM (reads per kilobase per million mapped reads) normalized log₂ transformed counts. Red represents high expression and green represents low expression. Each row represents a gene

Fig. 3

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 (S1-S2, S1-S3, S2-S3) of Sollux. d GO classification among three contrasts (G1-G2, G1-G3, G2-G3) of Gaoyou

Fig. 4

KEGG pathway categories of differentially expressed genes at 5–7 DAF, 15–17 DAF and 25–27 DAF