QTL Mapping And Time-series Transcriptomic Analysis For Oil Content In Seed Of Brassica Napus L.

Oilseed rape(Brassica napus L.)is an important oil crop in China.Improving oil content is one of the most important goals of rapeseed breeding.However,the gene network regulating oil synthesis is very complex,and its genetic and molecular mechanism has not been fully analyzed.In this study,a Recombinant Inbred Lines(RIL)population of 235 lines was used to construct a high-density genetic linkage map by using 60 K SNP chip.The oil content of RIL population in three different environments were determined and QTL mapping was performed.According to the results of genotyping,two lines of RIL with similar genetic background but different oil content were selected for RNA sequencing.The transcriptomes of six different developmental stages of their seeds were determined,and the gene expression patterns of different developmental stages were revealed.We found the candidate genes and the related network for oil accumulation.These results provide a certain foundation for high oil content breeding of oilseed.The main conclusions of the study are as follows:1.Construction of high-density genetic linkage map of Brassica napus L.: A total of 235 recombinant inbred lines were genotyped using 60 K SNP chip and a highdensity genetic linkage map was constructed.The map contained 7974 SNP markers and 225 SSR markers,respectively.Among them,3690 markers were anchored in the A subgenome and 4509 markers were anchored in the C subgenome.The total length of the map is 3077 c M,the shortest linkage group was 15.5c M,the longest was 166.9c M,and the average distance was 0.375 c M.This linkage map coverd 77.95% of the reference genome(Darmor-bzh).2.QTL mapping of oil content: The recombinant inbred line population was planted in three different environments,and the oil content was investigated.The QTLs for oil content were mapped by compound interval mapping(CIM).Five oil content related QTLs were identified,which were distributed in linkage group A09,C03,C04 and C05,respectively.QTL located on C03 was repeatedly detected in three environments.Based on gene functional annotation of these QTL regions,and screed19 genes related to oil synthesis,including 3 triglyceride synthesis genes,4 fatty acid degradation or extension genes,6 phospholipid metabolism or phospholipid signaling genes,1 carbohydrate metabolism gene and 5 transcription factors.Among them,the gene Bn LPAT2 located in q-Oil＿C03 is reported as one of the synthesis genes for triacylglycerol.The Bn PAH1 and Bn PAH2 is located in q-oil＿C05 and Q-oil＿C04regions,respectively,and are important genes in the synthesis of triacylglycerol and oil body formation.3.Variation analysis of the candidate genes in the QTL region: A total of 242 genes were identified with structure variation between two parents in QTLs confidence intervals,including 14 genes were lipid metabolism related genes.4.Transcriptome sequencing analysis: The transcriptome sequencing analysis was performed on seeds with different oil content at 16,23,30,37,44 and 51 days after pollination(DAP),and it was found that the gene expression difference from different seeds oil content,and the largest different at 30 DAP and 37 DAP.Combined with the oil content phenotype analysis of seeds at different developmental stages,it is revealed that 30 DAP to 37 DAP is the key period of oil content formation.Transcriptomes of different periods were compared,and it was found that the gene expression differences between high and low oil content materials were the largest during S3(30 and 37 DAP)and S4(37 and 44 DAP).Compared with the high oil content seeds,the genes of fatty acid synthesis pathway and photosynthesis-related pathway in the seeds of the two low oil content seeds were down-regulated during S3 and S4 respectively.5.Coexpression network analysis: Eighteen transcriptome data were used to construct the gene coexpression network.It was found that two modules were negatively correlated with oil content,and two modules were positively correlated with oil content.The positive correlation module is enriched in metabolic pathways such as oil synthesis,carbon fixation,sugar metabolism and amino acid metabolism,while the negative correlation module is enriched in genes related to fatty acid degradation,histone lysine methylation and DNA methylation.Further analysis of these modules revealed that Bn LTP6,Bn GPAT4 and Bn REM17 were the core genes related to oil synthesis.b In addition,six genes,Bn DGK5,Bn KCS4,Bn PLDALPHA2,Bn PAH2,Bn TAGL and Bn LTP6,were directly linked to each other,as well as to acyl lipid metabolism,fatty acid metabolism,carbohydrate metabolism,photosynthesis and transcription factors,and form potential molecular networks regulation seed oil content.In this research,combined with QTL mapping and transcriptome sequencing,we identitied several candidate genes controlling the seed oil content of rapeseed.Timeseries transcriptomic analysis reveals that from 30 DAP to 37 DAP is the most important stage for the different oil content seeds.Further more,the seed with high oil content probablely due to the constant expression level of the fatty acid biosynthesis and the photosynthesis genes during 30 to 37 and 37 to 44 DAP respectively.The candidate genes could provide molecular markers for the cultivation of high oil content varieties,and the transcriptome dynamic analysis provides resources for the study of developmental seed and mechanism of seed oil content.