Identify Candidate Genes Of Oil Content QTLs In Brassica Napus L.

Rapeseed is an important oil crop,and its seed oil content is one of the continuing focuses of rapeseed breeding scientists.Through the unremitting endeavors,the oil content of rapeseed in China is obviously improved（from 38%to 45%）.But the oil content of Chinese cultivars is still lower than that of Canada,Australia,and Europe.Therefore,increasing seed oil content is still an important mission for rapeseed breeding in our country.Oil content is a quantitative trait.Recent technological advances by combining GWAS and transcriptome sequencing have made it possible to dig out new regulatory loci and genes which influence oil biosynthesis.In the previous researches,using two different populations,we identified high quality QTLs by combining linkage maps and GWAS.Seven over-lapping QTLs were identified in both mapping methods,which covered an average confidence interval of 183 kb and explained the phenotypic variation of 10.23 to 24.45%per each.Among these QTLs,2 QTLs on A7 and A2chromosomes were shorter and the most contributed,and selected for further functional gene identification:1.According to transcriptome data and QTLs data,we choose BnLEC1.A07（Bna A07G0126300）and BnMOD1.A07（Bna A07G0055500）from chromosome A7,and BnCCI1（Bna A02G0413900ZS）,BnHVA22b（Bna A02G0400700ZS）,BnHMG1（Bna A02G0221800ZS）from chromosome A2 as the candidate genes.BnLEC1.A07,BnMOD1.A07,BnCCI1,BnHMG1 were high-level-expressed in variety 61616,and BnHVA22b was high-level-expressed in variety 51070.2.We designed specific primers to verify gene expression in 15d and 25d seeds.RT-PCR result shows that the expression of BnLEC1.A07 and BnMOD1.A07 both were higher in the seed of 61616.According to the transcriptome data,there were no sequence differences between the coding area of BnLEC1.A07 and BnMOD1.A07 two lines.The promoter sequences of genes in two lines were cloned and named P_BnLEC161616,P_BnLEC151070,P_BnMOD161616,P_BnMOD151070,respectively. Sequence analyzes showed that there were many sequence differences between P_BnLEC161616 and P_BnLEC151070,while there were only two base differences between P_BnMOD161616 and P_BnMOD151070.The LUC and GUS plasmids which contain promoters were constructed.We transformed plasmids into Agrobacterium tumefacien respectively and injected into tobacco leaves.In vivo imaging and GUS staining experiments were performed to compare the promoter expression strength.The results showed that the activity of P_BnLEC161616 was stronger than that of P_BnLEC151070,but the activities of P_BnMOD161616 and P_BnMOD151070 were too low to be compared.3.The c DNA of rapeseed varieties 61616 and 51070 were used as templates to clone genes BnCCI1, BnHMG1 and BnHVA22b,respectively.We compared sequence with reference genome sequence, and we found that BnCCI1 and BnHMG1 were mutant in coding regions,and the coding area of BnHVA22b had no difference with reference genome sequence.Next,we constructed GFP vector connected with genes and transformed Arabidopsis thaliana.For each gene,we obtained more than 15 over-expression plants.We analyzed gene expression of T₀generation plants and seed oil content of T₁ generation.The result shows that seed oil content of BnHVA22b overexpression plants were lower than WT,which is possibly the candidate gene influencing seed oil content.In conclusion,we analyzed the promoter sequence differences of oil content related genes in different varieties,and find a new candidate gene which may influence seed oil content.Our experiments laid a foundation for further research on new genes of oil content and analysis of oil biosynthesis.