Genetic Mechanism Study Of Seed Oil Synthesis And Construction Of EMS Mutant Library In Brassica Napus L.

Rapeseed(Brassica napus L.;AACC,2n = 38)is an important oil crop in China,which is the third largest source of vegetable oil in the world after soybean oil and palm oil.Improving seed oil content(SOC)is one of the important goals of rapeseed breeding.At present,the SOC of the main winter rapeseed varieties in China is about 40%,which is 5-8 percentage points lower than that of foreign high-quality varieties.Breeders have collected a large number of germplasm resources with SOC of more than 50%,even reaching 60%.Therefore,there is great potential for cultivating high SOC rapeseed varieties suitable for popular cultivation.Fatty acid synthesis and accumulation in plant seeds is a complex metabolic regulatory network involving multiple metabolic pathways and hundreds of genes.However,the oil biosynthesis mechanism of rapeseed is still unclear,which greatly hinders the genetic improvement of rapeseed oil.In this study,the genetic and molecular mechanisms of oil biosynthesis in rapeseed were studied by multi-omics association analysis.Besides,an EMS mutant library was constructed to screen mutants related to SOC and fatty acid,which provided germplasm resources for studying oil synthesis and breeding in Brassica napus.The main results are summarized as follows:A rapeseed natural population with 505 inbred lines was constructed,and genomic variation map was obtained by resequence.Multi-year multi-location SOC phenotypic data of natural populations were collected,and the genome-wide association study(GWAS)in different environments was performed using a linear mixed model(LMM).In summary,27 signals were detected in at least two environments(in different years,locations or groups).The effect of significant QTL loci showed that very few diversities were all synergistic alleles at these loci,and most of the synergistic loci have not been well utilized in breeding;the frequencies of synergistic alleles in different subgroups are different,but they are basically derived allele,meaning they are selected during breeding.These results suggesting that many of SOC associated signals were under artificial selection during breeding,but some OC-improving QTLs were not well utilized.Transcriptome sequencing was performed on 309 20 DAF and 274 40 DAF seeds.Transcriptome association analysis were performed with SOC,and observed 605 and 148 transcriptome-wide significant genes in 20 DAF and 40 DAF,respectively.Gene ontology(GO)enrichment analysis showed that many OC-associated genes were involved in stress related pathways at 20 DAF and 40 DAF genes were mainly concentrated to seed coat development.Furthermore,we performed Independent Component Analysis(ICA)-based decomposition of expression profiles of 20 DAF and 40 DAF,then 146 and 141 components were identified as independent signatures of seed transcriptome.In 20 DAF,only one component(termed M65)was significantly correlated with SOC,which was enriched in stress and hormone stimulus pathways.In 40 DAF,three components(termed M79,M103 and M139)were significantly correlated with OC.Among which,M139 was enriched in stress related categories and flavonoid related categories,and M103 was enriched in regulation of cell population proliferation plant organ development.Furthermore,the correlation between OC-related QTLs and OC-related modules showed that M65 and M139 was associated with OC-related QTL q OC.A09.5,and M79 was associated with OC-related QTL q OC.A05.3,q OC.C05.2 and q OC.C05.3.Based on the candidate gene screening platform for association analysis established in Brassica napus,the candidate genes related to SOC are ranked.The candidate genes of QTL q OC.A05.3 and q OC.C05.3 was identified as PMT6.Based on the mutant materials and transgenic overexpression materials of Arabidopsis and rapeseed,it was determined that the gene PMT6 negatively regulates the accumulation of SOC.An EMS mutant population with ～100,000 M2 lines was generated using Zhongshuang 11 as the parent line.The EMS-induced genome-wide mutations in M2-M4 plants were assessed.The average number of mutations including single nucleotide polymorphisms and insertion/deletion in M2-M4 was 24,576,33,507 and 29,266,respectively.The effects of the mutations on gene function were predicted in M2-M4 mutants,respectively.We screened the seeds from 98,113 M2 lines and 9,415 SOC and fatty acid mutants were identified.We further confirmed 686 mutants with altered SOC and fatty acid in advanced generation(M4 seeds).Seven representative M4 mutants with increased oleic acid were resequenced and the potential causal variations in FAD2 and ROD1 genes were identified.This study generated and screened a large scale of Brassica napus EMS mutant population and the identified mutants could provide useful genetic resources for the study of oil biosynthesis and genetic improvement of SOC and fatty acid composition of B.napus in the future.In this study,through a multi-omics correlation analysis of rapeseed germplasm resources with significant differences in SOC,27 QTLs / genes for regulating SOC were obtained,and 686 SOC and fatty acid mutants were selected by constructing a large EMS mutant library.This will not only promote functional genomics of lipid metabolism in Brassica napus,but also provide new ideas for genetic improvement of SOC in Brassica napus.