Fine Mapping And Transcriptomic Analysis Of Oleic Acid In Brassica Napus

Rapeseed is one of the important oilseed crops worldwide and is the major source of edible oil in China.The fatty acid composition in rapeseed seed is of great significance to the quality of edible oil.Rapeseed oil with a high C18:1 content is less susceptible to oxidation during storage,frying and food processing and is thus desirable for its thermal stability,at same time high oleic rapeseed have great advantages to human healthy,thus make it important for edible oil.So far high oleic germplasm is mainly depend on the mutation of FAD2 so that we need search more genetic resource to get high oleic germplasm.In this study,we applied GWAS and linkage mapping analysis to mapping QTL with oleic acid and try to uncover the mechanism of high oleic by transcriptome analysis in developing seed at different stages.The main result of this study are as follows:1.A novel QTL with high oleic acid identified by association and linkage mappingAn association panel with 375 rapeseed low erucic acid inbred lines was employed for association analysis of C18:1 content and were genotyped using the60K SNP array chips.A total of 19 SNPs significantly associated with C18:1 content were identified.Of these significant SNPs,17 SNPs were located on A9,and the most significant SNP was Bn-A09-p31549208(-log₁₀（P）=6.97),which explained 10.11%phenotypic variance.Two B.napus inbred lines with significant difference in C18:1content were used as the parents of the DH population.In the ZD-DH population,we used a high-density SNP-based genetic map which length was 2879.7 c M for QTL mapping.In total,six QTLs were identified on six chromosomes（A2,A3,A4,A5,A9and C1）,which explained 1.1%-86.3%of the phenotypic variation individually.Among the QTLs,a major locus with the largest effect,OLEA5 on A5 was detected,which explained 67.18%、86.30%and 69.26%of the phenotypic variation in the three seasons.With the allele-specific marker,it was confirmed that the OLEA5,is indeed the Bna A.FAD2.a.In addition to OLEA5,another major QTL,OLEA9,was detected on A9,which explained 11.25%,5.72%and 6.29%of the phenotypic variation in the three seasons,and the allele from ZP1 increased the C18:1 content by 2.14%（additive effect）on average.OLEA9 were located on chromosome A9,which is overlapped with the locus detected in GWAS analysis.2.The effect and verification of the novel QTL OLEA9To examine the effects of OLEA5 and OLEA9 on C18:1 content,a closely linked insertion/deletion polymorphism（In Del）marker Bn A129 was developed.The DH population was then classified into four groups according to the genotypes of allele-specific marker YQ-FAD2a-1 for OLEA5 and Bn A129 for OLEA9.The average C18:1 content in the group with both additive effect alleles was 77.07%,12.48%higher than the group without additive effect alleles.For OLEA5,the average C18:1content in two groups with respective alleles were significantly higher than the groups with no alleles,with the C18:1 content increased by 9.22%-10.13%.For OLEA9,the average C18:1 content in two groups with respective alleles were significantly higher than the groups with no alleles,with the C18:1 content increased by 2.35%-3.26%.Our results thus showed that C18:1 can be further increased by minor locus OLEA9.We set to construct a new bilateral mapping population using two parental lines without the interference of OLEA5.One of the two parental lines is ZP1,with 66.1%of C18:1,and another ZD12,with a significantly lower C18:1（63.8%）.A segregating F₂ population（ZD-F2）then were generated.In an F₂sub-population sampled from the ZD-F2,166 individual plants could be classified into three groups with the marker Bn A129.Both AA and BB exhibited a continuous distribution of C18:1 with an overlapping each other,although the average content in AA（67.63%）was significantly higher than BB（64.68%）.The distribution of AB group was even wider.These data thus established that OLEA9 is responsible for the segregation of C18:1content in the F₂population.3.Fine mapping of the novel locus OLEA9We developed new markers by resequencing the two parents and developed a BC₃F₂ NIL population.A local linkage map consisting of fifteen markers was subsequently constructed.After screening among 5184 BC₃F₂ individuals with flanking markers,161 recombinant plants between the markers of Bn A144 and Bn A153 were obtained.SNP markers were developed within the interval of approximately 260 kb between marker Bn A144 and Bn A153.Subsequently,these SNP markers together with previously used markers were employed to genotype 161BC₃F₂ individuals and some recombinant progeny.The locus of OLEA9 was delimited to an interval of approximate 76 kb between SNP1 and SNP7 on A9chromosome.4.Candidate genes analysisWe compared the relative gene expression in developing seed for each of the 12genes in candidate region using two NILs.The expression levels of Bna A09g39520D and Bna A09g39570D were dramatically increased in H-NIL compared with L-NIL in almost all developing stage of seed except 42dap,while Bna A09g39490D was decreased dramatically in H-NIL compared with L-NIL.Furthermore,Bna A09g39570D had much higher fold change between two NIL lines（log₂=1.46）at21dap compared with other stages.Arabidopsis mutants of the three genes were collected and the fatty acid composition in matured seeds of the mutants were determined.In the A.thaliana mutant of AT3G62010,a homolog for Bna A09g39570D,the oleic acid was significantly higher than other two mutants and wild type,suggesting that Bna A09g39570D gene was most likely the candidate gene underlying OLEA9.5.Transcriptomic analysis uncovers the mechanisms of high oleic acid synthesisWe performed transcriptomic analysis in developing seeds from two NIL lines at six developmental stages,namely ovary,14,21,28,35,42 days after pollination（dap）and mature seed.All the expressed genes number increased slowly and stable during14-35dap and decreased suddenly after 35dap,when mature seed have the lowest expressed genes number.We suppose 21-35dap is a key period for fatty acid synthesis.There are 816 up-regulated and 1574 down-regulated differentially expressed genes during all stages and 28dap have the most number of DEGs.For the GO analysis of DEGs at 28dap,carbohydrate process was the most important one in biological process and mostly the expression level with fold changes between-2 to 3,so we suppose the loci may function by fine tuning.By KEGG analysis,we found metabolism pathway,linoleic pathway,linolenic pathway and lignin pathway were enriched mostly.The genes involved in linoleic pathway and linolenic pathway were lipoxygenase so that the loci may work by influencing lipid oxidation process.By analysis all DEGs using Mapman,the transcription factor,protein modification and degradation have more DEGs in regulation pathway and down-regulated genes occupied more than up-regulated genes;P450 cytochrome and glucosidases have most genes in enzyme family overview.In conclusion,the novel loci may regulate the fatty acid content by fine tuning lipid oxidation and carbohydrate process under the control of hormone and transcription factor.