Functional Analysis Of Seed Fatty Acid Reducers And Identification Of Genes Related To Seed Oil Content In Rapeseed (Brassica Napus L.)

Rapeseed(Brassica napus L.)is one of the most important oil crops in China.One of the main disadvantages of domestic rapeseed is the relatively low seed oil content(SOC).Therefore,elevating SOC is an important target for domestic rapeseed breeding.A large number of previous studies to increase rapeseed SOC focused on the upregulation of key genes in the lipid biosynthesis pathway.However,the repression of the genes involving the lipid degradation is an alternative strategy to increase SOC.GDSL lipases may play important role in lipid degradation.It was reported that over expressing of five GDSL-type Seed fatty acid reducers(SFARs)could reduce SOC in Arabidopsis,and by contrary,knocking-out these genes resulted in a significant increase of SOC in Arabidopsis.Knocking out the SFAR orthologs in Brassica oilseeds might give rise to a similar effect,which means considerable economic value.However,rapeseed(Brassica napus)is a typical polyploid crop consisting of two subgenomes,and its SFAR orthologs in different subgenomes might have different expression pattern and function.On the other hand,SOC is a typical quantitative trait which is controlled by multiple loci.Most of the SOC QTLs that were identified so far were resulted from the genetic populations generated by two parents with SOC difference,i.e.,the identification of the QTLs was limited by the allelic variation of the two parents.In recent years,we resequenced the genomes of a large germplasm collection,paving the way to identify more SOC QTLs in a population with large genetic diversity.In this study,we created bnsfars mutants using targeting induced local lesions in genomes(TILLING)and CRISPR/Cas9 technologies,and verified the effect of BnSFARs on SOC of rapeseed;We searched for the genes that regulate the SOC of rapeseed through genome-wide association study(GWAS)on a genetic diverse population containing 290 core germplasm accessions of rapeseed.The main results are described as the following:1)There were 222 BnGDSLs across the Brassica napus genome,which were evenly distributed in the A and C subgenomes.BnGDSLs of the A and C subgenomes were divided into 6 and 4 subfamilies,respectively,and there was an obvious collinearity relationship between BnGDSLs and AtGDSLs.Through sequence alignment,12 genes highly homologous to AtSFARs(AtSFAR1-AtSFAR5)were selected as candidate BnSFARs.The expression patterns of each BnSFAR subfamily were different from each other,while the expression patterns of homologous copies within the same BnSFAR subfamily were roughly similar.Analysis of the SNPs across BnSFARs in a rapeseed genetic population consisting of 870 accessions revealed that the SNPs distributing in BnSFAR1 and BnSFAR4 were significantly correlated(P<0.05)with SOC,and the SNPs distributing in BnSFAR1,BnSFAR4 and BnSFAR5 were significantly correlated(P<0.05)with oleic acid content of seeds.2)Homozygous single mutants,double mutants of bnsfar1 and bnsfar4 were created through ethyl methanesulfonate(EMS)mutangensis and TILLING analysis.The SOC of the homozygous bnsfar4 single mutants(bnsfar4.C03a,bnsfar4.A06a,bnsfar4.A06b and bnsfar4.Cnnb)was not significantly different from that of the wild type with the same genetic background.However,the SOC of homozygous bnsfar4double mutants(bnsfar4.A06a bnsfar4.C03a and bnsfar4.A06b bnsfar4.Cnnb)was8.7%-12.1%higher than that of the wild-type plant,which was statistically significant.Unlike bnsfar4,the SOC of homozygous bnsfar1 double mutant(bnsfar1.Ann bnsfar1.C04)was not significantly different from that of the wild type.Because EMS caused a large number of random mutations in the whole genome,the SOC of TILLING mutants was significantly lower than that of the control plants without EMS treatment.3)Four paralogs of BnSFAR4 and two paralogs of BnSFAR5 were knocked out using CRISPR/Cas9 technology,and the respective bnsfar4 quadruple mutants(bnsfar4.A06a bnsfar4.C03a bnsfar4.A06b bnsfar4.Cnnb)and bnsfar5 double mutants(bnsfar5.A03 bnsfar5.C07)were created.Compared with the wild-type seeds,the SOC of T₃ and T₄ bnsfar4 mutants was increased by 9.7%-14.5%and 12.9%,respectively,while the SOC of T₃ and T₄ bnsfar5 mutants were increased by 10.4%and 11.2%,respectively.Except for the T₄ seeds of the bnsfar4 mutant,thousand seed weight of the other mutants did not change significantly compared with the wild type,and the germination rate of the mutant seeds and the growth after germination were not negatively affected.However,the oil body area in the bnsfar4 seed cells was significantly larger than that of the wild type,and the reduction of the mutant SOC during the late stage of seed development and the early stage of germination was significantly slower than that of the wild type.4)The SOCs of 290 rapeseed core accessions at two locations were measured,and the respective GWAS was performed.There was a significant correlation between the SNPs distributing across 35.25-35.79 Mbp on chromosome C07(Chr C07)and SOCs(-log^p₁₀>5).There was a linkage disequilibrium(coefficient of determination R²=0.68)between the SNP at the position Chr C07?35249208 and the gene Bna C07g30920D(Bna.PTL.C07).Bna.PTL.C07 is a member of the patatin-like lipase(PTL)gene family.There were six SNPs in the 5'regulatory region of Bna.PTL.C07that were significantly correlated with the SOCs(P<0.05),two of which were located in the CAAT-box.Taken together,we analyzed BnGDSL family in polyploidy Brassica napus,and created bnsfars mutants as pre-breeding materials in which BnSFAR function was effectively blocked.The study demonstrates the advantages of CRISPR/Cas9 over TILLING in creating loss-of-fucntion mutants.In addition,new locus which regulates SOC formation was identified in a genetically diverse population.Our study provided an example for precise breeding toward a high rapeseed SOC.