Mining Seed Weight Candidate Genes By Intergrated GWAS And WGCNA Analysis In Brassica Napus L.

Brassica napus is an important oil-bearing crop in China.Increasing yield is an important goal of current rapeseed breeding research.Thousand-seed weight(TSW)is one of the important composition of rapeseed seed yield.In this study,a collection of 496 rapeseed germplasm resources were used as the association panel,and were genotyped using the high-throughput 60 K SNP array.The population’s TSW phenotype in three environments(14NJ,15 TZ,16TZ)was investigated,and genome-wide association analysis(GWAS)was performed to detect significant loci for TSW in Brassica napus.Utilizing the seed and silique transcriptome data of two Brassica napus cultivars,ZS 11 and ZY 821,a weighted gene co-expression network analysis(WGCNA)was performed to detect hub genes significantly related to the seed weight phenotype.The strategy of integrating GWAS and WGCNA analysis was adopted to identify the TSW loci and underlying candidate genes in Brassica napus,and the major results are listed below:1.Mining the large TSW germplasm resources of rapeseedThe TSW phenotype of 496 rapeseed was investigated in three environments(14NJ,15 TZ,16TZ).Statistical analysis showed that the TSW phenotypes varied widely.The mean value ranged from 3.51 g to 4.27 g,the coefficient of variation ranged from 0.16 to0.21,and the broad sense heritability was 63.12%.The correlation analysis of the silique traits showed that TSW was positively and significantly correlated with silique length,but was weakly correlated with seed number per silique.Given the phenotypic data of three environments,these accessions,including 2012-8998,Jia 904,WH-59 and CY19PXW-65,had a stably large TSW phenotype,with an average value of 5.63,5.59,5.09 and 5.06 g,respectively.Meanwhile,the silique length of these accessions was significantly longer than the average value.These materials can be used as donor parents to improve seed weight of rapeseed.2.TSW loci detected by GWAS analysisThe GWAS of TSW was performed based on the phenotypic data and 19,167 SNP markers.Six and 61 loci were detected with MLM and GLM,which explained 28.92% and47.08% of the phenotypic variance,respectively.Combining the common loci between two models,62 significant loci were obtained and accounted for 47.31% of the phenotypic variance.These loci were distributed on all chromosomes of the genome,with the largest number of 9,8,and 7 loci detected on A07,A03,and C06,respectively.The most significant locus Bn-scaff＿17526＿1-p1066214 was detected on C09,and accounted for5.55% and 15.26% of the phenotypic variance in MLM and GLM,respectively.Among them,22 loci overlapped with previously reported QTLs,of which 9 loci were verified by at least two populations.The remaining 40 loci were newly detected,and several of them had large effects and were detected in multiple environments.3.TSW hub genes identified by WGCNA analysisWGCNA analysis was performed based on the seed and silique transcriptome of two accessions ZS11 and ZY821,which had difference in TSW,and 13 co-expression modules were constructed.Correlation coefficient method showed that the purple and magenta modules were most significantly correlated with the seed weight phenotype.The Arabidopsis homolog of two purple module hub genes Bna A06g00850 D and Bna A07g36220 D,ETHEL and TITAN,respectively,were reported to regulate seed size by regulating the endosperm cell development.Bna A08g01500 D and Bna A05g08020 D were hub genes in the magenta module,and their Arabidopsis homologous genes ABA2 and ABI5 were reported to regulate seed development through the abscisic acid signaling pathway.Besides,homolog of both magenta module genes Bna Cnng73760 D and Bna C03g25530 D in Arabidopsis,BRM,was reported to remodel the chromatin state to influence the seed maturation process.4.Candidate gene mining based on GWAS and WGCNA analysisSeventeen TSW candidate genes were identified by integrating information from GWAS and WGCNA analysis.13 candidate genes were predicted for GWAS loci,including homologs of Arabidopsis well-known seed weight genes,like NPC6,DGAT,TTG2 and AGL61.Furthermore,two of them,Bna A07g36220 D and Bna C03g22530 D were important genes in purple and magenta module,respectively.These Arabidopsis homologous genes regulate seed size through the zygotic and maternal tissue pathways.The results are helpful for analyzing the genetic architecture of TSW of rapeseed and lay a foundation for studying the regulation mechanism and guiding the genetic improvement of seed weight.