Genome-and Transcriptome-wide Association Studies Provide Insights Into The Genetic Basis Of Natural Variation Of Thousand-seed Weight In Brassica Napus

Rapeseed is an important oil crop in China,but with the impact of low-priced imported rapeseed on the domestic rapeseed market,the economic benefits of rapeseed in China are difficult to guarantee,which in turn leads to a substantial decline in rapeseed production and planting area.Thousand-seed weight is one of the three factors that affect the yield of rapeseed.Increasing thousand-seed weight is also one of the main ways to increase rapeseed yield.By locating the SNP locus that regulate rapeseed thousand-seed weight and predicting and validating thousand-seed weight candidate genes,the genetic basis and molecular mechanism of thousand-seed weight of rapeseed were clarified,and a theoretical basis for improving the thousand-seed weight of rapeseed was provided.In this study,genome-wide association study was carried out on the thousand-seed weight phenotype data of 505 natural populations of Brassica napus L.in Wuhan(WH2013,WH2014,WH2016,WH2017),Hefei(HF2016,HF2017)and Chengdu(CD2017).The transcriptome data from seeds of 20 days after flowering of 309 copies of Brassica napus L.and 40 days after flowering of 274 copies of Brassica napus L.and thousand-seed weight phenotype data were used for transcriptome-wide association study.Combining the results of GWAS,TWAS and transgenic technology,the location of the significant association locus of thousand-seed weight,the prediction of the candidate genes of thousand-seed weight,and the preliminary verification of the function of candidate genes were realized.In this study,the thousand-seed weight data of natural population containing 505 Brassica napus L.varieties in 7 environments were sorted,analyzed by variance,and related analysis.The results showed that the average range of the thousand-seed weight ranged from 3.33 g to 3.71 g,and the total thousand-seed weight of the group at 1.43 g-6.38 g,the generalized heritability rate is 0.94.The above results indicate that the thousand-seed weight phenotype is abundant in the entire population.Thousand-seed weight frequency distributions in the 7 environments are normally distributed,and the correlation of the thousand-seed weight phenotype data at the same location is strong.Genome-wide association study was performed using 505 thousand-seed weight phenotypic data from 7 environments of the natural population of Brassica napus L.and10,620,048 SNP markers in the genome.A total of 304 SNPs with a significant correlation of thousand-seed weight were located and integrated into 127 locus.There were 32 locus repeatedly detected in many environments,12 locus were the same as those after BLUP analysis of thousand-seed weight phenotype data,and 7 locus overlaped with those reported by previous researchers.Combining GWAS results,gene annotations and expression levels,four candidate genes were predicted: Bna A07.KCR(Bna A07g26670D),Bna A07.EXPA1(Bna A07g28080D),Bna A09.IAA30(Bna A09g39680D)and Bna A10.GIF2(Bna A10g00680D).Transcriptome-wide association study was performed using transcriptome data from seeds of 20 days after flowering of 309 copies of Brassica napus L.and 40 days after flowering of 274 copies of Brassica napus L.and thousand-seed weight phenotype data of Brassica napus L.,and a total of 21 genes were significantly correlated.Combining GWAS mapping results and TWAS predicted gene results and gene annotations,it is speculated that Bna A07.ATHIR1(Bna A07g28360D),Bna A07.FTSH1(Bna A07g26630D),Bna A07.YCF32(Bna A07g26680D),Bna C06.FTSH 1(Bna C06g28800D),Bna C06.YCF32(Bna C06g28840D),Bna C07.MIOX4(Bna C07g40160D)gene may regulate the thousand-seed weight of Brassica napus L..Through verification,the results showed that the thousand-seed weight of the T0 generation mature seeds of the partial overexpression materials of Bna A07.KCR,Bna A07.EXPA1,Bna A09.IAA30,Bna A10.GIF2 was significantly higher than that of WT.The relative expression levels of genes Bna A07.KCR,Bna A07.EXPA1,Bna A10.GIF2 in pod of some T0 overexpression plants 25 days after flowering were significantly higher than those of WT.The oil content of genes Bna A07.KCR,Bna A09.IAA30,Bna A10.GIF2 in mature seeds of some T0 overexpressed plants was significantly higher than that of WT,and the oil content of some Bna A07.EXPA1 overexpressed plants was significantly lower than WT.The fatty acid composition of mature seeds of some T0 overexpression plants of genes Bna A07.KCR,Bna A07.EXPA1,Bna A09.IAA30,Bna A10.GIF2 was consistent,specifically oleic acid(C18:1),arachidonic acid(C20:1)were significantly higher than WT,soft acid(C16:1),stearic acid(C18:0),linoleic acid(C18:2),linolenic acid(C18:3),arachidonic acid(C20:0)were significantly lower than WT.The above results indicate that the 4 candidate genes may indeed regulate the thousand-seed weight of Brassica napus L..Because the phenotype of T0 overexpressing transgenic plants is not yet stable,the four genes regulating the thousand-seed weight of rapeseed need further verification.At present,most of the research on rapeseed thousand-seed weight is at the level of QTL mapping.This study uses multi-omics association analysis to locate the SNP loci of rapeseed thousand-grain weight and predict genes related to thousand-seed weight,and then reuse transgenic technology validates candidate genes.The research results will provide a theoretical basis for the analysis of the genetic basis and molecular mechanism of the thousand-seed weight of rapeseed,and it is also of great significance for improving the yield of rapeseed and increasing the economic benefits of rapeseed.