Genome-wide Association Analysis Of Flowering Time And Development Of Flowering Gene Markers In Brassica Napus L.

Rapeseed is the second largest oil crop in the world and has extensive economic impact in many countries.As a major oil consumer in the world,the self-sufficiency rate of edible vegetable oil in China is less than 40%.In order to improve the self-sufficiency rate of edible vegetable oil in China,using a large number of winter fallow areas in southern China to plant early-maturing rape varieties on a large scale can reduce the seasonal contradiction between rape and rice production and increase rape yield.With the advancement of rice-rice-rape cropping pattern and the requirement of rapeseed mechanized production,breeding early-maturing rapeseed varieties is an important research goal of rapeseed breeding in southern China at the present stage.In this study,we investigated the flowering time of 109 inbred lines of Brassica napus,and investigated the simplified genome sequencing of 100 inbred lines.The whole genome association analysis was carried out by using the mixed linear model,and then the phenotypic differences among SNP alleles were analyzed.The candidate genes were predicted by analyzing the candidate interval located at the SNP locus significantly associated with flowering time,the molecular markers of candidate genes were developed,and the effect was evaluated combined with the molecular markers developed by predecessors at flowering time.The main results are as follows:(1)Phenotypic variation of flowering time of Brassica napus: the variation range of flowering time of 109 samples was 124 ～ 180 days,and the coefficient of variation was6.48%.Among them,the variation range of flowering time of 100 samples used for association analysis was 124 ～ 166 days,and the coefficient of variation was 5.64%.It shows that there is a wide range of variation in the flowering time of the selected experimental samples.(2)Population structure and relative kinship analysis: the results of ADMIXTURE and PCA analysis were basically the same,and 100 Brassica napus were divided into 4subgroups.The results of relative kinship analysis showed that the relative kinship of64.36% of the associated population was 0,95% of the associated population was less than or equal to 0.3,indicating that most of the samples in the associated population had no kinship or weak kinship.It can avoid the false positive in the results of association analysis due to the existence of kinship.(3)Genome-wide association analysis of flowering time: using SNP genotyping and filtered 28097 SNP markers,the whole genome association analysis of flowering time of this population was carried out by Q+K model.It was found that 69 SNP loci exceeded the Bonferroni threshold(-lg P > 3),which were distributed on A01,A03,A06,A07,A08,A10,C02,C03,C04,C07 chromosomes,which could explain 13.42% phenotypic variation.(4)Analysis of phenotypic mean difference among SNP alleles: using levene test and t-test to analyze the flowering time between 18568 SNP alleles obtained by further filtered,it was found that a total of 674 SNP loci with p value less than 0.01.(5)Candidate gene analysis: based on the published genome sequencing results(Darmor.v4.1)of Brassica napus,a total of 23 flowering-related genes were screened within the 300 kb range of the upstream and downstream GWAS significant association sites,and 13 SNPs were found in 6 flowering-related genes on both sides of the SNP loci with p < 0.01.(6)Development and application of flowering gene markers: according to the sequence polymorphism of BnFRI.A10,BnEBS.A01 and BnCLPS3.A03,three pairs of functional markers were developed.109 experimental samples were tested by using the developed markers and the flowering period markers provided by Huazhong Agricultural University.(7)The analysis of the effect of developing marker and applying marker: the samples with genotype + of ID-FRI.A10 marker flowered 8.05 days earlier than the samples with genotype-,the samples with genotype C of EBS.A01-DraⅢ marker flowered 6.02 days earlier than that of samples with genotype T,and the samples with genotype A-A-TG-TGG of CLPS3.A03 marker flowered 20.20 days earlier than the samples with genotype A-C-TG-TGG.The samples with genotype + of FLC.C2-1,FLC.C2-2 and FLC.A10-2markers flowered 4.7d,4.6d and 8.2d earlier than those with genotype-,respectively.(8)Analysis of pyramiding effect of markers: the samples with genotype A/T-C-T-T of EBS.A01-DraⅢ × CLPS3.A03 marker combination flowered 23.9 days later than those with genotype G/A-A-TG-TGG.The samples with genotype(-,-,-)of ID-FRI.A10 ×FLC.C2-2 × FLC.A10 marker combination flowered 24.8 days later than those with genotype(+,+,+).