Genome-wide Association Study Identifies Candidate Genes For Cadmium Tolerance In Brassica Napus

Brassica napus is the main edible oil source in China.In recent years,the Cd pollution have seriously affected the B.napus yield and quality in Southwest China and central China which was the main B.napus producing areas.Discovering Cd tolerance genes and innovating Cd tolerance B.napus germplasm can guide the production safe of B.napus.In this study,a scientific and effective evaluation method for Cd tolerance in B.napus was developed.Then we identified the Cd tolerance germplasms in472 B.napus core collections using the method.Cd tolerance genes were discovered using genome-wide association study?GWAS?and bioinformatics method,to provide Cd tolerance gene resources and markers for molecular breeding of Cd tolerance in B.napus.The main research contents and results are as follows:1.Identfying the Cd tolerance germplasms in 472 B.napus core collections.Firstly,we developed a scientific and effective evaluation method for Cd tolerance in B.napus.Then,the Cd tolerance phenotyping of 472 B.napus core collections were studied using the method,and the Cd tolerance germplasms were identified in this population.The results showed that the B.napus relative radicle length?RRL?in germination stage was the most sensitive to Cd stress.The RRL of 6 B.napus exhibited large varation under 30mg/L Cd stress.Finally,we developed a scientific and effective evaluation method for Cd tolerance in B.napus which phenotyped Cd tolerance by RRL under 30mg/L Cd stress.The concentration of 30mg/L Cd stress can inhibit the radicle length?RL?of all 472 B.napus cultivars.The RRL of 472 B.napus cultivars under Cd stress were exhibited extensive variation,and the maximum variation coefficient was 40.28%.The RRL were significantly different among different ecotypes,breeding periods and geographic origins.The RRL of semi-winter ecotype cultivars were significantly greater than other ecotype cultivars.Cultivars from 1950-1970 and 2001-2010 breeding period performed highest RRL,while accessions from 1981-1990 breeding period performed lowest RRL.And the Asia accessions were higher but not significant RRL than Europe and Oceania accessions.Consequently,we identified 12 excellent Cd tolerant germplasms and 13 Cd sensitive germplasms amount the 472 B.napus cultivars according the RRL index.2.Discovering the SNPs and genes associated with Cd tolerance by GWAS in B.napus.GWAS were performed with genotype data which obtained by 60 K SNP chip and Cd tolerance index RRL of472 B.napus cultivars.The results showed that 12 SNPs distributed in A03,A05,C03 and C06chromosomes?and C06 contained two SNP peaks?were associated with RRL.We also discovered a number of haplotypes included A03_Hap7,A03_Hap8,A05_Hap3,C03_Hap3,C06_1Hap1,C06_1Hap3 and C06_2Hap4 associated with Cd tolerance in these five association loci.And 6 of 10 Cd tolerance genes include BnaA3g21020 D,BnaC03g47480D,BnaC6g36720 D,Bna C6g36750 D,BnaC6g36930D and BnaC6g37320 D were verified by qRT – PCR amount the candidate Cd tolerance genes.3.Analysis of Heavy Metal transporting ATPase?HMA?genes for cadmium tolerance in B.napus.We identified the HMA genes in B.napus and discovered the Cd tolerance HMA genes in B.napus by homology seraching of the 8 known HMA genes of Arabidopsis thaliana.The results showed that a total of 23 HMA genes were identified in B.napus,belonging to P₁B-1,P₁B-2,P₁B-4 three subgroups.The 23 HMA genes anchored on 11 chromosomes with uneven distribution.and existing uneven duplication in B.napus genome.The new variant of GICCSME and GICCPSE in B.napus genomes which were different from the A.thaliana HMA genes functional site GICC?T/S?SE,might be also associated with new function of the corresponding genes.Finally,we identified 18 candidate Cd tolerance HMA genes,12 of them were homologous with AtHMA1-4,respectively.And 6 HMA genes were homologous with AtHMA5,but were significantly different from AtHMA5 in some characteristics..