| Cadmium is a potentially toxic heavy metal.Rapeseed(Brassica napus L.),a vegetable and oilseed crop,might also be a Cd hyperaccumulator,but there is little information on this trait in rapeseed.We evaluated Cd accumulation in different oilseed cultivars and employed a genome-wide association study(GWAS)to identify quantitative trait loci(QTLs)related to Cd accumulation.A total of 419 B.napus accessions and inbred lines were genotyped with a 60 K Illumina Infinium SNP array of Brassica.RNA-seq is applied to study the gene expression of B.rape,B.oleracea and new-type B.napus after exposing to Cd.The DEGs were compared with the candidate gene detected by GWAS,and the potential candidate genes related to Cd accumulation were screened out.The main results of this study are as follows:1.The 419 rapeseed accessions were cultivated in 5 mg L-1 Cd2+ in the hydroponic solutions at the seedling stage.Shoot and root Cd concentrations and Cd translocation were compared after 3-weeks.The broad-sense heritability(h2)values for Cd concentration in shoot and root,and Cd translocation were 85.3%,78.1%,and 69.8%,respectively.Unimodal distribution patterns were observed for all traits among these accessions in the 3 environments independently.The results indicated that these Cd-related traits for a given genotype are stable.2.There were large genotypic variations in the Cd concentration and translocation across the 3 environments.The average root Cd concentration ranged from 1689.99 ± 192.31 to 1882.31 ± 238.39 μg g-1 DW.The average shoot Cd concentration ranged from 210.65 ± 20.9 to 257.46 ± 22.1 μg g-1 DW.The average Cd translocation coefficient ranged from 0.37 ± 0.041 to 0.40 ± 0.04(Table 1).3.We discovered 98 significantly(-Log(P)> 4.0 or P < 0.0001)associated SNPs of shoot and root Cd concentration and Cd translocation.32.7%(32/98)of the SNPs were verified in more than 1 environment(including BLUP),which indicates high reliability.The 98 significant SNPs constituted 35 QTLs and explained 3.49-7.57% of the phenotypic variation in oilseed varieties.4.We identified Cd-related orthologous genes of A.thaliana in B.napus,which located within 500 kb of each SNPs.We obtained 32 candidate Cd accumulation-related genes compared to the B.napus reference genome.The physical distance between the significant SNPs ranged from 0.33 kb to 497.97 kb.totally,7,10 and 15 genes were identified for shoot Cd concentration,root Cd concentration,and Cd translocation,respectively.Candidate genes were found,orthologous to NRAMP6,IRT1,CAD1,PCS2 and others.5.RNA-seq were performed with root samples of B.campestris,B.oleracea and newtype B.napus after cadmium exposing,109 candidate genes were found.Among them,67 from newtype B.napus,33 from B.campestris and 29 from B.oleracea.Some of them were homologous genes to IRT1,PCR1,HMA1,HMA4,CAD1 and the glutathione-S-transferase family including GSTF7,GSTAU8,GSTAU13 and GSTAU19 in Arabidopsis thaliana.6.The differentially expressed genes from RNA-seq were aligned with the candidate regions of GWAS.A total of 74 DEGs located into the region.3 genes response to cadmium stress.Bna A09g29540(GSTU13)is a candidate gene associated with SCC traits;Bna C07g39340(FSD1)is a candidate gene associated with RCC traits;Bna A09g32780 is a CT related candidate gene encoding sucrose-6F-phosphate phosphorylase.There were different copy point to the same gene such as IRT1,CAD1,NRT1.8,ARF1,ASP1,FSD1,GAPC2,GSTF7,GSTU13,GSTU19 and PCK1 by GWAS and RNA-seq.These genes may play a role in cadmium accumulation in rapeseed.Some candidate genes related to cadmium accumulation were found by RNA-seq and GWAS in this study.Some of them were validated in both methods.These genes will be helpful for cloning the genes related to cadmium accumulation in rapeseed in future. |
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