Genome wide association study of seed phytate concentration and content in Brassica napus under contrasting phosphate conditions

Objectives The study was conducted to unravel the significant SNP (single nucleotide polymorphism) loci, candidate genes and favorable haplotypes affecting the seed phytate concentration (PA_Conc) and seed phytate content (PA_Cont) in oilseed rape (Brassica napus L.) under contrasting phosphorus (P) application rates, in order to provide elite genes for the breeding of low phytate varieties of B. napus.

Methods Field experiments were conducted to determine the PA_Conc and PA_Cont in the seed of an association panel of 403 B. napus accessions under sufficient (P₂O₅, 90 kg/hm²) and deficient (P₂O₅, 0 kg/hm²) P conditions. The whole genome sequencing (WGS) technology was used to develop more than 5.3 million SNPs in the association panel. The genome-wide association study was conducted to identify the significant SNPs associated with seed PA_Conc and PA_Cont by GLM (general linear model) and MLM (mixed linear model) using TASSEL 5.0 software, and the candidate genes were predicted. Bcftools was used to extract the SNP in the BnaA07.MRP13, and Haploview 4.2 was used for haplotype analysis.

Results In the association panel, the PA_Conc varied from 17.52–81.32 mg/g and PA_Cont in 5 seeds varied from 0.30–0.99 mg at sufficient P supply, while the two parameters varied from 17.47–49.05 mg/g and from 0.33–0.85 mg at P deficient supply. Compared with those at the sufficient P supply, the average seed PA_Conc and PA_Cont of the association panel at deficient P supply reduced by 13.5% and 10.8%, respectively. Fifty-six SNPs were identified to be significantly associated with seed PA_Conc and seed PA_Cont in B. napus by GLM, which were distributed on 17 of the 19 chromosomes (excluding A08 and C03) and explained the phenotypic variation of 7.33% to 14.28%. Chromosome A03 had the largest number of significant SNPs (9). Twenty-three SNPs were identified to be significantly associated with PA_Conc and PA_Cont in the seed of B. napus by MLM, and they were distributed on eleven chromosomes including A03, A05, A06, A07, A09, C01, C02, C04, C05, C06, and C07, and they explained the phenotypic variation of 9.60% to 14.28%. Among them, 8 SNPs were identified on chromosome A03. Fourteen candidate genes, such as MRP, PLC, PIP5K, SULTR4, in phytate synthesis pathway of Arabidopsis, were identified by the analysis of the genes in the interval between 300 kb up and downstream of the significant SNP. Candidate gene association study and haplotype analysis of BnaA07.MRP13 determined a high phytate haplotype “BnaA07.MRP13ContHap1” (TTTA) and a low phytate haplotype “BnaA07.MRP13ContHap2” (CCCT). Eighteen percent of the varieties (72/403) in the association panel were low phytic acid haplotypes, with an average PA_Cont in 5 seeds of 0.56 mg, which was significantly lower than that of high phytate haplotypes (0.62 mg).

Conclusions Seed PA_Conc and seed PA_Cont in the association panel of B. napus under both sufficient and deficient phosphorus supplies had extensive genetic variation. The GLM and MLM models detected 56 and 23 SNPs significantly associated with seed PA_Conc and PA_Cont in B. napus, respectively, and 14 candidate genes were predicted to be associated with the PA_Conc and PA_Cont. The high and low phytate haplotypes associated with BnaA07.MRP13 gene will be helpful for the genetic improvement of the seed PA_Conc and PA_Cont in B. napus.