Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in Brassica rapa

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.