Marker integration and development of Fluidigm/KASP assays for high-throughput genotyping of radish

Radish (Raphanus sativus L.) is a representative root crop of the Brassicaceae family and is important to the vegetable seed industry in East Asia. Due to its agronomic importance, various molecular markers, genetic maps, genomic resources, and genome assemblies of radish have been developed during the past decade. Marker integration and comparative mapping using these resources will accelerate genetic improvements in radish cultivars. With the goal of establishing a marker-based high-throughput genetic analysis tool, we integrated 3765 nonredundant genetic markers into the Rs1.0 reference genome and converted them into 1182 single nucleotide polymorphism (SNP) markers via whole-genome resequencing data of the mapping parents ‘WK10039’ and ‘WK10024’. A genetic map covering 721.3 cM with 768 framework loci was constructed by analyzing these SNP conversion markers in the F2 mapping population, which was composed of 93 individuals. Comparison of this map with the Rs1.0 reference genome and other linkage maps showed the physical and genetic correlations of the markers. To develop a high-throughput genotyping system for large accessions or populations with smaller numbers of SNPs, 674 Fluidigm and 68 kompetitive allele-specific PCR (KASP) markers were validated. Application of the 68 KASP assays to 127 commercial cultivars enabled successful identification and classification of genotypes; 11 KASP markers constituted the minimum marker set. The SNP markers used to construct the genetic maps will be a useful resource in research on radish and should lead to low-cost, accurate, and high-throughput genotyping platforms.