Development and validation of diagnostic SNP markers for quality control genotyping in a collection of four rice (Oryza) species

Morphological identification of closely related rice species, particularly those in the Oryza AA genome group, presents major challenges and often results in cases of misidentification. Recent work by this group identified diagnostic single nucleotide polymorphic (SNP) markers specific for several rice species and subspecies based on DArTseq next-generation sequencing technology ("DArTseq"). These SNPs can be used for quality control (QC) analysis in rice breeding and germplasm maintenance programs. Here, we present the DArTseq-based diagnostic SNPs converted into Kompetitive allele-specific PCR (KASPar or KASP) assays and validation data for a subset of them; these can be used for low-cost routine genotyping quality control (QC) analysis. Of the 224 species/subspecies' diagnostic SNPs tested, 158 of them produced working KASP assays, a conversion success rate of 70%. Two validation experiments were run with 87 of the 158 SNP markers to ensure that the assays amplified, were polymorphic, and distinguished the five species/subspecies tested. Based on these validation test results, we recommend a panel of 36 SNP markers that clearly delineate O. barthii, O. glaberrima, O. longistaminata, O. sativa spp. indica and japonica. The KASP assays provide a flexible, rapid turnaround and cost-effective tool to facilitate germplasm curation and management of these four Oryza AA genome species across multiple genebanks.