KASP Markers for Identifying Roselle (Hibiscus sabdariffa L.) Key Varieties Based on Genetic Polymorphisms Revealed by ddRAD-Seq

Variety characterization is crucial in the seed trade, particularly for protecting variety rights. However, the identification of roselle (Hibiscus sabdariffa L.) varieties, known for their beneficial effects on human health and high processing potential, has traditionally relied on morphological traits due to limited genetic information. To investigate genetic polymorphisms of roselle germplasms and to develop breeder-accessible genotyping tools, this study first phenotyped a roselle collection from diverse geographical origins for the selection of core varieties, and then utilized double-digest restriction-associated DNA sequencing (ddRAD-seq) to identify 53,746 single nucleotide polymorphisms (SNPs) across 17 core varieties. Cluster analysis of the SNP data effectively grouped varieties with similar genetic backgrounds. From this genetic information, we selected nine SNPs as a toolkit to simplify core variety discrimination. These SNPs were then converted into breeder-friendly kompetitive allele-specific PCR (KASP) markers, facilitating the classification of an additional 54 roselle accessions. In conclusion, this research contributes novel insights into the genetic relationships among roselle varieties, and establishes a robust framework utilizing ddRAD-seq and KASP markers for improved genetic resource identification and application in breeding programs.