Development of Single-Nucleotide Polymorphism (SNP) Markers in Sesame (Sesamum indicum L.).

Sesame (Sesamum indicum L.) is an important oilseed crop with considerable agronomic potential, widely cultivated across tropical and subtropical regions. Despite its substantial economic value, genomic research in sesame has progressed more slowly than in major staple crops. The advent of high-throughput single-nucleotide polymorphism (SNP) markers has markedly advanced sesame breeding by enabling the development of dense genetic linkage maps, genome-wide association studies (GWAS), and marker-assisted selection (MAS). This chapter presents detailed protocols for SNP discovery and validation in sesame using next-generation sequencing (NGS) approaches, specifically genotyping-by-sequencing (GBS) and double-digest restriction-site associated DNA sequencing (ddRAD-seq). It provides step-by-step methods for extracting high-quality DNA from tissues rich in polysaccharides and polyphenols, constructing sequencing libraries, performing bioinformatic filtering, and validating SNPs using Kompetitive allele specific PCR (KASP) and high-resolution melting (HRM) analysis. Particular emphasis is placed on troubleshooting common challenges related to DNA quality, restriction enzyme digestion efficiency, and the optimization of bioinformatic parameters to achieve a sufficient number of reliable SNP calls across the sesame genome.