Development and application of KASP assays to differentiate between Sorghum bicolor, halepense, and their hybrids

<h4>Background</h4>Sorghum bicolor and Sorghum halepense can readily hybridize, creating difficulty in identification. No genetic tools exist to accurately distinguish S. bicolor, S. halepense, and their hybrids. Detecting hybridization is essential to monitor crop-to-weed introgression. This study utilizes a single nucleotide polymorphism (SNP) in the internal transcribed spacer region between S. bicolor and S. halepense. This SNP was utilized in Kompetitive allele-specific PCR (KASP) assay to identify S. bicolor, S. halepense, and their hybrids.<h4>Results</h4>KASP assays were successful in accurately differentiating between S. bicolor, S. halepense, and their hybrids. The KASP assay performed as well as Oxford Nanopore sequencing for measuring SNP frequency and thus is a perfect proxy for genotyping. Greenhouse crosses confirmed crop-to-weed introgression, with S. halepense being more receptive to interspecific pollen. Known and unknown samples assayed displayed misidentification in germplasm lines and significant hybrid frequency in naturally occurring biotypes. Synteny analyses revealed duplications of the ITS region in S. halepense.<h4>Conclusion</h4>We developed a novel KASP assay targeting a conserved SNP that accurately distinguishes between S. bicolor, S. halepense, and their hybrids. This assay was validated through Oxford Nanopore sequencing, greenhouse crosses, and diverse germplasm and natural collections. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.