A simple and cost-effective SNP genotyping assay for marker-assisted selection of wilt resistance in castor breeding

Castor is an industrial crop with diverse applications, including biodiesel production. However, its cultivation faces significant challenges from both biotic and abiotic stresses, with Fusarium wilt being a major disease. SNP markers linked to Fusarium wilt resistance and genotypic assays based on Kompetitive Allele Specific PCR (KASP™) method are available in castor. However, the cost of introducing resistance genes into preferred lines through marker-assisted selection (MAS) is prohibitive, particularly when using KASP based genotyping. In this study, we developed an agarose-based Mismatch Amplification Mutation Assay (Agarose-MAMA) as a cost-effective SNP genotyping method for selecting wilt resistance in castor breeding programs using standard laboratory equipment and reagents. We selected two SNP markers, Rc_29806–125126 on chromosome 6 and Rc_29706–482910 on chromosome 8, which are linked to wilt resistance in castor for designing and optimizing the MAMA assay. A comparative analysis of KASP and MAMA assays demonstrated similar performance in allele discrimination and genotyping accuracy. Furthermore, we successfully applied the Agarose-MAMA assay for MAS of wilt resistance in three breeding populations segregating for this trait. This approach circumvents the need for expensive equipment for SNP genotyping, thereby enabling broader adoption in research settings with limited resources. Overall, this study highlights the potential of Agarose-MAMA assay as a practical tool for SNP-based MAS, not only in castor but also in other crops. • Protocol for ‘Mismatch Amplification Mutation Assay’ (MAMA) was standardized. • Agarose-MAMA assays for three SNP loci linked to wilt resistance in castor were developed. • The results of MAMA assays were comparable with KASP assays. • Agarose-MAMA assays have potential as a practical tool for SNP-based MAS in plants.