Genetic Diversity and Relationships Among Newly Developed Cowpea Mutant Lines Using SSR Markers and Agro‐Morphological Traits

ABSTRACT Cowpea ( Vigna unguiculata [L.] Walp.) is a drought‐resilient and protein‐rich legume of major importance in arid and semiarid regions, including Eswatini, where it contributes to food, feed, and nutritional security. The objective of this study was to assess the genetic diversity and relationships among 30 cowpea genotypes, comprising 24 advanced mutant lines, three founder parents, and three local checks, using polymorphic SSR markers and agro‐morphological traits to identify promising genotypes for breeding and production. Genotyping with 10 SSR markers detected 54 alleles across all genotypes, with alleles per locus ranging from 3 to 8 and polymorphic information content values between 0.33 and 0.78. The most informative markers—SSR6800, SSR6844, and SSR6239—recorded the highest allelic diversity and discrimination power. Field evaluation across two sites revealed significant ( p < 0.05) genotype × location interactions for phenotypic traits. Elite mutant lines NKL9P7, BRR4P11, SHR9P5, and NKL9P7‐2 exhibited higher grain yields ranging from 2255.8 to 3158.8 kg/ha. Cluster analysis based on phenotypic traits grouped genotypes into two major clusters, whereas SSR markers resolved them into three clusters. A combined tanglegram indicated differences in grouping patterns, although SHL2P4 and SHL3P7‐2 consistently maintained stable positions, underscoring their potential as reference genetic stocks. The highest genetic distances were observed among NKL9P7‐2, SHR3P4, SHL7P1, NKR193, NKR2P9, NKR10P5, and BRR11P2, suggesting their suitability as parents for hybridization and genetic improvement. Overall, this study demonstrates substantial genetic and phenotypic diversity among cowpea genotypes in Eswatini, providing a strong foundation for targeted breeding and yield improvement.