An annual pulse crop cowpea (Vigna unguiculata (L.) Walp.), commonly named southern pea, is a nourishing constituent for the human diet and fodder. Gamma rays are a potent mutagenic agent to stimulate genetic variation with better characteristics, improving the yield relating traits in crops. Hence, the present study focused on exploring genetic variation between three generations in the mutant populations of cowpea through SCoT markers. The mutant populations of three successive generations, M1, M2 and M3, were induced by different doses [200, 400, 600, 800, 1000 and 1200 Gray (Gy)] gamma irradiation. The results depict that the quantitative characters were reduced by increasing the dosage of gamma irradiation in the M1 generation. In contrast, the second and third generation of plants showed a significant increase in yield and yield contributing traits than control and the maximum increase was noticed at 200 Gy and 400 Gy. Days to first flowering was delayed in irradiated plants than control of M1 generation. In contrast, in consecutive generations (M2 and M3), the early first flowering was noticed at 400 Gy and late flowering was observed at 800 Gy compared respectively to control and other doses. Seed yield per plant mean value was increased at 200 Gy in both generations (M2 and M3); it may produce new genotypes to desirable traits such as yield and quality. SCoT markers were used to explore genetic variation at the genomic level of mutant populations and screened with eight primers. Among them, seven primers showed amplification of 222 bands, in which 133 bands showed polymorphism. The polymorphic bands varied from 3.03–96.07%. The genetic variation, such as the number of different alleles (Na), effective number of alleles (Ne), Shannon’s information index (I), expected heterozygosity (He) and unbiased expected heterozygosity (uHe) showed an average value of 1.352 ± 0.092, 1.278 ± 0.027, 0.293 ± 0.023, 0.184 ± 0.016, and 0.194 ± 0.016, respectively. AMOVA depicted significant genetic variation between all generations and indicated a total of 95% within populations and 5% among population variation by the marker used. The present investigations prominently showed that the variations induced by gamma irradiation were inherited from successive generations of the improvement in cowpea quantitative traits. This investigation gives acceptable proof that the SCoT markers are a valuable tool to identify the genetic variation among the three generations of cowpea.
