Gamma ray-induced phenotypic variability and mid-density SNP-based mutation characterization in forage sorghum (Sorghum bicolor L. Moench) genotype IS 33,871

Sorghum is an important dual-purpose cereal crop cultivated for both grain and forage, particularly in arid and semi-arid regions. However, limited genetic variability constrains the improvement of forage biomass and related agronomic traits. This study evaluated the effectiveness of gamma ray-induced mutagenesis for generating phenotypic and molecular variability in forage sorghum genotype IS 33,871. Four sorghum genotypes were initially assessed for radiosensitivity to estimate the 50% growth reduction dose (GR 50 ), and the relatively tolerant genotype IS 33,871 was selected for bulk irradiation at 300 Gy. Evaluation of the M 2 population identified a chlorophyll mutation frequency of 1.15%, confirming successful mutation induction. Substantial variation was observed for agro-morphological and biomass-related traits. Biomass fresh weight, biomass dry weight, stem girth, disease score, and panicle length exhibited high broad-sense heritability coupled with high genetic advance, indicating strong potential for effective phenotypic selection. Correlation, principal component, hierarchical clustering, and standardized deviation (Z-score) analyses identified elite mutants combining superior biomass accumulation, favourable plant architecture, and reduced disease severity. Mid-density SNP genotyping of 270 M 2 individuals identified 390 homozygous mutant-specific SNPs representing 115 polymorphic loci across 16 mutants. The induced mutation spectrum was dominated by transition substitutions, although substantial variation in mutation load was observed among individual mutants. Functional annotation associated the identified SNPs with genes involved in growth, development, stress signalling, metabolism, and cellular regulation. These findings demonstrate that integrating phenotypic evaluation with SNP-based molecular characterization provides an effective strategy for identifying valuable genetic resources and accelerating forage sorghum improvement through mutation breeding.