Late Leaf Spot (LLS), caused by Nothopassalora personata, is a major fungal disease that significantly reduces peanut (Arachis hypogaea L.) production, particularly in Sub-Saharan Africa, including Uganda. This disease lowers the quality of the produce and leads to severe yield losses. Despite control measures, genetic resistance in local peanut cultivars remains limited, and effective management strategies are not well established. Therefore, genetic improvement using LLS resistance is crucial for enhancing peanut productivity. This study aimed to characterize and profile resistance of 25 ICRISAT peanut genotypes, introduced from Malawi and evaluated under Ugandan environmental conditions, using Simple Sequence Repeat (SSR) markers. Genomic DNA was extracted from leaf samples using an optimized Cetyltrimethylammonium Bromide (CTAB) protocol, quantified via spectrophotometry, and amplified by Polymerase Chain Reaction (PCR). Eight SSR markers previously associated with LLS resistance were used to assess their ability to differentiate resistant genotypes. Results revealed that five markers (PM375, PMc588, pPGPseq2B10, pPGPseq2F5, pPGseq17F6) were strongly correlated with LLS resistance, while one marker (pPGseq13A7) was monomorphic. Nine genotypes (36%) carried at least one resistance allele, with five genotypes (20%) classified as resistant, containing four or more resistance alleles, including ICGV-SM 16605, ICGV-SM 16615, ICGV-SM 16602, ICGV-SM 16613, and ICGV-SM 16637. Multivariate analyses, including scatter plots, Unweighted Pair Group Method with Arithmetic Mean (UPGMA) phylogeny, and heat-map clustering, clearly separated genotypes into resistant, tolerant, and susceptible groups. This study highlights the potential of Marker-Assisted Selection (MAS) in identifying and utilizing resistant genotypes for breeding LLS-resistant peanut cultivars in Uganda. Identifying these resistant genotypes offers a valuable resource for improving disease resistance and enhancing sustainable peanut farming production in Uganda and other regions of Sub-Saharan Africa.
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