Genetic diversity of hot pepper (Capsicum annuum L.) germplasm from a selected site in Ethiopia using inter simple sequence repeat (ISSR) marker

Abstract Hot pepper ( Capsicum annuum L .) is a major vegetable crop worldwide. Identifying C. annuum germplasm diversity is vital for breeding, provides alleles for better yield, fruit size, disease resistance, and stress tolerance. Molecular marker provides a more stable, environment-independent approach for assessing genetic diversity compared with morphological and biochemical markers. Inter simple sequence repeat (ISSR) markers were selected due to their high reproducibility, cost-effectiveness, and ability to detect high polymorphisms without requiring prior genome sequence data. This study was assessed the genetic diversity of 53 Ethiopian C. annuum accessions and cultivars using ISSR markers. A total of 53 hot pepper accessions and cultivars were sourced from Ethiopian Biodiversity Institute (EBI). Genomic DNA was extracted using a slightly modified CTAB method. The quality and quantity of extracted genomic DNA was assessed by agarose gel electrophoresis and NanoDrop spectrophotometry. Twelve ISSR primers were screened; four primers (UBC-810, UBC-812, UBC-880, and UBC-881) with the highest polymorphism and reproducibility were selected for analysis. PCR was performed using a Biometra 2000 T3 master cycler with 25 µL reaction volume. The bands were scored as binary data: “1” for presence and “0” for absence. Population genetic diversity and structure were analyzed using standard clustering and population-genetic software. The primers UBC-810, UBC-812, UBC-880, and UBC-881 generated clear and reproducible amplification patterns (6–8 bands/primer). All 29 scorable bands were 100% polymorphic. The Mirab Gojjam population shows the highest genetic diversity (H = 0.31), indicating a richer gene pool and broader genetic base than the other populations and cultivars. Lowest genetic diversity occurred in Semen Gondar (H = 0.11), and cultivar groups (H = 0.07), likely due to domestication. Most of the ISSR diversity was partitioned among individuals within populations (55.30%), with the remaining variation distributed among populations (44.70%), indicating substantial population differentiation. ISSR markers are effectively in detecting genetic diversity within and among hot pepper ( C. annuum ) accessions. The result of this study identified key populations for conservation and provided baseline data to support breeding programs aimed at improving C. annuum varieties using diverse germplasm. Future studies should integrate expanded marker paneles, co-dominant loci, broader samoling, and environmental variables to resolve C. annuum genetic structure and better inform conservation and breeding.