<B>PCR Optimization Prior to Genetic Diversity Assessment of Sesame (Sesamum Indicum L.) Genotypes Using Inter-Primer Binding Site (IPBS) Markers</B>

Abstract

This study aimed to accurately and reliably determine the genetic diversity among sesame (Sesamum indicum L.) genotypes, which is an important oil crop. To achieve this, Polymerase Chain Reaction (PCR) conditions based on Inter-Primer Binding Site (IPBS) molecular markers were optimized to reveal genetic variation, which forms the basis of plant breeding programs. For the methodology, DNA was isolated from fresh sesame leaves grown under controlled conditions using the CTAB method and analyzed from 50 local sesame lines. Since PCR success is directly dependent on the specificity of the primers and reaction parameters like temperature, the gradient temperature PCR method was applied using 22 different iPBS primers to determine the optimal annealing temperatures. According to the findings, 20 out of the 22 primers successfully generated polymorphic bands, revealing genetic diversity. Determining the optimal PCR conditions was critical for identifying the binding temperature at which iPBS primers exhibited the highest polymorphism. For example, Primer 2277 showed high amplification and activity at 47.6 and 50.9°C, while Primer 2218 was highly active at 50.9°C. This optimization establishes a precise molecular foundation that will contribute to future sesame breeding programs.