Optimization of PCR protocol for SSR marker based genetic diversity assessment of Citrus genus in Nepal

The Citrus genus thrives in a wide variety of landscapes in Nepal. Understanding the genetic diversity of these cultivars can be a critical stride towards conservation and sustainable agricultural practices. The present study revolves around the optimization of the PCR protocols for Simple Sequence Repeats (SSR) marker-based genetic diversity assessment with the aim to facilitate the study of understanding the genetics of Citrus species. To achieve the aim, the PCR protocol was checked for the optimum result with the varying concentrations of DNA, MgCl2, primer, dNTPs, and Taq polymerase and different cycling conditions. The Cetyltrimethyl ammonium bromide (CTAB) extraction method was used to extract DNA from the ground preserved leaves of the selected sample, and the SSR-PCR products were visualized on a 4% agarose at 70 V for one and a half hour. The optimized PCR reaction conditions that gave clear, scorable, and sharp bands were found to be 25 ng DNA, 3.0 mM MgCl2 , 0.2 mM dNTPs, 0.4 µM primers, and 2 units Taq DNA polymerase, and the best PCR cycling conditions consisted of initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at primer-specific temperature (47-61°C) for 1 minute, elongation at 72°C for 30 s, and final elongation at 72°C for 4 minutes. Finally, Citrus species genotypes were successfully assessed for the genetic diversity using thus optimized SSR-PCR protocol, which will help in finding genetically diverse superior cultivars for off-season production.