Optimization of PCR protocol for ISSR marker based genetic diversity assessment of acid lime [Citrus aurantifolia (Christm.) Swingle] germplasm in Eastern Nepal

Abstract

Acid lime [ Citrus aurantifolia (Christm.) Swingle] is a valuable commercial fruit crop grown in Nepal's Terai to high hills which has high economic, cultural and medicinal importance. Due to low quality planting materials and poor orchard management, production and productivity of acid lime are extremely low in Nepal. The present study aimed at optimization of Inter-Simple Sequence Repeat (ISSR)-polymerase chain reaction (PCR) reaction and cycling conditions for PCR amplification and genetic diversity assessment of acid lime cultivars from eastern agro-ecological zone, Nepal. Five different parameters [ viz. Template DNA, MgCl 2 , Deoxynucleotide triphosphate (dNTPs), Primers and Taq DNA polymerase] were used in the ISSR-PCR reaction optimization. Moreover, 4 different cycling conditions were assessed for the determination of the optimum range for ISSR-PCR profiling. The optimized PCR reaction conditions were found to be 25 ng DNA, 3.0 mM MgCl 2 , 0.4 mM dNTPs, 0.4 µM Primers and 1.5 Unit Taq DNA polymerase and best PCR cycling condition consisted of initial denaturation of 94°C for 5 min followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 45 s, elongation at 72°C for 2 min and final elongation of 7 min at 72°C. The results from this study were successfully used for ISSR-PCR based genetic diversity assessment of Nepalese acid lime genotypes to find out the elite cultivars of Eastern Nepal.