DNA isolation and optimization of PCR protocol for ISSR analysis of Girardinia diversifolia: A medicinal and economic plant species from Nepal Himalaya

Girardinia diversifolia (Link) Friis is a perennial herb commonly known as Himalayan giant nettle which belongs to the family Urticaceae. The plant has cultural, medicinal and economic importance. The plant contains high concentration of polysaccharides, polyphenols and secondary metabolites which obstructs the process of isolation of the Deoxyribonucleic Acid (DNA) and inhibits downstream Polymerase Chain Reaction (PCR) amplifications. This study protocol developed a DNA extraction protocol from leaf-tissue based on the Cetyltrimethylammonium bromide, and optimized the PCR protocol for Inter Simple Sequence Repeat (ISSR) analysis. Genomic DNA extraction process was conducted using modified Doyle and Doyle method to obtain good quality DNA. The method yielded 445 ng/µL of DNA, where the purity ranged from 1.8-2.0 indicating minimum contamination of metabolites. The optimum condition for ISSR analysis was established using 4 mM MgCl2 , 0.6 mM dNTPs, 2.0 U  Taq polymerase, 50 ng template DNA, and 0.7 µM primer. PCR program was optimized in the sequence of denaturation at 94°C for 3 min, subsequently followed by 45 cycles at 94°C for 30 s, annealing temperature at 45°C for 30 s, extension at 72°C for 2 min, and final extension at 72°C for 10 min. The modified technique was found to be ideal for isolation of genomic DNA and optimization of PCR process for ISSR analysis of G. diversifolia. The results of the research are beneficial for future molecular characterization and genetic diversity analysis of allied taxa.   Key words: Girardinia diversifolia, Himalayan nettle, DNA isolation.