Optimization of RAPD-PCR conditions for the study of genetic diversity in Nepal’s <i>Swertia chirayita</i> (Roxb. Ex Fleming) H. Karst

Abstract

Of the 30 species (including five varieties) of the genus Swertia in Nepal, nine have been reported to possess medicinal properties. Among these, S. chirayita is the most valuable species, with high demand in domestic and international markets. Nepal's S. chirayita and related species are being recklessly exploited for commercial purposes. Two problems that have emerged with this lucrative market are (a) adulteration and fraudulent labeling of S. chirayita, and (b) depletion of S. chirayita and allied species from their natural habitats. To address the problem of adulteration and conservation, we studied molecular genetic diversity in S. chirayita populations and developed a molecular diagnostic tool for the purposes of authentication. We studied intra-specific genetic diversity in S. chirayita using Polymerase Chain Reaction (PCR)-based Random Amplified Polymorphic DNA (RAPD) technique. As a preliminary step, we identified optimal RAPD-PCR reaction and cycling conditions by varying PCR reaction parameters such as concentration of template DNA, MgCl 2 , dNTPs, primer, Taq DNA polymerase and RAPD-PCR programs. The optimized PCR reaction and cycling conditions were then used in subsequent RAPD profiling experiments for the study of genetic diversity within S. chirayita populations from various geographical locations. Genetic diversity characterization of S. chirayita populations at the molecular level would furnish information with significant applications in the conservation and sustainable utilization of S. chirayita and its allied species in Nepal.

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

“…For the assessment of genetic diversity, a variety of (PCR)-based molecular marker techniques are now available. Rand Amplified Polymorphism DNA (RAPD), Inter-Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR) markers are the most widely used molecular markers in diversity testing (Shrestha et al, 2010a).…”

Genetic diversity in Nepalese population of Swertia chirayita (Roxb. Ex Fleming) H. Karst based on inter-simple sequence repeats (ISSR) markers

“…The result indicated that ITS fragment can be used in identification as a barcoding marker for Swertia. The optimization of the RAPD-PCR conditions (Shrestha et al, 2011) and genetic diversity of 34 accessions of S. chirayita including six outlier species were assessed in Nepal (Shrestha et al, 2013). Of the total 285 amplified bands scored for S. chirayita, 263 (92.8%) were polymorphic.…”