Genetic Diversity analysis of Swertia Chirayita ( Robx. Ex FLEM) Karst Populations of nepal using Polymerase Chain Reaction based Random Amplified Polymorphic DNA ( RAPD- PCR) Technique

Abstract

Among the 150 species of Swertia distributed globally, 30 species have been reported to be found in Nepal. Of these 30 species, nine species are reported to have the medicinal properties. Swertia chirayita is the most valuable species, although other species are also being traded as adulterants. Owing to its high medicinal importance and demand, Swertia chirayita populations in wild habitats are being overexploited beyond its regeneration capacity. Present molecular investigation was undertaken in order to 1) understand the level of genetic diversity within and between various S. chirayita populations collected from various districts of Nepal using Random amplified polymorphic DNA (RAPD) technique and 2) generate species specific/population specific RAPD markers for authentication purposes. Thirty four accessions of S. chirayita along with the 6 allied species were analyzed using 26 RAPD primers. Of the total 285 amplified bands scored for S. chirayita, 263 bands (92.28%) were polymorphic. Seven primers revealed nine RAPD markers that were specific to various S. chirayita populations. Genetic relationship among various S. chirayita accessions was performed by using binary data matrix created for presence or absence of RAPD bands. Cluster analysis using RAPD (qualitative) data was performed in NTSYS statistical package by pair wise similarity matrices generated by Simple Matching, Jaccard’s and Dice coefficient of similarity. From comparisons of similarity matrices and phenograms, Jaccard’s coefficient of similarity in constructing phenogram using UPGMA module was revealed to be the best for deducing the genetic relationship among S. chirayita accessions. It revealed two major clusters with distinct delineation of allied species out of the clusters reflecting rich genetic diversity among populations under study. Principal Coordinate Analysis (PCO) substantiated the results of phenograms. The genetic interrelationship between the populations were clarified by the assessment of Nei’s genetic identity and distance using POPGENE 1.32 that revealed S. chirayita populations of Sankhuwasabha and Terathum as closest (0.9489, identity) and Kaski and Sankhhuwsabha as most distant (0.7078, identity). Assessment of genetic variation within populations estimated with Percent Polymorphism, Shannon’s diversity index and Nei’s gene diversity reflected the highest within diversity for the population of Nagarjun > Phulchowki> Terathum> Sankhuwasabha > Kaski. The mean genetic differentiation (GST) between populations over all loci was observed to be 0.5929 with low average gene flow (Nm) of 0.3433 reflecting genetic purity of this vulnerable species. Survival of a species depends on the maintenance of genetic variability within and among population that help to adapt and acclimatize in the new selection pressures brought about by environmental changes. It can be concluded that this genetic diversity observed according to different geographical gradient needs to be preserved and sustainably utilized based on their chemical attributes.

Key words: Swertia chirayita, RAPD, genetic diversity, population genetic, molecular marker