M.Tech in Biotechnology, IIT Mandi, Kamand campus, Mandi 175005 (Himachal Pradesh)
Dr. Moinak Banerjee
Scientist F, Human Molecular Genetics, Rajiv Gandhi Centre For Biotechnology, Thiruvananthapuram 695014
Insects account for a large proportion of the biodiversity on the planet with over 900,000 described and many more yet to be known. The insect Hyblaea peura Cramer, commonly called as teak defoliator, is a pest of major concern as it is involved in complete defoliation of teak trees during the early part of the growing season, which leads to huge loss of timber. Molecular markers are essential for categorizing living creatures and the fingerprints obtained are helpful in differentiating them morphologically; they are also used to assess intra- and inter-species genetic diversity. Genetic diversity refers to the variation of genes within a species and between species in a population. Ultimately, genetic diversity can be attributed to changes in the sequence of bases in the DNA. Sequence alterations in a gene can result from mutation, natural selection or even by sexual reproduction that causes recombination in organisms. Selection acts on a pool of current genetic variation and consequently facilitates evolution. The species level is generally regarded as the most natural choice for considering genetic diversity. In the present study, several individuals of the Hyblaea peura were investigated with the aim of detecting intra-species genetic diversity. These were examined with the use of Inter-simple Sequence Repeat (ISSR), random amplified polymorphic DNA (RAPD) and DNA bar-coding methodologies. The objective of the study was to develop a system of PCR-based markers for assessment of genetic diversity in population genetic studies and establish a DNA barcode of Hyblaea peura. ISSR and RAPD are the key markers to establish diversity of the unknown species chosen for this study as they don’t require prior knowledge of sequence data. Insect DNA samples were isolated and quantified by Nanodrop and PCR protocol was optimized for ISSR and random primers by trouble shooting with annealing temperature reproducibility. The optimized protocol was implemented for 11 randomly selected samples of Hyblaea puera and bar-coding was done to establish the diversity of unknown species. Further the genetic diversity was assessed by population genetic tools such as POPGENE and TFPGA. DNA bar-coding showed that the samples belonged to insect as they showed significant sequence identity with cytochrome oxidase I gene of insects.
Keywords: ISSR, Bar-coding, RAPD, Population genetics, PCR, Genetic code