Molecular ecology of endemic lower vertebrates of Western Ghats

This include development of species specific microsatellite markers, PCR optimization and analysis of these markers to investigate general population genetic questions of endemic lower vertebrates.

Antimicrobial peptides from frog skin secretions

Amphibian granular gland transcriptomes persist in stimulated skin secretions opens up new possibilities in highthroughput peptidome analysis and characterization. It is established that the organization of amphibian skin peptide biosynthetic precursors is both highly ordered and highly conserved between different taxa. In general the hydrophobic aminoacid residue rich signal peptide is followed by a region rich in aminoacid residues, a typical dibasic propeptide convertase processing sites and finally a single copy of bioactive peptide. Many variations exists from these general scheme such as processing site witin the acidic domain, C-terminal extensions following the mature bioactive peptide, glycine amide donors flanking the C-terminals and tandem repeat sequences found in the biosynthetic precursors of skin opiate peptides and bradykinins. However, alignment of either nucleic acid sequences or open reading frame aminoacid sequences of cloned biosyntheitic precursor transcripts from many species of amphibians, reveals regions of both highly conserved nucleic acid and aminoacid sequences specifically within signal peptide domains but also nucleic acid sequence domains within both 3’ and 5’ nontranslated regions. This has permitted design of general PCR primer sets that are either class, family, or genus specific within the amphibians. Thus armed with a skin secretion-derived cDNA library from an unstudied species and a general primer set, one can shot gun amplify and clone secretion peptide transcripts in the total absence of any specific peptide primary structural data. These techniques have been tried and obtained 21 new peptides having broad spectrum antimicrobial activity. Their charascterisation and structure function studies are in progressing.

DNA Barcoding of amphibians of Western Ghats

DNA barcoding is a standardized approach by using a short DNA sequence from a uniform locality of the genome that can be used to help discover, characterize, and distinguish species, and to assign unidentified individuals to species.The present work aims to explore the potential of CO1 mtDNA sequences for differentiating species. mtDNA sequences of CO1 of ten well known amphibian species were used initially to compare the suitability of a marker for barcode purposes. Protocols followed in the present work include DNA isolation, PCR amplification and sequencing of CO1. For DNA isolation, we have standardized a non-invasive protocol by dissecting less than 25 mg tissue from the toe of the adult frog and tail fin from the tadpole. Universal primers were used to amplify the mitochondrial genes. Sequences were subjected to NCBI BLAST search for obtaining nucleotide-nucleotide homology and species confirmation. Sequences were aligned and constructed phylogenetic trees by using Bioedit and MEGA softwares. Neighbor joining(NJ)) method was employed to construct phylogenetic trees. We found that CO1 gene is best suitable for differentiating amphibian species in Western Ghats. Based on this, we have generated CO1 gene sequences of amphibians of Western Ghats and aligned against the reference sequences and identified their taxonomy. Presently we have barcoded 18 species including the endangered frog Ansonia ornata from western ghats .

Conservation genetics of Indirana frogs of Western Ghats biodiversity hotspot (Indo-Finnish Collaborative project)

The aim of this project is to increase our understanding of the biodiversity and processes generating it on one hand, and to produce information useful for delimiting management and conservation units, including possible identification of yet unknown (i.e. cryptic) species, on the other. These goals will be targeted focusing on diversity mapping and population genetics of the amphibians of the Western Ghats (India), one of the most important biodiversity hotspots of the world. By developing novel microsatellite markers of frogs in endemic genus Indirana, we will investigate the genetic relationships and diversity among and between different populations and species in different parts of Ghats. Apart of seeking genetically unique lineages and signatures indicative of genetic problems (i.e. inbreeding, loss of variability due to drift), we will also resolve the phylogenetic (evolutionary) relationships between different Indirana taxa and populations with the aid of DNA-sequence and microsatellite data. In addition, the degree of population differentiation attainable due to neutral evolutionary processes (i.e. drift, migration) as measured by FST-index in neutral microsatellite loci will also be studied. Apart of producing results advancing our understanding of fundamental scientific issues, the studies on diversity of frogs have potential to pave road for making conservation strategies.