DNA BARCODE DATABASE GENERATION AND PHYLOGENY ANALYSIS OF THE LOACHES OF WESTERN GHATS

DNA barcoding is designed to provide accurate and automated species identification through molecular species identification for fish and other animals. Currently, DNA barcoding tool is potentially used in the food industry, diet analyses, forensic sciences and in preventing illegal trade and poaching of endangered species. In the present study, DNA barcoding of five species of loaches of Western Ghats were analyzed. The five species of loaches includes Lepidocephalus thermalis (Cobitidae), Nemachelilus triangularis, N.guentheri, N.semiarmatus and Bhavania australis (Balitoridae).

The fishes were collected from various locations of Western Ghats (Tamil Nadu and Karnataka State),  DNA was extracted and purity of DNA was ascertained with UV spectrophotometer at 260mm and 280 mm, the purity recorded was 1.60 -2.0 (Quality of DNA was good). DNA barcoding was carried out with the amplification of Cytochrome C oxidase subunit 1 (COI) gene primer using Polymerase Chain Reaction (PCR). The PCR products were sequenced for the  five species and the sequences were compared with global database (NCBI) using BLAST. The sequences were submitted to GenBank and the accession numbers provided by GenBank for Lepicephalus thermalis were KP744350, KP744351, KP744352 and KP744353, for Nemacheilus triangularis were KP772689, KP772690 and KP772691, for N. guentheri KP772692, KP772693, KP772694 and KP772695, for                      N. semiarmatus were KP823400, KP 823401 and KP 823402 and for Bhavania australis were KP823397, KP823398 and KP823399. The sequences of the five species loaches were used for constructing phylogenic tree and compared with the related species from the global databases.

The phylogenic relationship among the species was clearly established and closely related species were clustered under the same node while dissimilar species were clustered under separate nodes. The average Kimura two parameters (K2P) distances within  species (conspecific),  within genera (congeneric),  within family (confamilial) and between families  were 0.507%, 6.638%, 2.426 and 2.450 respectively