Genetic characterization, controlled breeding and development of transgenic varieties of puntius denisonii (day, 1865).

Abstract

Puntius denisonii, a beautiful ornamental fish indigenous to the Western Ghats, which has been indiscriminately exploited from the different rivers of Kerala has been recently declared to be vulnerable by the IUCN. The population structure and genetic diversity of P. denisonii has not yet been studied and documented. Many previous attempts to breed this fish in captivity have yielded negative results. The increasing demand for this fish to decorate aquariums worldwide could be satisfied only by developing controlled breeding techniques and larval rearing of its fry. In the present study, the present population structure of P. denisonii has been studied combining both phenotypic and genotypic techniques. Fishes were collected from Irrity, Chaliyar and Periyar rivers of Kerala. Truss network analysis was conducted and the size adjusted morphometric variables were subjected to Principal Component Analysis and Canonical Variance Analysis. Scatter diagram and Dendrogram was plotted using PCA and CVA loadings. The Irrity and the Chaliyar populations were grouped on the positive sector of the PC and CV component showing morphological similarities between the two populations while the Periyar population was placed in the negative sector of the component separated far from the other two. The PC scores were used to find out the variables showing maximum variation between fishes collected from different rivers. RAPD PCR was conducted after isolating DNA from the fins of different populations of P. denisonii. Universal random primers were screened and the primers that produced reproducible bands were selected. Popgene analysis of the binary data yielded the genetic structure of different populations of P. denisonii. Number and percentage of polymorphic loci, Nei's (1973) gene diversity, Shannon's Information index Lewontin (1972), Nei's Unbiased Measures of Genetic Identity and Genetic distance and Dendrogram Based Nei's (1978) Genetic distance using UPGMA --Modified from NEIGHBOR procedure of PHYLIP Version 3.5 were studied. The results obtained supports the truss analysis in that the Irrity and Chaliyar populations in Northern Kerala are genetically more similar while that of Periyar population in Central Kerala are distinct. P. denisonii was successfully induced bred under controlled conditions with synthetic hormone preparations Ovaprim and WOVA-FH. Stress during transport and handling was minimized and live feed was supplemented to enhance maturation of the broodstock. The whole developmental sequence starting from fertilized eggs to hatching was photographed and documented. It took 29-30 hours for the eggs to hatch at 280C. Rearing of fry was successfully accomplished under laboratory conditions. In an attempt to develop transgenic varieties of P. denisonii, pCMV-GFP was electroporated into newly fertilized eggs, maintained in hypoosmolar electroporation buffer. The electroporation parameters that yielded best results were 20V, 3 bursts at 1 second interval. Fin clips were taken from the transgenic individuals reared for a period of 6 weeks. Dot blot test was positive showing integration of the GFP gene in P. denisonii, eventhough expression was not detected under blue or UV light. The genetic and phenotypic data of P. denisonii populations in the present study will aid as a base line for formulating conservation procedures to protect the genetic diversity of wild ones. Stock identification studies are recommended for more concise information on each population. Moreover, the larval rearing and controlled breeding techniques along with the genetic diversity studies will help to design captive breeding programs and enhance the production of hatchery bred ones to meet increasing demand. Further research is recommended for generating transgenic lines with uniform GFP expression.