GENETIC CHARACTERIZATION, CONTROLLED BREEDING AND DEVELOPMENT OF TRANSGENIC VARIETIES OF PUNTIUS DENISONII (DAY, 1865).
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Date
2010-12-10
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KUFOS
Abstract
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.