Restriction fragment length polymorphism of mitochondrial DNA and phylogenetic relationships among five species of Indian freshwater turtles

Abstract

DNA-based identification of species for phylogenetic analysis as well as forensic identification is widely being carried out with the help of polymerase chain reaction (PCR). In this study, a successful effort has been made to identify 5 species of Indian freshwater turtles, including 3 hard-shell turtles (Geoemydidae), i.e. Kachuga dhongoka, K. kachuga and Geoclemys hamiltoni, and 2 species of soft-shell turtles (Trionychidae), i.e. Aspideretes gangeticus and Lissemys punctata punctata, by using a well-optimized PCR-RFLP method. The analysis of nucleotide sequence variations in the PCR-amplified mitochondrial cyt-b genes (encoding cytochrome b) from the 5 species revealed its usefulness in the taxonomic differentiation of these species. On the basis of cyt-b sequence data and the PCR-RFLP pattern, a phylogeny was developed to resolve the genetic relationships between these species, living in the same habitat type. In comparison, the PCR-RFLP of mitochondrial 16S rDNA genes appeared less decisive in analysing phylogenetic relationships or even in species differentiation. Further, the molecular method (PCR-RFLP) developed here is simple, rapid, reliable and reproducible; hence it can be routinely applied for species identification, essential for conservation and management of endangered chelonian species.