Species identification of Kachuga tecta using the cytochrome b gene

A DNA technique has been established for the identification to species level of tortoises. The test on the shell of the animal was used to identify samples from the species Kachuga tecta. A total of 100 tortoise shell specimens collected from the National Council of Agriculture (COA), Taiwan, were used in this study. Primer pairs were designed to amplify partial DNA fragments of cytochrome b within the mitochondrial genome. The DNA data showed that among the 100 samples, there were four distinct haplotype DNA sequences, within which there were a total of 90 variable sites. Between haplotypes I and II, there was only 1 nucleotide difference at position 228. Between haplotypes I and III, 65 nucleotide differences were observed; haplotypes I and IV, 62 nucleotide differences; and haplotypes III and IV, 56 nucleotide differences were observed. There were 66 and 63 nucleotide differences between haplotypes II and III and haplotypes II and IV respectively. All four haplotypes were compared with the DNA sequences held at the GenBank and EMBL databases. The most similar species were K. tecta (haplotype I and II), Morenia ocellata (haplotype III) and Geoclemys hamiltonii (haplotype IV), and their respective mtDNA similarities were 99.5%, 99.3%, 89.9% and 99.5%. However, as haplotype III was only 89.9% homologous with M. ocellata, it would seem that this haplotype shows only a limited relationship with a similar species registered currently in these databases. The method established by this study is an additional method for the identification of samples protected under Convention International Trade in Endangered Species (CITES) and will improve the work for the preservation of the endangered species.