Species identification of rhinoceros horns using the cytochrome b gene.

Material suspected of originating from species of Rhinoceros is frequently seized by forensic organizations investigating trade in endangered species. At present identification of the species is possible by DNA sequencing of the material, such as powdered rhinoceros horns. The unambiguous identification of rhino products using a 402 bp fragment of cytochrome b gene was investigated. This DNA sequence may not only assist in the identification of the unknown sample, but can be used to determine the phylogenetic relationships of rhinoceros species. Sequences of suspect rhinoceros horns were compared with the sequences registered in GenBank. The maximum value of genetic distance among white rhinoceros was 0.0176, and 0.0333 among black rhinoceros. In the comparison among rhinoceros species, the greatest genetic distance was between black and Indian rhinoceros (0.1564). The rhinoceros sequences extracted from GenBank and 13 samples in this study were clustered and separated from other mammals. Holstein cow was used as an out-group and was clustered with cattle in the phylogenetic tree. The results of this phylogenetic study also showed that there were four major branches among rhinoceros species from a common origin. The amplification of the 402 bp fragment of the cytochrome b gene was found to be able to detect rhinoceros DNA even in the ratio of 1:19 with Holstein cow DNA. In the initial identification of species from unknown powdered material, all the unknown samples were found to be from rhinoceroses. In phylogenetic analysis, the results supported the morphological hypothesis. The method used in this study can be applied in the identification of processed products of rhinoceros horns, such as sculptures, daggers, powders or even mixture powdered prescriptions.