Crocodiles are the largest species

Crocodiles are the largest species among the members of class Reptilia in vertebrates and are one of the last living links to the era when dinosaurs lived. Until the early 1950s, these species were numerous in the tropics of Africa, the Americas, Asia and Australia. In the recent years many of crocodilian species are considered endangered due to their habitat destruction and illegal poaching for their lucrative products.34 and 35 According to Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, crocodilian species were grouped in Appendix I and II. A significant illegal trade of crocodilians has engrossed a lot of attention and therefore the species deserve priority for conservation. In order to conserve these antiquities many countries have developed legal policies for prohibition of poaching and hunting.28 However, illegal trade still exists in many parts of world which appears to be a challenging task to proscribe. Moreover, the biological materials are often degraded quality, which do not lead to conclusive identification through conventional methods.6 and 39 In this perspective, development of novel methods for the identification of these species is inevitable towards their conservation.

Mitochondrial (mt) DNA is valuable tool for species identification from highly degraded samples.1 and 11 Many of universal mt DNA markers such as cytochrome b, 18 and 38 12S rRNA, 17 16S rRNA, 14 and noncoding D-Loop region 28 and 40 have shown their potential targets for animal species identifications. Within crocodiles a single mtDNA marker i.e. cytochrome b (cyt b) has been studied as a potential source for species identification. 21, 22, 23, 25 and 27 However, the efficiency of other regions for forensic identification of crocodiles has not been evaluated and thus these markers also need to be studied in forensic analyses.

The 16S rRNA gene, which encodes the mitochondrial large ribosomal subunit (mt LSU) in animals, has been employed extensively to explore phylogenetic relationships in anthropods at most phylogenetic levels,8 familial level2 and the genus level and below.5, 7 and 15 The wide range in utility of 16S at various taxonomic levels suggests that it could be useful marker for forensic identifications as well.

In this paper, we develop novel primer sets to amplify partial 16S ribosomal RNA region for discriminating the crocodile species. The primers were optimized and checked for their efficiency in six species i.e. Crocodylus palustris, Crocodylus porosus, Gavialis gangeticus, Crocodylus niloticus, Crocodylus siamensis and Crocodylus crocodilus which represent the three existing families of Crocodylia.