Molecular identification of crocodile species using novel primers for forensic analysis

Abstract

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.

Complete mitochondrial genome sequences of three Crocodylus species and their comparison within the Order Crocodylia

“…For LA-PCR two sets of primers, LA-F1: 5′ GTA TGA AAG GCT AAA CGA GAA TCT ATC TGT CTC 3′; LA-R1: 5′ GGT AGG ACG TAG CCT ATG AAG GCA GTT GCT AT 3′ and LA-F2: ATA GCA ACT GCC TTC ATA GGC TAC GTC CTA CC 3′; LA-R2: TTA ATA ATC ACT AGG GTT CAA GAT ATT TGG GC 3′ were designed utilizing the sequences of cytochrome b (Meganathan et al, 2009b) and NADH dehydrogenase subunit 2 (Meganathan, unpublished) of C. palustris and C. johnstoni. The LA-PCR was performed using LA PCR Kit ver.…”

DNA typing in wildlife crime: recent developments in species identification

“…There is a range of universal primer sets for mammalian species, such that a sample from any mammalian species will generate the same size product after amplification using the polymerase chain reaction (PCR) [62,63,71,79,[81][82][83]. The PCR product is then generally used in a sequencing reaction to determine the full sequence [29,30,64,72,[75][76][77][84][85][86][87][88][89][90]. This sequence is compared to those registered on a DNA database such as GenBank.…”

Population genetics implications for the conservation of the Philippine Crocodile Crocodylus mindorensis Schmidt, 1935 (Crocodylia: Crocodylidae)

“…Molecular genetic methodology has been used to address taxonomic issues, assess genetic variability and inbreeding, track gene flow and detect hybridization, all in an effort to conserve genetically healthy populations and aid in the identification of ecologically significant units (Fleischer 1998). The use of nuclear DNA (nucDNA) and mitochondrial DNA (mtDNA) sequence data in crocodilian research has increased our understanding of genetic variability (Flint et al 2000;Ray et al 2004;Russello et al 2007), hybridization (FitzSimmons et al 2002;Ray et al 2004;Cedeño-Vásquez et al 2008), differences between individuals (Farias et al 2004), populations (Vasconcelos et al , 2008 and species (Li et al 2007;Gatesy & Amato 2008;Meganathan & Dubey 2009;Meganathan et al 2010). Microsatellites have been used to investigate population structure and gene flow in wild populations of Morelet's Crocodile Crocodylus moreletii Duméril & Bibron, 1851 (Dever & Densmore 2001;Dever et al 2002), American Alligator Alligator mississippiensis Daudin, 1802 (Glenn et al 1998;Davis et al 2002) and Black Caiman Melanosuchus niger Spix, 1825.…”