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.
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References
An J, Lee MY, Min MS et al (2006) Molecular genetic approach for species identification of mammals and sex determination of birds in a forensic case of poaching from South Korea. Forensic Sci Int 167:59–61. doi:10.1016/j.forsciint.2005.12.031
Bravi CM, Liron JP, Mirol PM et al (2004) A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene. Leg Med 6:246–251. doi:10.1016/j.legalmed.2004.06.003
Brochu CA (2003) Phylogenetic approaches toward crocodilian history. Annu Rev Earth Planet Sci 31:357–397. doi:10.1146/annurev.earth.31.100901.141308
Budowle B, Garofano P, Hellman A et al (2005) Recommendations for animal DNA forensic and identity testing. Int J Legal Med 119:295–302. doi:10.1007/s00414-005-0545-9
Caine L, Lima G, Pontes L et al (2006) Species identification by cytochrome b gene: casework samples. Int Congr Ser 1288:145–147. doi:10.1016/j.ics.2005.09.152
Cook C, Smith M, Telford M et al (2001) Hox genes and the phylogeny of the arthropods. Curr Biol 11:759–763. doi:10.1016/S0960-9822(01)00222-6
Cummings MP, Otto SP, Wakeley J (1999) Genes and other samples of DNA sequence data for phylogenetic inference. Biol Bull 196:345–350. doi:10.2307/1542967
Dasmahapatra KK, Mallet J (2006) DNA barcodes: recent successes and future prospects. Heredity 97:254–255. doi:10.1038/sj.hdy.6800858
Gatesy J, Baker RH, Hayashi C (2004) Inconsistencies in arguments for the supertree approach: supermatrices versus supertrees of Crocodylia. Syst Biol 53:342–355. doi:10.1080/10635150490423971
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
Hsieh HM, Chiang HL, Tsai LC et al (2001) Cytochrome b gene for species identification of the conservation of animals. Forensic Sci Int 122:7–18. doi:10.1016/S0379-0738(01)00403-0
Irwin DM, Kocher TD, Wilson AC (1991) Evolution of the cytochrome b gene of mammals. J Mol Evol 32:128–144. doi:10.1007/BF02515385
Kocher TD, Thomas WK, Meyer A et al (1989) Dynamics of mitochondrial DNA evolution in mammals: amplification and sequencing with conserved primers. Proc Natl Acad Sci USA 86:6196–6200. doi:10.1073/pnas.86.16.6196
Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163. doi:10.1093/bib/5.2.150
Miles LJ, Lance SL, Isberg SR, et al (2008) Cross-species amplification of microsatellites in crocodilians: assessment and applications for the future. Conserv Genet. doi:10.1007/s10592-008-9601-6
Paabo S, Painer H, Serre D (2004) Genetic analyses from ancient DNA. Annu Rev Genet 38:645–679. doi:10.1146/annurev.genet.37.110801.143214
Roos J, Agarwal RK, Janke A (2007) Extended mitogenomic phylogenetic analyses yield new insight into crocodilian evolution and their survival of the Cretaceous-Tertiary boundary. Mol Phylogenet Evol 45:663–673. doi:10.1016/j.ympev.2007.06.018
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Sanderson MJ, Shaffer HB (2002) Troubleshooting molecular phylogenetic analyses. Annu Rev Ecol Syst 33:49–72. doi:10.1146/annurev.ecolsys.33.010802.150509
Santiapillai C, de Silva M (2001) Status, distribution and conservation of crocodiles in Sri Lanka. Biol Conserv 97:305–318. doi:10.1016/S0006-3207(00)00126-9
Stuart BL, Hayes B, Manh BH et al (2002) Status of crocodiles in the U Minh Thuong Nature Reserve Southern Vietnam. Pac Conserv Biol 8:62–65
Su B, Wang YX, Lan H et al (1999) Phylogenetic study of complete cytochrome b genes in musk deer (Genus Moschus) using museum sample. Mol Phylogenet Evol 12:241–249. doi:10.1006/mpev.1999.0616
Teletchea F, Maudet C, Hanni C (2005) Food and forensic molecular identification: update and challenges. Trends Biotechnol 23:359–366. doi:10.1016/j.tibtech.2005.05.006
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. doi:10.1093/nar/22.22.4673
Verma SK, Singh L (2003) Novel universal primers establish identity of an enormous number of animal species for forensic application. Mol Ecol Notes 3:28–31. doi:10.1046/j.1471-8286.2003.00340.x
Verma SK, Prasad K, Nagesh N et al (2003) Was elusive carnivore a panther? DNA typing of faeces reveals the mystery. Forensic Sci Int 137:16–20. doi:10.1016/S0379-0738(03)00277-9
Yang DY, Speller CF (2006) Co-amplification of cytochrome b and D-loop mtDNA fragments for the identification of degraded DNA samples. Mol Ecol Notes 6:605–608. doi:10.1111/j.1471-8286.2006.01370.x
Acknowledgements
The authors are thankful to Madras Crocodile Bank Trust (MCBT), Centre for Herpetology, Mamallapuram, Tamilnadu, India and National Chambal Sanctuary Project, Agra, Uttar Pradesh, India for providing the biological samples for the study. The authors thank Dr. T. S. Vasulu, Human Anthropology Unit, Indian Statistical Institute, Kolkata for his valuable suggestions in statistical analyses. The research was supported by Directorate of Forensic Sciences, Ministry of Home Affairs, Government of India.
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Meganathan, P.R., Dubey, B. & Haque, I. Molecular identification of crocodile species using novel primers for forensic analysis. Conserv Genet 10, 767–770 (2009). https://doi.org/10.1007/s10592-008-9658-2
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DOI: https://doi.org/10.1007/s10592-008-9658-2