Mitochondrial 12S rRNA gene sequence analysis, a tool for species identification
DOI:
https://doi.org/10.22120/jwb.2019.107094.1066Keywords:
Species identification, 12S rRNA, bioinformatics, polymerase chain reactionAbstract
Biodiversity is under threat worldwide. Many species of animals, birds and reptiles have experienced considerable decline in their populations within the past two centuries due to illegal trade, poaching and habitat destruction. The biodiversity in India is under threat due to growing wildlife crime. In this study a segment of mitochondrial 12S rRNA was used to develop an easy and standard protocol to identify a number of species from variety of samples. Polymerase chain reaction (PCR) was done after DNA extraction and quantification. PCR products were then sequenced bi-directionally and aligned to a database non reductant nucleotide sequence. It was then confirmed that the sequences aligned specifically to mitochondrial sequences from expected species of origin. Bioinformatics tools were then applied for phylogenetic and genetic diversity analysis. Result of this study showed that universal primer could amplify partial mitochondrial 12rRNA gene from all studied animals, birds and reptiles. Based on the sequences obtained, a Neighbour joining tree was constructed using K2P model. This molecular technique together with bioinformatics tools provides a reliable and fast method for species identification and taxonomic classification.
References
Arif I.A., Khan H.A. 2009. Molecular markers for biodiversity analysis of wildlife animals: a brief review, Animal Biodiversity Conservation 32: 9-17.
Barbar M.E., Hussain T., Wajid A., Nawaz A., Nadeem A., Sha S.A., Shahid M.A., Ahmad N., Javed K., Abdullah M. 2015. Mitochondrial cytochrome b and d-loop sequence based genetic diversity in Mareecha and Bareela camel breeds of Pakistan. Journal of Animal and Plant Sciences 25: 591-594.
Birstein V.J. and de Salle R. 1998. Molecular phylogeny of Acipenserinae. Molecular Phylogenetic Evolution 9: 141–155 .
Dratch P., Shafer J., Hoesch R. and Espinoza E. 1996. Comparison of electrophoretic and chromatographic methods for analysis of deer haemoglobins. International Society for Animal Genetics. 27: 17–42.
Felsenstein, J .1985. Confidence limit on phylogenies: an approach using the boot strap. Evolution. 39: 783-791.
Friedheim, S. (2016). Comparison of Species Identification Methods, DNA Barcoding versus Morphological Taxonomy. Biology, 301(54): 74-86.
Galtier, N., Nabholz, B., Glémin, S., & Hurst, G. D. D. (2009). Mitochondrial DNA as a marker of molecular diversity: a reappraisal. Molecular Ecology, 18(22), 4541–4550. doi:10.1111/j.1365-294x.2009.04380.x
Girish P.S., Anjaneyulu A.S.R., Viswas K.N., Anand M., Rajkumar N., Shivkumar B.M., Bhaskar S. 2004. Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Science, 66: 551–556.
Gupta A, Bhardwaj A, Supriya, Sharma P, Pal Y,Mamta., and Kumar S. 2015. Mitochondrial DNA- a Tool for Phylogenetic and Biodiversity Search in Equines. Journal of Biodiversity & Endangered Species S1:006.
Gupta A.R., Patra R.C., Das D.K., Gupta P.K., Swarup D., Saini M.2008. Sequence characterization and polymerase chain reaction, restriction fragment length polymorphism of the mitochondrial DNA 12S rRNA gene provides a method for species identification of Indian deer. Mitochondrial DNA. 19: 394–400.
Hall T.A .1999. BioEdit: a user-friendly biological sequence alignment editor and analysis programme for windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98.
Hayashi J.I., Tagoshira Y., Yoshida M.C. 1985. Absence of extensive recombination between inter and intra species mtDNA in mammalian cells. Experimental Cell Research, 160: 387-395.
Irwin D.M., Kocher T.D., Wilson A.C. 1991. Evolution of the cytochrome b gene of mammals Journal of Molecular Evolution, 32: 123-144.
Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16: 111-120.
Kocher T.D., Thomas W.K., Meyer A., Edwards S.V., Paabo, S., Villablaned F.X., Wilson A.C. 1989. Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America. 86, 6196-6200.
Kumar V. P., Kumar D., Goyal S.P. 2014. Wildlife DNA forensic in curbing illegal wildlife trade: specie identification from seizures. International Journal of Forensic Science & Pathology, 2: 38–42.
Martinez I., Yman I.M. 1998. Species identification in meat products by RAPD analysis. Food Research International, 31: 459-466.
Melton T., Holland C. 2007. Routine forensic use of the mitochondrial 12S ribosomal RNA gene for species identification. Journal of Forensic Sciences, 52: 1305-1307.
Nakamura, M., Ishibashi, Y., & Syuiti, A. (2002). Novel primer sets for species-specific amplification of the mitochondrial 12S rRNA genes in four Japanese woodpeckers (Picidae, Piciformes). Molecular Ecology Notes, 2(4), 419–421.
Panday R., Jha D.K., Thapa N., Pokharel B.R., Aryal N.K. 2014. Forensic Wildlife Parts and their Product Identification and Individualization using DNA Barcoding. The Open Forensic Science Journal, 7:6-13.
Panprommin D., Soontornprasit, K., Pangeson, T. 2019. Comparison of three molecular methods for species identification of the family Cichlidae in Kwan Phayao, Thailand. Mitochondrial DNA Part A, 30(1): 184-190
Pereira F., Carneiro J., Amorim A. 2008. Identification of species with DNA- based technology: current progress and challenges. Recent Patents on DNA & Gene Sequencو. 2:187-199.
Saitou N., Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406-425
Sambrook J., Russel D.W. 2001. Molecular cloning: A laboratory manual. 3rd ed., Cold spring harbor laboratory press, New York, pp. 2000.
Tamura K., Slecher G., Peterson D., Filipski A., Kumar S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30: 2725-2729.
Veerkar E.L.C., Nijman L.J., Boutaga K., Lenstra J.A. 2002. Differentiation of cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA. Meat Science, 60: 365-369.
Vos P., Hogers R., Bleeker M., Reijans M., Lee T., Hornes M. 1995. AFLP a new technique for DNA finger printing. Nucleic Acids Research. 23: 4407-4414.
Wolf C., Rentsch J., Hubner P. 1999. PCR-RFLP analysis of mitochondrial DNA: a reliable method for species identification. Journal of Agricultural and Food Chemistry, 47: 1350-1355.
Yang L., Tan Z., Wang D., Xue L., Guan M., Huang T., Li R. 2014. Species identification through mitochondrial rRNA genetic analysis. Scientific Reports. 4: 4089.
Zhang Y.P., Ryder O.A. 1994. Phylogenetic relationships of bears (the Ursidae) inferred from mitochondrial DNA sequences. Molecular Phylogenetics and Evolution, 3: 351-359.
JWB applies the 