Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Species and Sex Identification of the Korean Goral (Nemorhaedus caudatus) by Molecular Analysis of Non-invasive Samples

  • Kim, Baek Jun (Conservation Genome Resource Bank for Korean Wildlife (CGRB), College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Lee, Yun-Sun (Conservation Genome Resource Bank for Korean Wildlife (CGRB), College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • An, Jung-hwa (Conservation Genome Resource Bank for Korean Wildlife (CGRB), College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Park, Han-Chan (Conservation Genome Resource Bank for Korean Wildlife (CGRB), College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Okumura, Hideo (Wildlife Ecology Laboratory, Department of Wildlife Biology, Forestry and Forest Products Research Institute) ;
  • Lee, Hang (Conservation Genome Resource Bank for Korean Wildlife (CGRB), College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Min, Mi-Sook (Conservation Genome Resource Bank for Korean Wildlife (CGRB), College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University)
  • Received : 2007.11.01
  • Accepted : 2008.04.15
  • Published : 2008.09.30
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Abstract

Korean long-tailed goral (Nemorhaedus caudatus) is one of the most endangered species in South Korea. However, detailed species distribution and sex ratio data on the elusive goral are still lacking due to difficulty of identification of the species and sex in the field. The primary aim of this study was to develop an economical PCR-RFLP method to identify species using invasive or non-invasive samples from five Korean ungulates: goral (N. caudatus), roe deer (Capreolus pygargus), feral goat (Capra hircus), water deer (Hydropotes inermis) and musk deer (Moschus moschiferus). The secondary aim was to find more efficient molecular sexing techniques that may be applied to invasive or non-invasive samples of ungulate species. We successfully utilized PCR-RFLP of partial mitochondrial cytochrome b gene (376 bp) for species identification, and sex-specific amplification of ZFX/Y and AMELX/Y genes for sexing. Three species (goral, goat and water deer) showed distinctive band patterns by using three restriction enzymes (Xbal, Stul or Sspl). Three different sexing primer sets (LGL331/335 for ZFX/Y gene; SE47/48 or SE47/53 for AMELX/Y gene) produced sex-specific band patterns in goral, goat and roe deer. Our results suggest that the molecular analyses of non-invasive samples might provide us with potential tools for the further genetic and ecological study of Korean goral and related species.

Keywords

Acknowledgement

Supported by : Seoul National University

References

  1. Aasen, E., and Medrano, J.F. (1990). Amplification of the zfy and zfx genes for sex identification in humans, cattle, sheep and goats. Biotechnology 8, 1279-1281 https://doi.org/10.1038/nbt1290-1279
  2. Adams, J.R., Leonard, J.A., and Waits, L.P. (2003). Widespread occurrence of a domestic dog mitochondrial DNA haplotype in southeastern USA coyotes. Mol. Ecol. 12, 541-546 https://doi.org/10.1046/j.1365-294X.2003.01708.x
  3. An, J. (2006). Development and charaterization of microsatellite markers for endangered Korean goral (Nemorhaedus caudatus raddeanus) and its molecular phylogenetic status. Ph.D. thesis, Seoul national university, Seoul, Korea
  4. Bellemain, E., and Taberlet, P. (2004). Improved non-invasive genotyping method: application to brown bear (Ursus arctos) feces. Mol. Ecol. Notes 4, 519-522 https://doi.org/10.1111/j.1471-8286.2004.00711.x
  5. Gerloff, U., Schlotterer, C., Rassmann, K., Rambold, I., Hohmann, G., Fruth, B., and Tautz, D. (1995). Amplification of hypervariable simple sequence repeats (microsatellites) from excremental DNA of wild living bonobos (Pan paniscus). Mol. Ecol. 4, 515-518 https://doi.org/10.1111/j.1365-294X.1995.tb00247.x
  6. Goossenss, B., Waits, L.P., and Taberlet, P. (1998). Plucked hair samples as a source of DNA: reliability of dinucleotide microsatellite genotyping. Mol. Ecol. 7, 1237-1241 https://doi.org/10.1046/j.1365-294x.1998.00407.x
  7. Hattori, K., Burdin, A.M., Onuma, M., Suzuki, M., and Ohtaishi, N. (2003). Sex determination in the sea otter (Enhydra lutris) from tissue and dental pulp using PCR amplification. Can. J. Zool. 81, 52-56 https://doi.org/10.1139/z02-219
  8. Hutton, J., and Dickson, B. (2000). Endangered species, threatened convention: the past, present and future of CITES. (London: UK, Earthscan Publications)
  9. IUCN (1996). 1996 IUCN red list of threatened animals. Gland, Switzerland: IUCN
  10. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., and Wilson, A.C. (1989). Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86, 196- 200
  11. Ministry of Environment of Korea (2002). New Technique for the Restoration of Endangered Species in Korea. National Institute of Environmental Research, Korea
  12. Ministry of Environment of Korea (2004). Wildlife Protection Act, Ministry of Government Legislation, Korea
  13. Murata, K., and Masuda, R. (1996). Gender determination of the Linne's two-toed sloth (Choloepus didactylus) using SRY amplified from hair. J. Vet. Med. Sci. 58, 1157-1159 https://doi.org/10.1292/jvms.58.12_1157
  14. Palomares, F., Godoy, J.A., Piriz, A., O'Brien, S.J., and Johnson, W.E. (2002). Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx. Mol. Ecol. 11, 2171-2182 https://doi.org/10.1046/j.1365-294X.2002.01608.x
  15. Parson, W., Pegoraro, K., Niederstatter, H., Foger, M., and Steinlechner, M. (2000). Species identification by means of the cytochrome b gene. Int. J. Legal. Med. 114, 23-28 https://doi.org/10.1007/s004140000134
  16. Pfeiffer, I., and Brenig, B. (2005). X- and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep (Ovis aries) and European red deer (Cervus elphus). BMC Genet. 6, 16 https://doi.org/10.1186/1471-2156-6-16
  17. Pfeiffer, I., Burger, J., and Brenig, B. (2004). Diagnostic polymorphisms in the mitochondrial cytochrome Ä gene allow discrimination between cattle, sheep, goat, roe buck and deer by PCRRFLP. BMC Genet. 5, 30 https://doi.org/10.1186/1471-2156-5-30
  18. Shaw, C.N., Wilson, P.J., and White, B.N. (2003). A reliable molecular method of gender determination for mammals. J. Mamm. 84, 123-128 https://doi.org/10.1644/1545-1542(2003)084<0123:ARMMOG>2.0.CO;2
  19. Taberlet, P., and Luikart, G. (1999). Non-invasive genetic sampling and individual identification. Biol. J. Linn. Soc. 68, 41-55 https://doi.org/10.1111/j.1095-8312.1999.tb01157.x
  20. Walsh, S., Metzger, D., and Higuchi, R. (1991). Chelex-100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10, 506-513
  21. Yamamoto, K., Tsubota, T., Komatsu, T., Katayama, A., Murase, T., Kita, I., and Kudo, T. (2002). Sex identification of Japanese black bear, Ursus thibetanus japonicus, by PCR based on amelogenin gene. J. Vet. Med. Sci. 64, 505-508 https://doi.org/10.1292/jvms.64.505
  22. Yamauchi, K., Hamasaki, S.I., Miyazki, K., Kikusui, T., Takeuchi, Y., and Mori, Y. (2000). Sex determination based on fecal DNA analysis of the amelogenin gene in sika deer (Cervus Nippon). J. Vet. Med. Sci. 62, 669-671 https://doi.org/10.1292/jvms.62.669