Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
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Genetic Variation of Mitochondrial DNA in Duroc (Sus Scrofa) Using Single Stranded Conformation Polymorphism Analysis

Single Stranded Conformation Polymorphism 분석에 의한 돼지 Duroc 품종의 미토콘드리아 DNA 유전적 변이

  • Cho, I.C. (National Jeju Agricultural Experiment Station, R.D.A.) ;
  • Jung, Y.H. (National Jeju Agricultural Experiment Station, R.D.A.) ;
  • Jung, J.K. (National Jeju Agricultural Experiment Station, R.D.A.) ;
  • Seong, P.N. (National Jeju Agricultural Experiment Station, R.D.A.) ;
  • Kim, B.W. (Division of Applied Life Science, Gyeongsang National University) ;
  • Lee, J.G. (Division of Applied Life Science, Gyeongsang National University) ;
  • Jeon, J.T. (Division of Applied Life Science, Gyeongsang National University)
  • 조인철 (농촌진흥청 제주농업시험장) ;
  • 정용환 (농촌진흥청 제주농업시험장) ;
  • 정진관 (농촌진흥청 제주농업시험장) ;
  • 성필남 (농촌진흥청 제주농업시험장) ;
  • 김병우 (경상대학교 응용생명과학부) ;
  • 이정규 (경상대학교 응용생명과학부) ;
  • 전진태 (경상대학교 응용생명과학부)
  • Published : 2003.12.31
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Abstract

The mitochondrial DNA(mtDNA) D-loop region was amplified from Duroc(Sus scrofa) by polymerase chain reaction(PCR). The oligonucleotide primer used to amplify the Sus scrofa mtDNA D-loop region was designed using tRNA-Pro and tRNA-Phe sequence in mtDNA regions highly conserved in many other animal species. There were 1,145 base pairs(bp) in the D-loop region. The middle of the region contained 10 tandem repeat of an 10-bp Sus scrofa-specific sequence, TACACGTGCG. We designed primers for PCR-mediated single stranded conformation polymorphism(SSCP) analysis that amplified a 345 bp fragment, which contained the most variable region according to our sequencing data. SSCP analysis of denatured amplification products was carried out by polyacrylamide(8%) gel electrophoresis followed by ethidium bromide staining. The SSCP analysis identified two band patterns(A and B) and comparision of these two nucleotide sequences identified 21 base substitutions. These results show that SSCP analysis of the D-loop region is useful for detecting the genetic polymorphism.

돼지 Duroc 품종의 mitochondria DNA D-loop전체 유전자를 증폭하기 위하여 많은 동물에서 고도로 상동성이 높은 tRNA-Pro와 tRNA-Phe 염기서열 일부를 이용하여 oligonucleotide primer를 제작하였다. 그 결과 Duroc 품종의 D-loop 전체 유전자는 1,145 base pairs 였으며, 그 중간위치에 10bp의 Sus Scrofa-specific sequence (TACACGTGCG)가 10개 존재하고 있었다. 돌연변이 검출을 위하여 가장 변이가 심한 지역을 primer 제작하여 345 bp의 DNA 단편을 증폭하였으며, Single Stranded Conformation Polymorphism(SSCP) 분석은 8% polyacrylamide gel에서 200 V, 16시간 전기영동하여 ethidium bromide (EtBr)로 10분간 염색하여 UV image analyzer로 관찰하였다. 그 결과 두 개의 서로 다른 밴드유형을 관찰하였으며, 21개 부위에서 염기서열 변이가 관찰되었다. 이러한 결과는 유전적 다양성 변이를 검출하는데 SSCP 분석이 유용한 도구라고 사료된다.

Keywords

References

  1. Arnason, U., Gullberg, A. and Widegren, B. 1991. The complete nucleotide sequence of the mitochondrial DNA of the fin whale, Balae noptera physalus. J. Mol. Evol. 33:556-568.
  2. Bjorn, M., Ursing, B. M. and Arnason, U. 1998. The Complete Mitochondrial DNA Sequence of the Pig(Sus scrofa). J. Mol. Evol. 47:302-306.
  3. Bowling, A. T., Valle, A. D. and Bowling, M. 2000. A pedigree-based study of mitochondrial D-loop DNA sequence variation among arabian horses. Animal genetics 31:1-7.
  4. Giuffra, E., Kijas, J. M. H., Amarger, V., Carlborg, A., Jeon, J.-T. and Andersson, L. 2000. The Origin of the Domestic Pig: Independent Domestication and Subsequent Introgression. Genetics 154:1785-1791.
  5. Honeycutt, R. L., Nedbal, M. A., Adkins, R. M. and Janecek, L. L. 1995. Mammalian mitochondrial DNA evolution: a comparison of the cytochrome b and cytochrome c oxidase II genes. J. Mol. Evol. 40:260-272. https://doi.org/10.1007/BF00163231
  6. Irwin, D. M., Kocher, T. D. and Wilson, A. C. 1991. Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32:128-144. https://doi.org/10.1007/BF02515385
  7. Jones, G. F. 1998. Genetic aspects of domestication, common breeds and their origin, pp. 17-50 in The Genetics of the Pig, edited by A. Ruvinsky and M. F. Rothschild. CAB International, Oxon, UK.
  8. Kim, K. I., Yang, Y. H., Lee, S. S., Park, C., Ma, R., Bouzat, J. L. and Lewin, H. A. 1999. Phylogenetic relationships of Cheju horse to other horse breeds as determined by mtDNA D-loop sequence polymorphism. Animal Genetics 30:102-108. https://doi.org/10.1046/j.1365-2052.1999.00419.x
  9. Kraus, F. and Miyamoto, M. M. 1991. Rapid cladogenesis among the pecoran ruminants: evidence from mitochondrial DNA sequences. Syst. Zool. 40:117-130.
  10. Lin, C. S., Sun, Y. L. and Liu, C. Y. 1999. Complete nucleotide sequence of pig(Sus scrofa) mitochondrial genome and dating evolutionary divergence within Artiodactyla. Gene 236:107-114. https://doi.org/10.1016/S0378-1119(99)00247-4
  11. Miyamoto, M. M. and Boyler, S. M. 1989. The potential importance of mitomitochondrial DNA sequence data to eutherian mammal phylogeny. In: Fernholm, B., Bremer, B., Jornvall, H. (Eds.), The Hierarchy of Life. Elsevier, Amsterdam, pp. 437-450.
  12. Miyamoto, M. M., Kraus, F. and Ryder, O. A. 1990. Phylogeny and evolution of antlered deer determined from mitochondrial DNA mitosequences. Proc. Natl. Acad. Sci. USA 87:6127-6131. https://doi.org/10.1073/pnas.87.16.6127
  13. Miyamoto, M. M., Kraus, F., Laipis, P. J., Tanhauser, S. M. and Webb, S. D. 1993. Mitochondrial DNA phylogenies within Artiodactyla. In: Slalay, F.S., Novacek, M.J., McKenna, M.C. (Eds.), Mammal Phylogeny. Springer, New York, pp. 268-281.
  14. Oh, M. Y. and Jung, Y. H. 2001. Mitochodrial DNA Polymorphism in Cheju and Tsushima Native Horse using SSCP analysis. Korean J. Genetics 23(1):35-43.
  15. Orita, Iwahana, Kanazawa, Hayashi and Sekiya. 1989. Detection of polymorphisms of human DNA by the gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA. 86:2766-2770.
  16. Ursing, B. M. and Arnason, U. 1998. The complete mitochondrial DNA sequence of the pig (Sus scrofa). J. Mol. Evol. 47:302-306 https://doi.org/10.1007/PL00006388
  17. Xu, X. and Arnason, U. 1994. The complete mitochondrial DNA sequence of the horse, Equus caballus: extensive heteroplasmy of the control region. Gene 148:357-362.
  18. Xu, X., Gullberg, A. and Arnason, U. 1996. The complete mitochondrial DNA (mtDNA) of the Donkey and mtDNA comparisions among four closely related mammalian species-pairs. J. Mol. Evol. 43: 438-446.
  19. Xu, X., Janke, A. and Arnason, U. 1996. The complete mitochondrial DNA sequence of the Greater Indian Rhinoceros, Rhinoceros unicornis, and the phylogenetic relationship among Carnivora, Perissodactyla, and Artiodactyla (+Cetacea). Mol. Biol. Evol. 13(9):1167-1173.
  20. Xu, X. and Arnason, U. 1997. The complete mitochondrial DNA sequence of the White Rhinoceros, Ceratotherium simum, and comparision with the mtDNA sequence of the indian Rhinoceros, Rhinoceros unicornis. Molecular Phylogenetics and Evolution. 7(2):189-194.