Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Development of Sequence Characterized Amplified Regions (SCAR) Showing for Cheju Native Horse

품종 특이성을 이용한 제주마 판별 표지인자 재발

  • Cho Byung Wook (Department of Animal Science, Miryang National University)
  • 조병욱 (밀양대학교 동물자원학과)
  • Published : 2005.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

This study was conducted to analyze genetic characteristics and to develop the specific marker for Cheju native horse (Coo) at the level of sequence characterized amplified regions (SCARs). We collected blood samples from Cheju native horse and Thoroughbred horse (Th) and obtained genomic DNA from the blood of 50 individuals randomly selected within the breeds. Seven hundred primers were chosen randomly and were used to examin the polymorphism and 40 kinds of primers showed polymorphic RAPD band patterns between two breeds. Thirty primers of them showed horse specific bands. With the primer MG 30, amplified band of 2.0 kb showed the specificity to Cheju native horse (Cnh). Additionally MG 53 detected the thoroughbred horse (Th) specific markers at size of 2.3 kb. As the next, 2.3 kb band from MG 53 was checked with the all individuals from all the breeds of this study, and it maintained the reproducible breed specificity to thoroughbred horse (Th). With this results, 2.3 kb band was cloned into plasmid vector and sequenced bidirectionally from both ends of the cloned fragment. With the obtained sequences 10 nucleotide extended primers including the original arbitray primer were designed as a SCARs primer. Finally, the primer with extended sequence showed the reproducible breed differentiation pattern and it was possible to identify Cheju native horse (Cnh) from other breeds. The SCARs marker 2.3 kb from MG 53 could be used to identify Cheju native horse (Cnh) for not only registration but also horse breeding programe.

본 연구는 RAPD 기법을 이용한 종 특이 marker 개발 및 이 marker의 SCAR marker로의 개발을 목표로 수행되었다. Random primer 700개에 대하여 PCR 수행결과, 품종간, 개체간에 많은 다형성이 관찰되었으며 품종특이적인 양상을 나타내는 MG30, MG53의 primer는 각각 2.0kb, 2.3kb의 위치에서 제주말과 더러브렛종의 특이적인 RAPD 단편을 나타내었다. 이들 단편들 중 품종 특이적인 단편을 클로닝한 후 random primer가 포함된 부분의 염기서 열을 결정하였다. 10 bp의 RAPD random primer에 10bp의 염기를 추가하여 SCAR primer를 제작하였다. SCAR marker의 수행결과 RAPD marker와 같은 2.3kb, 2.0kb의 크기에서 제주마와 더러브렛종에 특이적인 하나의 밴드가 증폭되었다. 따라서 이 Cnh-SCAR marker는 보다 안정적이고 재현성 있는 marker로서 사용이 가능하여 제주말의 판별에 유용하게 사용될 수 있을 것이다.

Keywords

References

  1. Bailey, E. and T. L. Lear. 1994. Comparison of Thoroughbred and Arabian horse using RAPD markers. Anim Genet. 25, 105-108 https://doi.org/10.1111/j.1365-2052.1994.tb00414.x
  2. Cargill, S. L., G. B. Anderson and J. F. Medrano. 1995. Development of a species-specific marker using RAPD analysis to distinguish between sheep and goats. Anim. Biotechnol. 6, 93-100 https://doi.org/10.1080/10495399509525837
  3. Cho, B. W., K. C. Hwang, H. K. Lee, K. J. Lee and J. Y. Han. 1997. Application of DNA markers to characterize cattle breed using random amplified polymorphic DNAs. Anim. Genet. and Breeding. 1, 39-48
  4. Hong, Y. H., J. C. Cheong, T. H. Kim, H. B. Kim, D. H. Yoon, H. S. Kim, B. W. Cho and J. Y. Han. 1998. Development of sequence characterized amplified regions (SCARs) showing Korean cattle (Hanwoo) specificity. Anim. Genet. and Breeding. 2, 107-114
  5. Hayash, K. 1991. PCR_SSCP: A simple and sensitive method for detection of mutations in genomic DNA. PCR Meth. Appl. 1, 34-38 https://doi.org/10.1101/gr.1.1.34
  6. Jeffrey, A. J., V. Wilson and S. J. Thein. 1985. Individual specific fingerprints of human DNA. Nature 316, 76 https://doi.org/10.1038/316076a0
  7. Ishida, N., T. Hasegawa, T. Oyunsuren and H. Mukoyama. 1996. PCR-RFLP analysis of the cytochrome b gene in horse mitochondrial DNA. Anim. Genet. 27, 359-363 https://doi.org/10.1111/j.1365-2052.1996.tb00979.x
  8. Kemp. S. J. and A. J. Teale. 1994. Randomly primed PCR amplification of pooled DNA reveal polymorphism in a ruminant repetitive DNA sequence which differentiated Bas indicus and Bas taurus, the major subspecies of domestic cattle. Anim Genet. 25, 82-88
  9. Kim, K. I., Y. H. Yang, S. S. Lee, C. Park, R. Ma, J. J. Bouzat and H. A. Lewin. 1999. Phylogenetic relationships of Cheju horses to other horse breeds as determined by mtDNA D-loop sequence polymorphism. Anim. Genet. 30, 102-108 https://doi.org/10.1046/j.1365-2052.1999.00419.x
  10. Micheli, M. R., R. Bova., E. Pascale and E. D' Ambrosio. 1994. Reproducibility of Random Amplified Polymorphic DNA (RAPD) analysis among laboratories. PCR Method. Appl. 2, 341-345
  11. Myers, R. M., T. Maniatis and J. S. Lerman. 1987. Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol. 155, 501-527 https://doi.org/10.1016/0076-6879(87)55033-9
  12. Paran, I. and R. Michelmore, 1993. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor. Appl. Genet. 85, 985-993
  13. Plotsky, Y., M. G. Kaiser and S. J. Lamonts. 1995. Genetic characterization of highly inbred chicken lines by two DNA methods: DNA fingerprinting and polymerase chain reaction using arbitrary primers. Anim. Genet. 26, 163-170 https://doi.org/10.1111/j.1365-2052.1995.tb03156.x
  14. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning of a laboratory manual. 2nd Ed. Cold Spring Habor Laboratory
  15. Welsh, J. and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitary primers. Nucleic Acids Res. 18, 7213-7218 https://doi.org/10.1093/nar/18.24.7213
  16. Williams, J. G. K., A. R Kubelik, J. A. Rafalski and S. V. Tingy. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531-6535 https://doi.org/10.1093/nar/18.22.6531
  17. Waugh, R. and W. Powell. 1992 using RAPD markers for crop improvement. TIBTECH 10, 186-191 https://doi.org/10.1016/0167-7799(92)90212-E