DOI QR코드

DOI QR Code

Development and Characterization of Chloroplast Simple Sequence Repeat markers in Pinus koraiensis

잣나무 엽록체 Simple Sequence Repeat 표지자 개발 및 특성 분석

  • Lee, Jei-Wan (Division of Forest Genetic Resources, National Institute of Forest Science) ;
  • Baek, Seung-Hoon (Division of Forest Genetic Resources, National Institute of Forest Science) ;
  • Hong, Kyung-Nak (Division of Forest Genetic Resources, National Institute of Forest Science) ;
  • Hong, Yong-Pyo (Division of Forest Genetic Resources, National Institute of Forest Science) ;
  • Lee, Seok-Woo (Division of Forest Genetic Resources, National Institute of Forest Science) ;
  • Ahn, Ji-Young (Division of Forest Genetic Resources, National Institute of Forest Science)
  • 이제완 (국립산림과학원 산림유전자원과) ;
  • 백승훈 (국립산림과학원 산림유전자원과) ;
  • 홍경낙 (국립산림과학원 산림유전자원과) ;
  • 홍용표 (국립산림과학원 산림유전자원과) ;
  • 이석우 (국립산림과학원 산림유전자원과) ;
  • 안지영 (국립산림과학원 산림유전자원과)
  • Received : 2015.07.10
  • Accepted : 2015.08.10
  • Published : 2015.12.31

Abstract

Novel cpSSR primers were developed based on the sequence information of the Pinus koraiensis chloroplast genome. A total of 30 cpSSR loci were detected in the chloroplast genome, and a total of 30 primer sets flanking those loci were designed. All primer sets were successfully amplified for chloroplast DNA in P. koraiensis. The cross-species transferability of the 30 primer sets was considerably high in P. pumila (100%) and P. paviflora (97%) belonging to the same Subgenus (Strobus) of P. koraiensis. Meanwhile, the transferability was relatively low (73%) in P. densiflora and P. sylvestris belonging to Subgenus Pinus. A total of 13 cpSSR loci out of the 30 loci were polymorphic in the Mt. Jumbong population of P. koraiensis. The mean of haploid diversity(H) was 0.512. The number of haplotypes(N) and the haplotype diversity($H_e$) were 25 and 0.992, respectively. Of the 25 haplotypes, 22 were unique in the analyzed population. The unique haplotypes differentiated 22 individuals (79%) from the total of 28 individuals. In conclusion, the novel cpSSR primers developed in this study would be applicable to other Pinus species, especially the subgenus Strobus, and provide a high level of polymorphism for the study of genetic variation of P. koraiensis.

본 연구에서는 잣나무 엽록체 DNA의 전체 염기서열을 기반으로 엽록체 SSR(chloroplast simple sequence repeat) 영역을 특이적으로 증폭하는 primer를 개발하고 그 특성을 분석하였다. 잣나무 엽록체 DNA에서 총 30개의 SSR 영역을 탐색하였으며, 이들 영역을 증폭하기 위한 30개의 primer를 제작하였다. 모든 primer가 잣나무를 대상으로 PCR 증폭이 가능하였다. 근연종에 대한 primer의 종간 전환률은 잣나무와 동일한 아속(Subgenus Strobus)에 속하는 눈잣나무(100%)와 섬잣나무(97%)에서 가장 높게 나타났다. 반면 소나무아속(Subgenus Pinus)에 속하는 소나무와 구주소나무에서의 종간 전환률은 73%로 비교적 낮게 나타났다. 점봉산 잣나무 집단을 대상으로 조사한 결과 13개의 유전자좌에서 다형성이 관찰되었으며, 평균 haploid 다양도(H)는 0.512로 계산되었다. 다형적 유전자좌로부터 조합된 haplotype의 수(N)는 25개로 확인되었고, haplotype 다양도($H_e$)는 0.992로 매우 높게 나타났다. 집단내 독특하게 관찰되는 haplotype은 22개(88%)로 전체 28개체 중에서 22개체(79%)를 식별하였다. 본 연구에서 개발한 cpSSR primer는 높은 종간 전환률을 나타냄에 따라 소나무속의 근연종, 특히 잣나무아속 수종에 활용 가능성이 높고, 잣나무 유전변이 분석을 위한 충분한 다형성을 제공하는 유용한 표지자로 판단된다.

Keywords

References

  1. Bang, K.H., Lee, J.W., Kim, Y.C., Kim, D.H., Lee, E.H., and Jeung, J.U. 2010. Construction of genomic DNA library of Korean ginseng (Panax ginseng C.A. Meyer) and development of sequence-tagged sites. Biological and Pharmaceutical Bulletin 33(9): 1579-1588. https://doi.org/10.1248/bpb.33.1579
  2. Deguilloux, M.F., Pemonge, M.H., and Petit, R. J. 2004. Use of chloroplast microsatellites to differentiate oak populations. Annals of Forest Science 61(8): 825-830. https://doi.org/10.1051/forest:2004078
  3. Demesure, B., Sodzi, N., and Petit, R.J. 1995. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology 4(1): 129-134. https://doi.org/10.1111/j.1365-294X.1995.tb00201.x
  4. Feng, F.J., Han, S.J., and Wang, H.M. 2006. Genetic diversity and genetic differentiation of natural Pinus koraiensis population. Journal of Forestry Research 17(1): 21-24. https://doi.org/10.1007/s11676-006-0005-5
  5. Feng, F.J., Zhao, D., Sui, X., and Sun, X.Y. 2011. Study on mating system of Pinus koraiensis in natural population based on cpSSR technology. In Advanced Materials Research 183: 700-704.
  6. Filiz, E. and Koc, I. 2014. Assessment of chloroplast microsatellite from pine family (Pinaceae) by using bioinformatics tools. Indian Journal of Biotechnology 13: 34-40.
  7. Grauda, L., Aravanopoulos, F., Bennadji, Z., Changtragoon, S., Fady, B., Kjaer, E.D., Loo, J., Ramamonjisoa, L., and Vendramin, G.G. 2014. Global to local genetic diversity indicators of evolutionary potential in tree species within and outside forests. Forest Ecology and Management 333: 35-51. https://doi.org/10.1016/j.foreco.2014.05.002
  8. Grivet, D., Heinze, B., Vendramin, G.G., and Petit, R.J. 2001. Genome walking with consensus primers: application to the large single copy region of chloroplast DNA. Molecular Ecology Notes 1(4): 345-349. https://doi.org/10.1046/j.1471-8278.2001.00107.x
  9. Hong, K.N., Kwon, Y.J., Chung, J.M., Shin, C.H., Hong, Y.P., and Kang, B.Y. 2001. Spatial genetic structure at a Korean pine (Pinus koraiensis) stand on Mt. Jumbong in Korea based on isozyme studies. Journal of Korean Forest Society 90(1): 43-54.
  10. Hong, Y.P., Kwon, H.Y., Han, S.U., Choi, W.Y., and Kim, Y.Y. 2005. Identification of true full sib progenies of Japanese red pine via cpSSR haplotyping. Journal of Korean Forest Society 94(3): 178-182.
  11. Hong, Y.P., Kwon, H.Y., and Kim, Y.Y. 2006. Distribution pattern of cpSSR variants in Korean populations of Japanese red pine. Journal of Korean Forest Society 95(4): 435-442.
  12. Hong, Y.P., Kim, Y.M., Ahn, J.Y., and Park, J.I. 2012. Mating system of seed orchard of Japanese red pines revealed by DNA markers. Journal of Korean Forest Society 99(3): 344-352.
  13. Hong, Y.P., Ahn, J.Y. Kim, Y.M., Hong, K.N., and Yang, B.H. 2013. Mating System in Natural Population of Pinus koraiensis at Mt. Seorak Based on Allozyme and cpSSR Markers. Journal of Korean Forest Society 102(2): 264-271.
  14. Kong, W.S. 2004. Species Composition and Distribution of Native Korean Conifers. The Korean Geographical Society 39(4): 528-543.
  15. Lee, J.W. 2010. Development of DNA markers for identification of Korean ginseng cultivars. Dongguk University, Ph. D. thesis paper. pp. 113.
  16. Lee, J.W., Kim, Y.C., Jo, I.H., Seo, A.Y., Lee, J.H., Kim, O.T., Hyun D.Y., Cha S.W., Bang K.H., and Cho, J.H. 2011. Development of an ISSR-derived SCAR marker in Korean ginseng cultivars (Panax ginseng C.A. Meyer). Journal of Ginseng Research 35(1): 52-59. https://doi.org/10.5142/jgr.2011.35.1.052
  17. Melotto-Passarin, D.M., Tambarussi, E.V., Dressano, K., De Martin, V.F., and Carrer, H. 2011. Characterization of chloroplast DNA microsatellites from Saccharum spp and related species. Genetics Molecular Research 10(3): 2024-2033.
  18. Mudunuri, S.B. and Nagarajaram, H.A. 2007. IMEx: imperfect microsatellite extractor. Bioinformatics 23(10): 1181- 1187. https://doi.org/10.1093/bioinformatics/btm097
  19. Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89(3): 583-590.
  20. Peakall, R.O.D. and Smouse, P.E. 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6(1): 288-295. https://doi.org/10.1111/j.1471-8286.2005.01155.x
  21. Potenko, V.V., and Velikov, A.V. 1998. Genetic diversity and differentiation of natural populations of Pinus koraiensis (Sieb. et Zucc.) in Russia. Silvae Genetica 47(4): 202-207.
  22. Powell, W., Morgante, M., McDevitt, R., Vendramin, G.G., and Rafalski, J.A. 1995. Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines. Proceedings of the National Academy of Sciences 92(17): 7759-7763. https://doi.org/10.1073/pnas.92.17.7759
  23. Provan, J., Powell, W., and Hollingsworth, P.M. 2001. Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends in Ecology & Evolution 16(3): 142-147. https://doi.org/10.1016/S0169-5347(00)02097-8
  24. Provan, J., Soranzo, N., Wilson, N.J., Goldstein, D.B., and Powell, W. 1999. A low mutation rate for chloroplast microsatellites. Genetics 153(2): 943-947.
  25. Provan, J., Soranzo, N., Wilson, N.J., McNicol, J.W., Forrest, G.I., Cottrell, J., and Powell, W. 1998. Gene pool variation in Caledonian and European Scots pine (Pinus sylvestris L.) revealed by chloroplast simple sequence repeats. Proceedings of the Royal Society of London B: Biological Sciences 265(1407): 1697-1705. https://doi.org/10.1098/rspb.1998.0491
  26. Raubeson, L.A. and Jansen, R.K. 1992. A rare chloroplast-DNA structural mutation is shared by all conifers. Biochemical Systematics and Ecology 20(1): 17-24. https://doi.org/10.1016/0305-1978(92)90067-N
  27. Richardson, B.A., Brunsfeld, S.J., and Klopfenstein, N.B. 2002. DNA from bird-dispersed seed and wind-disseminated pollen provides insights into postglacial colonization and population genetic structure of whitebark pine (Pinus albicaulis). Molecular Ecology 11(2): 215-227. https://doi.org/10.1046/j.1365-294X.2002.01435.x
  28. Sablok, G., Mudunuri, S.B., Patnana, S., Popova, M., Fares, M.A., and La Porta, N. 2013. ChloroMitoSSRDB 2.0: Open source repository of perfect and imperfect repeats in organelle genomes for evolutionary genomics. DNA Research 20(2): 127-133. https://doi.org/10.1093/dnares/dss038
  29. Schlotterer, C. and Tautz, D. 1992. Slippage synthesis of simple sequence DNA. Nucleic Acids Research 20(2): 211-215. https://doi.org/10.1093/nar/20.2.211
  30. Sia, E.A., Jinks-Robertson, S., and Petes, T.D. 1997. Genetic control of microsatellite stability. Mutation Research 383: 61-70. https://doi.org/10.1016/S0921-8777(96)00046-8
  31. Strauss, S.H., Palmer, J.D., Howe, G.T., and Doerksen, A.H. 1988. Chloroplast genomes of two conifers lack a large inverted repeat and are extensively rearranged. Proceedings of the National Academy of Sciences 85(11): 3898- 3902. https://doi.org/10.1073/pnas.85.11.3898
  32. Sugiura, M. 1992. The chloroplast genome. Plant Molecular Biology 19: 149-168. https://doi.org/10.1007/BF00015612
  33. Sun, X. and Feng, F. 2011. Development and Analysis on Microsatellite Sequence of Chloroplast DNA of Pinus koraiensis. In Bioinformatics and Biomedical Engineering,( iCBBE) 2011 5th International Conference. IEEE. pp. 1-4.
  34. Taberlet, P., Gielly, L., Pautou, G., and Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17(5): 1105- 1109. https://doi.org/10.1007/BF00037152
  35. Toth, G., Gaspari, Z., and Jurka, J. 2000. Microsatellites in different eukaryotic genomes: survey and analysis. Genome Research 10(7): 967-981. https://doi.org/10.1101/gr.10.7.967
  36. Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M., and Rozen, S.G. 2012. Primer3-new capabilities and interfaces. Nucleic Acids Research 40(15): e115-e115. https://doi.org/10.1093/nar/gks596
  37. Vendramin, G.G., Lelli, L., Rossi, P., and Morgante, M. 1996. A set of primers for the amplification of 20 chloroplast microsatellites in Pinaceae. Molecular Ecology 5(4): 595-598. https://doi.org/10.1111/j.1365-294X.1996.tb00353.x
  38. Wolfe, K.H., Li, W.H., and Sharp, P.M. 1987. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences 84(24): 9054-9058. https://doi.org/10.1073/pnas.84.24.9054