한국산림과학회지 (Journal of Korean Society of Forest Science)

  • 제105권2호
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  • Pages.186-192
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  • 2016
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  • 2586-6613(pISSN)
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  • 2586-6621(eISSN)
한국산림과학회 (Korean Society of Forest Science)
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차세대 염기서열 분석을 이용한 굴참나무(Quercus variabilis)의 microsatellite 마커 개발 및 특성 분석

Identification and Characterization of Polymorphic Microsatellite Loci using Next Generation Sequencing in Quercus variabilis

  • 백승훈 (국립산림과학원 산림유전자원과) ;
  • 이제완 (국립산림과학원 산림유전자원과) ;
  • 홍경낙 (국립산림과학원 산림유전자원과) ;
  • 이석우 (국립산림과학원 산림유전자원과) ;
  • 안지영 (국립산림과학원 산림유전자원과) ;
  • 이민우 (국립산림과학원 산림유전자원과)
  • Baek, Seung-Hoon (Division of Forest Genetic Resources, National Institute of Forest Science) ;
  • Lee, Jei-Wan (Division of Forest Genetic Resources, National Institute of Forest Science) ;
  • Hong, Kyung-Nak (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) ;
  • Lee, Min-Woo (Division of Forest Genetic Resources, National Institute of Forest Science)
  • 투고 : 2016.02.29
  • 심사 : 2016.04.04
  • 발행 : 2016.06.30
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초록

본 연구는 차세대 염기서열 분석방법을 이용하여 굴참나무의 microsatellite 마커를 개발하고 특성을 분석하기 위해 수행되었다. GS-FLX Titanium 차세대 염기서열 분석 장비를 이용하여 305,771개의 read를 얻었고, 117 Mbp의 데이터를 생산하였다. De novo assembly를 통하여 7,326개의 contig를 확보하였다. 크기가 500 bp 이상이 되는 contig는 2,921개로 나타났다. 그 중 microsatellite 영역을 포함하는 contig는 606개(20.75%)로 나타났으며, 총 microsatellite의 수는 911개로 확인되었다. 그 중 13개의 microsatellite 유전자좌에서 굴참나무 개체 간 다형성이 관찰되었다. 이들 microsatellite 유전자좌에 대하여 주왕산 집단에서 관찰된 유효 대립유전자수($A_e$)는 평균 4.966(2.439~7.515)로 나타났다. 평균 이형접합도 관측치($H_o$)와 평균 이형접합도 기대치($H_e$)는 각각 0.873(0.731~1.000)과 0.766(0.590~0.867)으로 나타났다. 다형성이 관찰된 모든 microsatellite 유전자좌에서 null 대립유전자는 관찰되지 않았으며, 마커 간 연관불평형은 나타나지 않았다. 따라서 본 연구에서 개발된 13개의 microsatellite 마커는 굴참나무 집단의 유전변이 분석에 유용할 것으로 사료된다.

This study was conducted to develop microsatellite markers in Quercus variabilis using next generation sequencing. A total of 305,771 reads (384 bp on average) were generated on a Roche GS-FLX system, yielding 117 Mbp of sequences. The de novo assembly resulted in 7,346 contigs. A total of 606 contigs (20.75%) including 911 microsatellite loci were derived from the 2,921 contigs longer than 500 bp. A total of 180 primer sets were designed from the 911 microsatellite loci and screened in eight Q. variabilis individual trees sampled from a natural stand to obtain polymorphic loci. As a result, a total of thirteen polymorphic microsatellite loci were selected and used for estimating population genetic parameters in the 54 individual trees. The mean number of effective alleles was 4.996 ranging from 2.439 to 7.515. The observed heterozygosity and the expected heterozygosity ranged between 0.731 and 1.000 with an average of 0.873 and from 0.590 to 0.867 with an average of 0.766, respectively. Null alleles were not detected in all loci. No significant linkage disequilibrium was detected after Bonferroni correction in all loci. In the near future, these novel polymorphic microsatellite markers will be used to study population and conservation genetics of Q. variabilis of Korea in more detail.

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참고문헌

  1. Botstein, D., White, R.L., Skolnick, M., and Davis, R.W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics. 32(3): 314.
  2. Brondani, R.P.V., Brondani, C., Tarchini, R., and Grattapaglia, D. 1998. Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla. Theoretical and Applied Genetics 97(5-6): 816-827. https://doi.org/10.1007/s001220050961
  3. Chatwin, W.B., Carpenter, K.K., Jimenez, F.R., Elzinga, D.B., Johnson, L.A., and Maughan, P.J. 2014. Microsatellite primer development for post oak, Quercus stellata (Fagaceae). Applications in Plant Sciences 2(10).
  4. Chen, D., Zhang, X., Kang, H., Sun, X., Yin, S., Du, H., Yamanaka, N., Gapare, W., Wu, H.X., and Liu, C. 2012. Phylogeography of Quercus variabilis based on chloroplast DNA sequence in East Asia: multiple glacial refugia and mainland-migrated island populations. PloS One 7(10): e47268. https://doi.org/10.1371/journal.pone.0047268
  5. Coburn, J.R., Temnykh, S.V., Paul, E.M., and McCOUCH, S.R. 2002. Design and Application of Microsatellite Marker Panels for Semiautomated Genotyping of Rice (L.). Crop Science 42(6): 2092-2099. https://doi.org/10.2135/cropsci2002.2092
  6. COP10, C.B.D. 2010. Strategic Plan for Biodiversity, 2011-2020. In Conference of the Parties to the Convention on Biological Diversity, Nagoya, Japan.
  7. Dakin, E.E. and Avise, J.C. 2004. Microsatellite null alleles in parentage analysis. Heredity 93(5): 504-509. https://doi.org/10.1038/sj.hdy.6800545
  8. Faircloth, B.C. 2008. msatcommander: detection of microsatellite repeat arrays and automated, locus-specific primer design. Molecular Ecology Resources 8(1): 92-94. https://doi.org/10.1111/j.1471-8286.2007.01884.x
  9. Gardner, M.G., Fitch, A.J., Bertozzi, T., and Lowe, A.J. 2011. Rise of the machines-recommendations for ecologists when using next generation sequencing for microsatellite development. Molecular Ecology Resources 11(6): 1093-1101. https://doi.org/10.1111/j.1755-0998.2011.03037.x
  10. Goudet, J. 2002. FSTAT 2.9. 3: a program to estimate and test gene diversities and fixation indices (updated from Goudet 1995).
  11. Guo, S.W. and Thompson, E.A. 1992. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 361-372.
  12. Kremer, A,, Casasoli, M., Barreneche, T., Bodenes, C., Sisco, P., Kubisiak, T., Scalfi, M., Leonardi, S., Bakker, E.G., Buiteveld, J., Romero-Severson, J., Arumuganathan, K., Derory, J., Scotti-Saintagne, C., Roussel, G., Bertocchi, M.E., Lexer, C., Porth, I., Hebard, F., Clark, C., Carlson, J., Plomion, C., Koelewijn, H., Villani, F., 2007. Fagaceae trees. pp. 161-187. In: C. Kol, ed. Genome Mapping and Molecular Breeding in Plants. Springer, Berlin Heidelberg.
  13. Lee, Y.N. 2006. New flora of Korea. Kyohaksa, Seoul, Korea. pp. 975.
  14. 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
  15. Pinto, L.R., Oliveira, K.M., Marconi, T., Garcia, A.A.F., Ulian, E.C., and De Souza, A.P. 2006. Characterization of novel sugarcane expressed sequence tag microsatellites and their comparison with genomic SSRs. Plant Breeding 125(4): 378-384. https://doi.org/10.1111/j.1439-0523.2006.01227.x
  16. Rousset, F. 2008. genepop'007: a complete re-implementation of the genepop software for Windows and Linux. Molecular Ecology Resources 8(1): 103-106. https://doi.org/10.1111/j.1471-8286.2007.01931.x
  17. Saeki, I., Hirao, A.S., and Kenta, T. 2015. Development and evaluation of microsatellite markers for Acer miyabei (Sapindaceae), a threatened maple species in East Asia. Applications in Plant Sciences 3(6): 1500020. https://doi.org/10.3732/apps.1500020
  18. Sonah, H., Deshmukh, R.K., Sharma, A., Singh, V.P., Gupta, D.K., Gacche, R.N., Rana, J.C., Singh N.K., and Sharma, T.R. 2011. Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium. Plos One 6(6): e21298. https://doi.org/10.1371/journal.pone.0021298
  19. Song, J.H., Kim, N.S., Yi, Y.S., Kim, Y.J., Song, J.M., and Yi, J.S. 2002. Genetic variation of Quercus variabilis in Korea based on RAPD marker analysis. Korean Journal of Genetics 24(2): 189-195.
  20. Tautz, D. 1989. Hypervariabflity of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Research 17(16): 6463-6471. https://doi.org/10.1093/nar/17.16.6463
  21. 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 Rresearch 40(15): e115-e115. https://doi.org/10.1093/nar/gks596
  22. Van Oosterhout, C., Hutchinson, W.F., Wills, D.P., and Shipley, P. 2004. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4(3): 535-538. https://doi.org/10.1111/j.1471-8286.2004.00684.x
  23. Varshney, R.K., Graner, A., and Sorrells, M.E. 2005. Genic microsatellite markers in plants: features and applications. Trends in Biotechnology 23(1): 48-55. https://doi.org/10.1016/j.tibtech.2004.11.005
  24. Wang, L., Wang, Z., Chen, J., Liu, C., Zhu, W., Wang, L., and Meng, L. 2015a. De Novo transcriptome assembly and development of novel microsatellite markers for the traditional chinese medicinal herb, Veratrilla baillonii Franch (Gentianaceae). Evolutionary Bioinformatics 11: 39-45.
  25. Wang, X., Li, J., and Li, Y. 2015b. Isolation and characterization of microsatellite markers for an endemic tree in East Asia, Quercus variabilis (Fagaceae). Applications in Plant Sciences 3(6): 1500032. https://doi.org/10.3732/apps.1500032
  26. Weir, B.S. 1979. Inferences about linkage disequilibrium. Biometrics 235-254.
  27. Welsh, J. and McClelland, M. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18(24): 7213-7218. https://doi.org/10.1093/nar/18.24.7213
  28. Xu, X.L., Xu, L.A., Huan, M.R., and Wang, Z.R. 2004. Genetic diversity of microsatellites (SSRs) of natural populations of Quercus variabilis. Yi chuan=Hereditas/Zhongguo yi chuan xue hui bian ji 26(5): 683-688.
  29. Zalapa, J.E., Cuevas, H., Zhu, H., Steffan, S., Senalik, D., Zeldin, E., McCown B., Harbut R., and Simon, P. 2012. Using nextgeneration sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences. American Journal of Botany 99(2): 193-208. https://doi.org/10.3732/ajb.1100394
  30. Zane, L., Bargelloni, L., and Patarnello, T. 2002. Strategies for microsatellite isolation: a review. Molecular Ecology 11(1): 1-16. https://doi.org/10.1046/j.0962-1083.2001.01418.x
  31. Zhang, L., Yan, H.F., Wu, W., Yu, H., and Ge, X.J. 2013. Comparative transcriptome analysis and marker development of two closely related Primrose species (Primula poissonii and Primula wilsonii). BMC Genomics 14(1): 1. https://doi.org/10.1186/1471-2164-14-1
  32. Zhou, J.Y., Guo, J.Z., and Yang, Z.S. 2003. Variation of peroxidase isozyme on natural populations of Quercus variabilis. Journal-Northwest Forestry University 18(2): 33-36.
  33. Zhu, H., Senalik, D., McCown, B.H., Zeldin, E.L., Speers, J., Hyman, J., Bassil, N., Hummer, P., Simon, W., and Zalapa, J.E. 2012. Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (Vaccinium macrocarpon Ait.). Theoretical and Applied Genetics 124(1): 87-96. https://doi.org/10.1007/s00122-011-1689-2

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