제초제 저항성 유전자와 기존 병 저항성 유전자가 연관된 형질전환 토마토 개체 선발 및 후대분석

Progeny Analysis and Selection of Tomato Transformants with patII Gene linked to Inherent Disease Resistance Gene

  • 투고 : 2011.04.07
  • 심사 : 2011.05.09
  • 발행 : 2011.08.30

초록

원예작물의 내병성 육종에 있어 $F_2$와 그 이후 분리 세대에서 유용하게 사용할 수 있는 선발방법의 모델 시스템을 개발하고자 하였다. 상용 토마토 품종인 '모모따로 요크'에 제초제 저항성 유전자를 도입한 후 획득된 형질전환체의 도입 유전자 수와 후대 분리비를 분석하고, 제초제 저항성 유전자와 기존 병 저항성 유전자가 연관된 개체를 선발하였다. 총 60개의 형질전환체 중에서 42개체에 Southern blot을 실시하여 도입 유전자 수를 확인하고, 제초제 저항성 유전자에 대한 분리비를 비교 분석하여 Southern 결과와 도입된 유전자의 표현형의 분리비가 일치하지 않으며 여러 개의 copy가 삽입된 대부분의 경우, 실제 도입된 copy 수보다 더 적은 copy 수를 가지는 것처럼 분리비를 나타내는 것을 알 수 있었다. 삽입된 제초제 저항성 유전자와 기존 병 저항성 유전자가 가깝게 연관된 개체를 찾기 위해 $T_1(F_2)$세대 개체에 대하여 two-stepwise screening 방법을 실시하여 위조병 저항성 I2 유전자(11번 염색체)와 제초제 저항성 patII 유전자가 대략 12-13cM으로 가깝게 연관된 개체(T-20)를 선발하였다.

This study was carried out to develop a model system using selection method for disease resistant plant breeding programs using a herbicide bialaphos-resistant patII gene as a gene-based marker. Spraying bialaphos could eliminate the susceptible plants from the segregating populations such as ${F_2}^{\prime}s$ and thereafter. Tomato cv. Momotaro-yoke was transformed with patII gene 60 independent transformants were acquired. Total 42 transformants were analyzed in transgene copy numbers by Southern blotting and the segregation ratios for the bialaphos resistance. Statistical analysis revealed that the transgene copy numbers and the segregation ratios were not always coincided, especially having the tendency of underestimating the real numbers of the transgenes in the multicopy lines. A two-stepwise screening method was applied to select $T_1$ tomato plants which linked the transgenic patII to a disease resistance gene (I2 and Ve). Based on the resistant to susceptible ratios, T-20 plant was finally selected due to the estimated linkage 12-13 cM between the patII gene to the I2 gene on chromosome 11. This newly developed system could be applied to any economical crop in breeding programs.

키워드

참고문헌

  1. Atibalentja, N. and D.M. Eastburn. 1997. Evaluation of inoculation methods for screening horseradish cultivars for resistance to Verticillium dahliae. Plant Dis. 81:356-362. https://doi.org/10.1094/PDIS.1997.81.4.356
  2. du Toit, L.J. and J.K. Pataky. 1999. Variation associated with silk channel inoculation for common smut of Sweet Corn. Plant Dis. 83:727-732. https://doi.org/10.1094/PDIS.1999.83.8.727
  3. James, V.A., C. Avart, B. Worland, J.W. Snape, and P. Vain. 2002. The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor. Appl. Genet. 104:533-561. https://doi.org/10.1007/s00122-001-0787-y
  4. Jung, S.M., H.K. Yun, K.B. Ma, G.C. Song, S.J. Park, and J.B. Kim. 2008. Influential factors for identifying levels of resistance and in vitro screening system against gray mold in grapevines. Kor. J. Hort. Sci. Technol. 26:432-439.
  5. Kuraya, Y., S. Ohta, M. Fukuda, Y. Hiei, N. Murai, K. Hamada, J. Ueki, H. Imaseki, and T. Komari. 2004. Suppression of transfer of non-T-DNA 'vector backbone' sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens. Mol. Breed. 14:309-320.
  6. Lee, J.D., J.B. Yoon, and H.G. Park. 2008. A CAPS marker associated with the partial restoration of cytoplasmic male sterility in chili pepper (Capsicum annuum L.). Mol. Breeding 21:95-104.
  7. Lim, S.H, S.H. Park, J.H. Kim, N.Y. Kim, S.Y. Won, S.M. Lee, K.S. Shin, H.J. Woo, D.H. Kim, and H.S. Cho. 2008. Analysis of junction between T-DNA and plant genome in insect resistance GM Chinese cabbage. K. J. Plant Biotech. 35:101-108. https://doi.org/10.5010/JPB.2008.35.2.101
  8. Murray, M.G. and W.F. Thompson. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8:4321-4325. https://doi.org/10.1093/nar/8.19.4321
  9. Sambrook, J. and D.W. Russell. 1989. Analysis of genomic DNA by southern hybridization. p. 9.31-9.46. In: Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  10. Shin, W.C., S.H. Baek, C.S. Seo, H.J. Kang, C.G. Kim, M.S. Shin, G.S. Lee, J.H. Harn, and H.S. Kim. 2006. Improvement of selection efficiency for bacterial blight resistance using SNP marker in rice. J. Plant Biotechnol. 33:309-313. https://doi.org/10.5010/JPB.2006.33.4.309
  11. Spencer, T.M., J.V. O'Brien, W.G. Start, T.R. Adams, W.J. Gordon-Kamm, and P.G. Lemaux. 1992. Segregation of transgenes in maize. Plant Mol. Biol. 18:201-210. https://doi.org/10.1007/BF00034949
  12. Steventon, L.A., I. Happstadius, P. Okori, and C. Dixelius. 2002. Development of a rapid technique for the evaluation of the response of Brassica napus to Verticillium wilt. Plant Dis. 86:854-858. https://doi.org/10.1094/PDIS.2002.86.8.854
  13. Vain, P., A.S. Afolabi, B. Worland, and J.W. Snape. 2003. Transgene behavior in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup. Theor. Appl. Genet. 107:210-217. https://doi.org/10.1007/s00122-003-1255-7
  14. Vain, P., V.A. James, B. Worland, and J.W. Snape. 2002. Transgene behavior across two generations in a large random population of transgenic rice plants produced by particle bombardment. Theor. Appl. Genet. 105:878-889. https://doi.org/10.1007/s00122-002-1039-5
  15. Viss, W.J., J. Pitrak, J. Humann, M. Cook, J. Driver, and W. Ream. 2003. Crown-gall-resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes. Mol. Breed. 12:283-295.
  16. Walters, D.A., C.S. Vetsch, D.E. Potts, and R.C. Lundquist. 1992. Transformation and inheritance of a hygromycin phosphotransferase gene in maize plants. Plant Mol. Biol. 18:189-200. https://doi.org/10.1007/BF00034948
  17. Yoon, J.B. and H.G. Park. 2001. Screening method for resistance to pepper fruit Anthracnose: Pathogen sporulation, inoculation methods related to inoculum concentrations and post-inoculation environment. J. Kor. Soc. Hort. Sci. 42:365-482.
  18. Yoon, J.B., D.C. Yang, W.P. Lee, S.Y. Ahn, and H.G. Park. 2004. Genetic resources resistant to anthracnose in the genus capsicum. J. Kor. Soc. Hort. Sci. 45:318-323.
  19. Yu, K., S.J. Park, and V. Poysa. 2000. Marker-assisted selection of common beans for resistance to common bacterial blight: Efficiency and economics. Plant Breeding 119:411-415. https://doi.org/10.1046/j.1439-0523.2000.00514.x