Skip to main content
Download PDF
  • Research Article
  • Published:

Real-time PCR for the detection of precise transgene copy number in durum wheat

Cellular & Molecular Biology Letters volume 16, Article number: 652 (2011) Cite this article

Abstract

Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.

This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

Abbreviations

FISH:

fluorescence in situ hybridization

GM:

genetically modified

HMW-GS:

high molecular weight glutenin subunits

MGB:

minor groove binder

PCR:

polymerase chain reaction

SDS:

Sequence Detection System

References

  1. De Preter, K., Speleman, F., Combaret, V., Lunec, J., Laureys, G., Eusson, B.H., Francotte, N., Pearson, A.D., De Paepe, A, van Roy, N. and Vandersompele J. Quantification of MYC-N, DDX I and NAG gene copy number in neuroblastoma using real time quantitative PCR assay. Mod. Pathol. 15 (2002) 150–166.

    Google Scholar 

  2. Mason, G., Provero, P., Vaira, A.M. and Accotto, G.P. Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotech. 24 (2002) 2–20.

    Google Scholar 

  3. Ingham, D.J., Beer, S., Money, S. and Hansen, G. Quantitative realtime PCR assay for determining transgene copy number in transformed plants. Biotechniques 31 (2001) 132–140.

    PubMed  CAS  Google Scholar 

  4. Li, Z., Hansen, J.L., Liu, Y., Zemetra, R.S. and Berger, P.H. Using real-time PCR to Determine transgene copy number in wheat. Plant Mol. Biol. Rep. 22 (2004) 179–188.

    Article  CAS  Google Scholar 

  5. Bubner, B. and Baldwin, I.T. Use of real-time PCR for determining copy number and zygosity in transgenic plant. Plant Cell Rep. 23 (2004) 263–271.

    Article  PubMed  CAS  Google Scholar 

  6. Yang, L., Ding, J., Zhang, C., Jia, J., Weng, H., Liu, W. and Zhang, D. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep. 23 (2005) 759–763.

    Article  PubMed  CAS  Google Scholar 

  7. Schmidt, M.A. and Parrot, W.A. Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Rep. 20 (2001) 422–428.

    Article  CAS  Google Scholar 

  8. Bubner, B., Gase, K. and Baldwin, I.T. Two-fold differences are the detection limit for determining transgene copy numbers in plants by realtime PCR. BMC Biotech. 4:14 (2004) 1–11.

    Google Scholar 

  9. Mackay, I.M., Arden, K.E. and Nitshe, A. Real-time PCR in virology. Nucleic Acid Res. 30 (2002) 1292–1305.

    Article  PubMed  CAS  Google Scholar 

  10. Mackay, I.M. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect. 10 (2004) 190–212.

    Article  PubMed  CAS  Google Scholar 

  11. Mayer, Z., Bagnara, A., Farber, P. and Geisen, R. Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods. I. J. Food Microb. 82 (2003) 143–151.

    Article  CAS  Google Scholar 

  12. Schnerr, H., Niessen, L. and Vogel, R.F. Real-time detection of the tri5 gene in Fusarium species by LightCycler™-PCR using SYBR-Green I for continuous fluorescence minitoring. I. J. Food Microb. 71 (2001) 53–61.

    Article  CAS  Google Scholar 

  13. Vaitilingom, M., Pijnenburg, H., Gendre, F. and Brignon, P. Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods. J. Agric. Food Chem. 47 (1999) 5261–5266.

    Article  PubMed  CAS  Google Scholar 

  14. Shewry, P.R., Halford, N.G., Tatham, A.S., Popineau, Y., Lafiandra D. and Belton, P.S. The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties. Adv. Food Nutr. Res. 45 (2003) 219–302.

    Article  PubMed  CAS  Google Scholar 

  15. Jones, H.D. Wheat transformation current technology and applications to grain development and composition. J. Cereal Sci. 41 (2005) 137–147.

    Article  CAS  Google Scholar 

  16. Blechl, A., Lin, J., Nguyen, S., Chan, R., Anderson, O.D. and Dupont, F.M. Transgenic wheats with elevated levels of Dx5 and/or Dy10 high-molecular-weight glutenin subunits yield doughs with increased mixing strength and tolerance. J. Cereal Sci. 45 (2007) 172–183.

    Article  CAS  Google Scholar 

  17. Barro, F., Rooke, L., Bekes, F., Gras, P., Tatham, A.S., Fido, R., Lazzeri, P.A., Shewry, P.R. and Barcelo, P. Transformation of wheat with high molecular weight subunit genes results in improved functional properties. Nat. Biotech. 15 (1997) 1295–1299.

    Article  CAS  Google Scholar 

  18. Rooke, I., Barro, F., Tatham, A.S., Fido, R., Steele, S., Békés, F., Gras, P., Martin, A., Lazzeri, P.A., Shewry, P.R. and Barcelo, P. Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes. Theor. Appl. Genet. 99 (1999) 851–858.

    Article  CAS  Google Scholar 

  19. Sangtong, V., Moran, D.L., Chikwamba, R., Wang, K.W., Woodman-Clikeman, M.J., Long, M., Lee, M. and Scott, P. Expression and inheritance of the wheat Glu-1Dx5 gene in transgenic maize. Theor. Appl. Genet. 105 (2002) 937–945.

    Article  PubMed  CAS  Google Scholar 

  20. Altpeter, F., Popelka, J.C. and Wieser, H. Stable expression of 1Dx5 and 1Dy10 high molecular weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutenin fraction in rye flour. Plant Mol. Biol. 54 (2004) 783–792.

    Article  PubMed  CAS  Google Scholar 

  21. Blechl, A.E. and Anderson, O.D. Expression of a novel high-molecularweight glutenin subunit gene in transgenic wheat. Nat. Biotech. 14 (1996) 875–879.

    Article  CAS  Google Scholar 

  22. Leon, E., Marín, S., Gimenez, M.G., Piston, F., Rodriguez-Quijano, M., Shewry, P.R. and Barro, F. Mixing properties and dough functionality of transgenic lines of a commercial wheat cultivar expressing the 1Ax1, 1Dx5 and 1Dy10 HMW glutenin subunit genes. J. Cereal Sci. 49 (2009) 148–156.

    Article  CAS  Google Scholar 

  23. Gadaleta, A., Blechl, A.E., Nguyen, S., Cardone, M.F., Ventura, M. and Blanco, A. Stable inheritance and expression of D-genome derived HMW glutenin subunit genes transformed into different durum wheat genotypes. Mol. Breed. 22 (2008) 267–279.

    Article  CAS  Google Scholar 

  24. Iglesias, V.A., Moscone, E.A., Papp, I., Neuhuber, F., Michalowski, S., Phelan, T., Spiker, S., Matzke, M. and Matzke, A.J.M. Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell 9 (1997) 1251–1264.

    Article  PubMed  CAS  Google Scholar 

  25. Srivastava, V., Vasil, V. and Vasil, I.K. Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.). Theor. Appl. Genet. 92 (1996) 1031–1037.

    Article  CAS  Google Scholar 

  26. Anderson, O.D., Greene, F.C., Yip, R.E., Halford, N.G., Shewry, P.R. and Malpica-Romero, J-M. Nucleotide sequences of the two high-molecular weight glutenin genes form the D-genome of a hexaploid bread wheat, Triticum aestivum cv. Cheyenne. Nucleic Acids Res. 17 (1989) 461–462.

    Article  PubMed  CAS  Google Scholar 

  27. Sharp, P.J., Kreis, M., Shewry, P.R. and Gale, M.D. Location of α-amylase sequences in wheat and its relatives. Theor. Appl. Genet. 75 (1988) 286–290.

    Article  CAS  Google Scholar 

  28. Kumpatla, S.P., Teng, W., Buchholz, W.G. and Hall, T.C. Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice. Plant Physiol. 115 (1997) 361–373.

    Article  PubMed  CAS  Google Scholar 

  29. Pawlowski, W.P. and Somers, D.A. Transgene inheritance in plants genetically engineered by microprojectile bombardment. Mol. Biotech. 6 (1996) 17–30.

    Article  CAS  Google Scholar 

  30. Matzke, A.J.M., and Matzke, M.A. Position effect and epigenetic silencing of plant transgenes. Curr. Op. Plant Biol. 1 (1998) 142–148.

    Article  CAS  Google Scholar 

  31. Meyer, P. and Heidmann, I. Epigenetic variants of a transgenic petunia line show hypermetilation in transgene DNA, an indication for specific recognition of foreign DNA in transgenic plants. Mol. Gene Genet. 243 (1994) 390–399.

    CAS  Google Scholar 

  32. Altpeter, F., Vasil, V., Srivastava, V. and Vasil, I.K. Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. Nat. Biotech. 14 (1996) 1155–1159.

    Article  CAS  Google Scholar 

  33. Blechl, A.E., Le, H.Q. and Anderson, O.D. Engineering changes in wheat flour by genetic transformation. Plant Phys. J. 152 (1998) 703–707.

    CAS  Google Scholar 

  34. Alvarez, M.L., Guelman, S., Halford, N.G., Lustig, S., Reggiardo, M.I., Ryabushkina, N., Schewry, P., Stein, J. and Vallejos, R.H. Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits. Theor. Appl. Genet. 100 (2000) 319–327.

    Article  CAS  Google Scholar 

  35. Uthayakumaran, S., Lukow, O.M., Jordan, M.C. and Cloutier, S. Development of genetically modified wheat to assess its dough functional properties. Mol. Breed. 11 (2003) 249–258.

    Article  CAS  Google Scholar 

  36. He, G.Y., Jones, H.D., D’Ovidio, R., Masci, S., Chen, M., West, J., Butow, B., Anderson, O.D., Lazzeri, P., Fido, R. and Shewry, P.R. Expression of an extended HMW subunit in transgenic wheat and the effect on dough mixing properties. J. Cereal Sci. 42 (2005) 225–231.

    Article  CAS  Google Scholar 

  37. Meyer, P. Understanding and controlling transgene expression. Trends Biotech. 13 (1995) 332–337.

    Article  CAS  Google Scholar 

  38. Callaway, A.S., Abranches, R., Scroggs, J. and Allen, G.C. and Thompson, W.F. High throughput transgene copy number estimation by competitive PCR. Plant Mol. Biol. Rep. 20 (2002) 265–277.

    Article  CAS  Google Scholar 

  39. Song, P., Cai, C.Q., Skokut, M., Kosegi, B.D. and Petolino, J.F. Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKER-derived transgenic maize. Plant Cell Rep. 20 (2002) 948–954.

    Article  CAS  Google Scholar 

  40. Pedersen, C., Zimny, J., Becker, D., Janhne-Gartner, A. and Lorz, H. Localization of introduced genes on the chrmomosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization. Theor. Appl. Genet. 94 (1997) 749–757.

    Article  CAS  Google Scholar 

  41. Rooke, L., Steele, S.H., Barcelo, P., Shewry, P.R. and Lazzeri, P. Transgene inheritance, segregation and expression in bread wheat. Euphytica 129 (2003) 301–309.

    Article  CAS  Google Scholar 

  42. Gautier, M.F., Cosson, P., Guirao, A., Alary, R. and Joudrier, P. Pouroindoline genes are highly conserved in diploid ancestor wheat and related species but absent in teraploid Triticum species. Plant Sci. 153 (2000) 81–91.

    Article  CAS  Google Scholar 

  43. Matzke, M.A., Aufsatz, W., Kanno, T., Mette, M.F. and Matzke, A.J. Homology-dependent gene silencing and host defence in plants. Adv. Gen. 46 (2002) 235–275.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Environmental and Agro-Forestry Biology and Chemistry, Section of Genetics and Plant Breeding, University of Bari Aldo Moro, Via Amendola 165/A-70126, Bari, Italy

    Agata Gadaleta, Angelica Giancaspro, Maria Francesca Cardone & Antonio Blanco

Authors
  1. Agata Gadaleta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angelica Giancaspro
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria Francesca Cardone
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Antonio Blanco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Agata Gadaleta.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gadaleta, A., Giancaspro, A., Cardone, M.F. et al. Real-time PCR for the detection of precise transgene copy number in durum wheat. Cell Mol Biol Lett 16, 652 (2011). https://doi.org/10.2478/s11658-011-0029-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.2478/s11658-011-0029-5

Key words

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us