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Zygosity identification in transgenic cotton (Gossypium hirsutum) by real-time quantitative PCR

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Abstract

Successful identification of homozygous and heterozygous transgenic plant with currently available techniques such as southern blot hybridization, dot blot hybridization, fluorescence in situ hybridization (FISH) and so on, demands tedious and time-consuming procedures with a high proportion of ambiguous results. Real-time PCR is a quantitative and extremely precise method with high throughput that could be applied to the analysis of large number of plants differing only by a factor of two in the amount of target sequences. In the present study, we determined zygosity level of transgenes in cotton [Gossypium hirsutum L.] with two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers. TPS, a single copy gene per haploid Gossypium hirsutum genome was used as the endogenous reference to estimate copy number of transgene. Both assays were accurate and reproducible in determination of the number of transgenes present in a cell line. These methods are standard curves and Delta delta C t method.

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Abbreviations

Bt:

Bacillus thuringiensis

ΔC t :

Normalized critical threshold

ΔΔC t :

Comparative threshold value

FAM:

5-Carboxyfluoroscein

TPS gene:

Trehalose 6-phosphate-synthase gene

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Acknowledgments

This study was funded by Zanjan University, Zanjan, Iran and was partly supported by Agricultural Biotechnology Research Institute of Iran, Karaj, Iran. We thank Dr Mohamad Reza Khoramizade for his assistance in conducting this research.

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Authors and Affiliations

  1. Institute of Industrial Biotechnology, Academic Center for Education, Culture and Research, Zanjan, Iran

    Mehri Javdi

  2. Research Center of Biotechnology and Physiology, Zanjan University, Zanjan, Iran

    Ali Haghnazari

  3. Agricultural Biotechnology Research Institute of Iran, Karaj, Iran

    Masoud Tohidfar & Behzad Ghareyazi

  4. Laboratory of Molecular Biology, Center of Molecular Pathobiology, Tehran, Iran

    Shahin Negari

Authors
  1. Mehri Javdi
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  2. Ali Haghnazari
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  3. Masoud Tohidfar
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  4. Shahin Negari
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  5. Behzad Ghareyazi
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Correspondence to Ali Haghnazari.

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Javdi, M., Haghnazari, A., Tohidfar, M. et al. Zygosity identification in transgenic cotton (Gossypium hirsutum) by real-time quantitative PCR. Euphytica 173, 185–191 (2010). https://doi.org/10.1007/s10681-009-0079-1

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  • DOI: https://doi.org/10.1007/s10681-009-0079-1

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