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

Javdi, M. and Haghnazari, A. and Tohidfar, M. and Negari, S. and Ghareyazi, B. (2010) Zygosity identification in transgenic cotton (Gossypium hirsutum) by real-time quantitative PCR. Euphytica, 173 (2). pp. 185-191.

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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 Ct method

Item Type: Article
Author Affiliation: Institute of Industrial Biotechnology, Academic Center for Education, Culture and Research, Zanjan, Iran
Subjects: Crop Improvement
Divisions: Other Crops
Depositing User: Syamala
Date Deposited: 24 Mar 2011 21:33
Last Modified: 24 Mar 2011 21:33
Official URL: http://dx.doi.org/10.1007/s10681-009-0079-1
URI: http://eprints.icrisat.ac.in/id/eprint/1706

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