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| Development of Endogenous Reference Gene PSG719 for The Detection of Transgenic Common Wheat (Triticum aestivum L.) |
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Abstract To quantitatively detect and analyze transgenes in genetically modified (GM) crops, development of endogenous reference genes with taxon-specificity, low copy number and no allelic variation in varieties is prerequisite. In this study, bioinformatic analyses of genes from database and literatures revealed that one pollen-specific protein gene, PSG719 (GenBank accession: FJ497025.1), from hexaploid wheat genome had high specificity for wheat and low copy number; based on its gene sequence specific primers were designed. These primers were used for qualitative PCR detection of 16 plant species including durum wheat and other closely related cereal species, and no PCR products were obtained from all the plant species except that from common wheat indicating a good taxon-specificity. And the homogeneity was verified on 16 widely different eco-systems wheat varieties by quantitative real-time PCRs. This gene was then used for subsequent qualitative and quantitative PCRs and relevant studies. In conventional qualitative PCRs, the limit of detection (LOD) was about 2 copies per wheat haploid genome per reaction. In the quantitative Real-time PCR assays, LOD and limit of quantitation (LOQ) were about 2 and 5 copies haploid genome, respectively, the latter of which was lower than other reported wheat endogenous reference genes. Furthermore, standard curves through five-fold serial dilutions corresponding to 10 000, 2 500, 625, 156 and 39 copies wheat haploid genome per reaction were performed by the above-developed PSG719 quantitative PCR system, and the results showed ideal slope (-3.395) and highly linear co-efficiency (R2=0.999) between Cross point (Cp) values and the amounts of DNA, indicating that an appropriate calibration curve could be constructed for quantitative detection of GM wheat. Taken together, these results indicated that the PSG719 gene was a new endogenous reference gene suitable for the specific detection and quantification of genetically modified materials in common wheat. The developed PSG719 system will facilitate the enforcement of genetically modified organism labeling for wheat in the near future.
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Received: 29 October 2014
Published: 17 March 2015
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