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Identification of WA-Type Three-Line Hybrid Rice with Real-Time Polymerase Chain Reaction (PCR) Method

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Abstract

A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R2-630 WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R2-630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P3, P4) and TaqMan fluorescence probe (P3-14) were designed based on the R2-630 DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F1 and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg2+ concentration is 3.5 mmol/L.

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Acknowledgments

This work was partly funded by molecular marker system of bio-resources General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. The authors would like to thank Dr. H.L. Yang from the China National Hybrid Rice Research and Development Center, Dr. W.X. Zhang from the Institute of Crop Germplasm Resource Chinese Academy of Agricultural Sciences (CAAS) and Dr. S.W. Huang from the China National Rice Research Institute for the donation of samples. We are grateful to Professor J. Leng and W. Li from the Institute of Bast-Fiber and L. Yu from Hunan Agricultural University for their critical reading and editing of the manuscript.

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Authors and Affiliations

  1. Institute of Bast-Fiber crops, Chinese Academy of Agricultural Sciences, Xianjiahu West Road 348, Changsha, China

    Y. Cheng

  2. Institute of Animal and Plant Quarantine, Chinese Academy of Inspection and Quarantine, Huixin West Road 241, Beijing, China

    H. Y. Chen, J. J. Mao, A. X. Cao & S. F. Zhu

  3. College of Bio-safety Science and Technology, Hunan Agricultural University, Furong District, Changsha, China

    Y. Cheng, B. D. Gao & J. J. Mao

  4. Hunan Entry-Exit Inspection and Quarantine Bureau, Xiangfu Middle Road 188, Changsha, China

    J. G. Zhu

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  1. Y. Cheng
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  2. B. D. Gao
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  3. H. Y. Chen
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  4. J. J. Mao
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  5. A. X. Cao
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  6. J. G. Zhu
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  7. S. F. Zhu
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Correspondence to Y. Cheng or S. F. Zhu.

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Cheng, Y., Gao, B.D., Chen, H.Y. et al. Identification of WA-Type Three-Line Hybrid Rice with Real-Time Polymerase Chain Reaction (PCR) Method. Appl Biochem Biotechnol 166, 819–829 (2012). https://doi.org/10.1007/s12010-011-9471-0

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  • DOI: https://doi.org/10.1007/s12010-011-9471-0

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