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Molecular authentication of Tetrastigma hemsleyanum from its adulterant species using ISSR, CAPS, and ITS2 barcode

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Abstract

Tetrastigma hemsleyanum is a rare and endangered herb, which is commercialized as the resource of anti-cancer drugs. Wild T. hemsleyanum plants are on the verge of extinction recently, there are increasing numbers of counterfeits on the market. In the present study, inter-simple sequence repeat (ISSR), Cleaved amplified polymorphic sequence (CAPS), and the internal transcribed spacer region II (ITS2) barcode were used for the first time for the authentication of T. hemsleyanum from its commonly counterfeits. ISSR analysis suggested that it was a useful method for distinguishing T. hemsleyanum from its adulterants of different genus. However, it was insufficient to distinguish T. hemsleyanum from those adulterants of the same genus. ITS2 of T. hemsleyanum and the commonly counterfeits were amplified and sequenced. The Neighbor-Joining tree constructed from the ITS2 sequences showed that T. hemsleyanum was clearly differentiated from all counterfeits samples. A mutation site in the ITS2 region of T. hemsleyanum had been found which could be recognized by the restriction endonuclease NcoI. T. hemsleyanum could be readily distinguished from counterfeits as the PCR products from T. hemsleyanum could be digested sufficiently by NcoI, while the PCR products from counterfeits could not be digested. The results indicated that CAPS and ITS2 barcode methods provided effective and accurate identification of T. hemsleyanum from all its adulterants, while ISSR could only distinguish T. hemsleyanum from its adulterants of different genus. The CAPS method developed in the present study will serve as a reliable tool for safe and effective use of T. hemsleyanum in the clinic application. It will also play an important role for the identification, management and conservation of this endangered species.

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Acknowledgments

This work was supported by the public welfare technology research projects of Zhejiang province (Grant No. 2013C32103), the Science and Technology Innovation Team Project of Ningbo Science and Technology Bureau, China (2015C110027), the agricultural research projects of Ningbo city (Grant No. 2014C10031), and the traditional Chinese Medical Science Technology Research Projects of Zhejiang province (Grant No. 2013ZA119).

Author Contribution

Xin Peng—Sequence alignment and analysis; Write the manuscript, Xiurong Wu—ISSR fingerprinting and statistical analysis, Qingyong Ji—Plant material sampling and pre-process, Ruikang Yang—DNA extraction and PCR amplification, Yulan Li—Restriction enzyme site analysis.

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Authors and Affiliations

  1. Institute of Biopharmaceutical Technology, Zhejiang Pharmaceutical College, Ningbo, 315100, Zhejiang, China

    Xin Peng, Xiurong Wu, Ruikang Yang & Yulan Li

  2. Lishui Academy of Agricultural Science, Lishui, 323000, China

    Qingyong Ji

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  1. Xin Peng
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  2. Xiurong Wu
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Correspondence to Yulan Li.

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Peng, X., Wu, X., Ji, Q. et al. Molecular authentication of Tetrastigma hemsleyanum from its adulterant species using ISSR, CAPS, and ITS2 barcode. Mol Biol Rep 43, 785–794 (2016). https://doi.org/10.1007/s11033-016-4023-x

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  • DOI: https://doi.org/10.1007/s11033-016-4023-x

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