汤欢+向丽+李西文+孙伟+王美娜+黄云峰+叶萌

[摘要] 兰科药用植物形态分类困难，此研究采用DNA条形码分子鉴定法从分子水平验证兰科药用植物的传统形态分类。以matK，psbA-trnH和ITS2序列作为DNA条形码对已进行了形态鉴定的49属135种163份兰科药用植物样品进行分子鉴定，经DNA提取，PCR扩增、双向测序及校对拼接后，将得到的序列在GenBank中进行BLAST比对，然后运用MEGA 7.0软件中的Neighbor-joining （NJ）法构建物种系统进化树。结果表明，163份样品均能成功提取DNA；matK，psbA-trnH和ITS2序列的PCR扩增效率分别为100%，100%，98.77%；共获得487条序列，其中345条序列在GenBank数据库中比对到了相应物种的序列，142条为新增序列；运用NJ法构建的兰科药用植物系统进化树中，基于matK序列所构建的物种系统进化树要优于基于psbA-trnH和ITS2序列所构建的系统进化树。matK，psbA-trnH和ITS2序列在鉴定兰科药用植物过程中互为补充，DNA条形码分子鉴定法可用于辅助兰科药用植物的分类鉴定。

[关键词] 兰科；DNA条形码；药用植物；分子鉴定

[Abstract] In this study，DNA barcoding was used to validate the traditional morphological classification of medicinal plants of Orchidaceae. The 163 samples of 135 species belong to 49 genera which have been confirmed by morphological identification were collected. Candidate sequences，including matK，psbA-trnH and ITS2 sequences，were amplified，bidirectionally sequenced，and assembled. All the sequences were blasted to GenBank database at NCBI，then analyzed using Neighbor-joining tree method by MEGA 7.0. The results showed that the DNAs of 163 samples were successfully extracted. The amplification efficiency of matK，psbA-trnH and ITS2 sequences were 100%，100% and 98.77%，respectively. The 487 sequences were obtained，345 sequences of which have matched corresponding sequences in the GenBank database and 142 sequences were new sequences. The topology of NJ tree which were constructed with the matK sequences was better than the trees of psbA-trnH and ITS2 sequences. In conclusion，the matK，psbA-trnH and ITS2 sequences were complementary and suitable for identification of medicinal plants of Orchidaceae. DNA barcoding can be used as an auxiliary means for identification of medicinal plants of Orchidaceae.

[Key words] Orchidaceae；DNA barcoding；medicinal plants；molecular identification

兰科Orchidaceae是世界性大科之一，包括地生、附生、菌类寄生等生活方式[1]，在全世界约有800属20 000～35 000种[2-4]，Flora of China中记载中国境内的野生兰科植物共有194属（中国特有属11个） 1 388种（中国特有种491个）[4]。兰科植物中的许多物种因其形态高度进化，物种之间仅有极细微的差异，从形态上对其进行鉴定较困难。

自双名法确立以来的250多年里，人类共鉴定和描述了约170万种生物[5]，但这仅仅占到分类学家预计物种数量的15%[6]。DNA条形码（DNA barcoding）是一种基于DNA序列进行生物物种鉴定的技术，即利用标准化的一个或者几个DNA片段进行序列分析，根据核苷酸序列差异，对物种进行快速和准确的鉴定[7-9]。……