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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。
Orchidaceae medicinal plants morphological classification difficulties, this study using DNA barcode molecular identification method from the molecular level to confirm the traditional morphology of Orchidaceae plant classification. The molecular identification of 163 species of Orchidaceae plants belonging to 49 genera with matK, psbA-trnH and ITS2 sequences as DNA barcodes was carried out. After DNA extraction, PCR amplification, two-way sequencing and proofreading, The obtained sequences were BLAST aligned in GenBank and phylogenetic tree was constructed using Neighbor-joining (NJ) method in MEGA 7.0 software. The results showed that all the 163 samples could be successfully extracted. The efficiency of PCR amplification of matK, psbA-trnH and ITS2 was 100%, 100% and 98.77%, respectively. A total of 487 sequences were obtained, of which 345 sequences were in GenBank database Compared to the sequence of the corresponding species and 142 for the new sequence; phylogenetic tree based on the matK sequence constructed by NJ method is superior to the phylogenetic tree based on psbA-trnH and ITS2 sequences The constructed phylogenetic tree. matK, psbA-trnH and ITS2 sequences complement each other in the identification of orchidaceous medicinal plants. The DNA barcode molecular identification method can be used to assist the identification and identification of orchidaceous medicinal plants.