基于ITS和ITS2序列的药用石斛DNA条形码鉴定研究

目的 探讨内转录间隔区（internal transcribed spacer,ITS）和内转录间隔区2(internal transcribed spacer 2,ITS2)片段作为DNA条形码鉴别我国41种药用石斛种类的可行性。方法 通过PCR扩增测序结合GenBank公共序列筛选分别获得我国41种药用石斛的核基因ITS和ITS2序列433条和410条，以Kimura-2-parameter(K2P)模型计算种间和种内遗传距离，以邻接法（neighbor-joining,NJ）构建系统发育树，并对ITS2二级结构进行预测分析。结果 基于遗传距离和NJ树分析结果均显示，ITS和ITS2作为DNA条形码可分别有效区分待测的30和31种药用石斛，物种鉴定效率分别为73.2%和75.6%。联合系统发育树和ITS2序列二级结构分析，药用石斛种类的鉴定效率可提升至87.8%。结论 ITS2作为DNA条形码对药用石斛的物种分辨率优于ITS，物种取样数量可显著影响DNA条形码对药用石斛的物种鉴定效率。结果丰富了我国石斛属植物的DNA条形码数据库，为保障药用石斛种质资源和药用安全提供了科学基础和技术...

DNA barcoding identification of medicinal Dendrobium based on ITS and ITS2 sequences

Objective To explore the feasibility of using internal transcribed spacer (ITS) and internal transcribed spacer 2 (ITS2) fragments as DNA barcodes to identify 41 species of medicinal Dendrobium in China. Methods By PCR amplification sequencing combined with GenBank public sequence screening, 433 ITS and 410 ITS2 sequences from 41 medicinal Dendrobium species nuclear genes were obtained. The intra- and inter-species genetic distances were estimated based on Kimura-2-parameter (K2P) model. The phylogenetic trees were constructed by neighbor joining (NJ) method. The secondary structures of ITS2 were also predicted and analyzed. Results The results of genetic distances calculation and NJ tree analysis revealed that ITS and ITS2 as DNA barcodes could effectively distinguish 30 and 31 tested medicinal Dendrobium species, with the species identification rates of 73.2% and 75.6%, respectively. The combination of phylogenetic tree construction and ITS2 sequence secondary structure analysis could increase the species identification rate of medicinal Dendrobium to 87.8%. Conclusion The identification rate of ITS2 as a DNA barcode for medicinal Dendrobium species tested in this study is better than that of ITS. Moreover, the success rate of species identification was found to be significantly affected by the number of species sampled. Our results not only enriched the DNA barcode database of Dendrobium plants in China, but also provided scientific basis and technical support for ensuring the germplasm resources and medicinal safety of medical Dendrobium.