目的 基于ITS2序列对天冬Asparagus cochinchinensis及其近缘种的样品进行分子鉴定，并通过HPLC特征图谱技术对不同产地天冬样品进行质量评价，为天冬的质量控制提供参考。方法 采用CTAB法提取19批天冬样品的DNA，进行PCR扩增、测序；从GenBank公共网站获取天冬及其近缘种的序列，通过MEGA 6.0软件分析序列特征、计算遗传距离、构建系统发育树；建立天冬HPLC特征图谱，结合相似度评价、聚类分析、主成分分析、正交偏最小二乘判别分析对天冬样品进行质量评价，筛选差异标志物。结果 天冬及其近缘种样品的ITS2序列长度为244～245 bp，GC含量在64.34%～64.75%，变异位点数为12；遗传距离结果表明，天冬种内平均遗传距离小于天冬及其相近基原的种间平均遗传距离；系统发育树聚类分析结果表明，19份样品聚为3类，具有良好的单系性，能较直观地将天冬及其相近基原的样品进行区分；从12批天冬样品的特征图谱中共标定了13个共有峰，指认了2个成分，分别为原薯蓣皂苷、伪原薯蓣皂苷；聚类分析将12批天冬样品分为2类，分类主要与种植、采收加工、经纬度、环境气候等因素有关；主成分分析结果表明，四川、广西产地的天冬质量较优；正交偏最小二乘判别分析筛选出峰1、7、12（原薯蓣皂苷）这3个质量标志差异物。结论 ITS2序列分析可作为区分天冬及其相近基原样品的分子鉴定方法，以及建立的HPLC特征图谱操作简单、重复性好；可为天冬药材的质量评价和控制提供参考。

Objective The ITS2 sequence was employed for the molecular identification of Tiandong (Asparagus cochinchinensis) and its related species. Through an analysis of the HPLC characteristic chromatogram, a comprehensive quality evaluation was conducted on A. cochinchinensis samples sourced from diverse origins. This study serves as a valuable reference for quality control of A. cochinchinensis. Methods The DNA from 19 batches of samples were extracted utilizing the CTAB method. Following this, the ITS2 sequence was amplified via PCR and sequenced. The sequences of A. cochinchinensis and its related species were obtained from the publicly accessible GenBank website. The MEGA 6.0 software was used to analyze the sequence characteristics, calculate genetic distances, and construct phylogenetic trees. The HPLC characteristic chromatogram was developed to serve as a foundation for quality assessment. This assessment was performed through a comprehensive approach combining similarity evaluation, HCA, PCA and OPLS-DA to evaluate the quality of A. cochinchinensis samples, with the aim of screening for the Q-Marker.Results The length of the ITS2 sequences in A. cochinchinensisand its related species was 244—245 bp and the GC content ranged from 64.34% to 64.75%. A total of 12 mutation sites were identified within these sequences. Genetic distance calculations indicated that the average intraspecific genetic distance within A. cochinchinensiswas smaller than the interspecific genetic distance between it and its related species. Phylogenetic tree clustering analysis effectively grouped the 19 batches of samples into three distinct classes, demonstrating good monophyly and facilitating the differentiation of A. cochinchinensis from its related species. In the HPLC characteristic chromatogram of 12 batches of A. cochinchinensissamples, a total of 13 common peaks were observed, with two peaks identified as protodioscin and pseudoprotodioscin. HCA divided these 12 batches into two categories, likely influenced by factors such as planting, harvesting and processing methods, as well as geographical factors like longitude, latitude and climate. PCA highlighted the relatively superior quality of A. cochinchinensissamples originating from Sichuan and Guangxi provinces. OPLS-DA identified three Q-Markers, corresponding to peak 1, peak 7, and peak 12 (protodioscin). Conclusion The ITS2 sequence can serve as a molecular identification method for distinguishing A. cochinchinensis from its related species. Moreover, the HPLC characteristic chromatogram established in this study is both simple and repeatable, offering a reference for the quality evaluation and control of A. cochinchinensis.

R286.12

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