川芎种质鉴定标记开发和系统发育研究

目的基于藁本属Ligusticum叶绿体基因组高频插入缺失区域开发InDel标记,同时结合通用条形码对道地川芎及其常用混伪品进行种质鉴定和系统发育研究。方法通过8个DNA通用条形码ycf1、matK、ITS2、rpoC1、rbcL、rpoB、trnK、psbA-trnH序列片段对采集的26个川芎样品及其常用混伪品进行了扩增和测序,采用了遗传距离统计法、barcoding gap分析法和构建系统发育树法进行亲缘关系和系统发育研究。同时利用InDel分子标记,采用构建进化树法对道地川芎及其混伪品物种进行分子鉴定。结果 rbcL片段保守位点最多(97.32%),且GC含量最高(44.9%)。rbcL+rpoB片段具有最小的平均种内遗传距离(0.002 5),psbA-trnH片段具有最大的平均种间遗传距离(0.429 2)。trnK片段和rbcL+rpoB片段具有最高的种间变异,psbA-trnH片段的"barcodinggap"区重叠度最小。采用的8对DNA通用条形码无法准确地鉴定道地川芎药材与其他混伪品物种。InDel分子标记的聚类分析中,24对InDel引物中的4对引物组合能准确地鉴定出道地川芎,并将道地川芎及其混伪品物种聚类为4个分支,其中一个分支为采集的道地川产川芎药材。结论 InDel标记对道地川芎及其常用混伪品的鉴定能力高于通用条形码。对于传统的通用DNA条形码,由于遗传成分差异大,无法区分川芎及其常用混伪品。新开发出的InDel分子标记可以有效地鉴定道地川芎及其常用混伪品,从分子水平上为川芎道地性研究提供有效手段。

Development of germplasm identification markers and phylogenetics analysis of Ligusticum chuanxiong

Objective In this study, InDel markers were developed based on the high frequency Insertion/Deletion region of chloroplast genome of Ligusticum. Germplasm identification and phylogenetic development of Ligusticum chuanxiong and its common adulterants were studied with universal barcode. Methods The 26 samples of L. chuanxiong and its common adulterants were amplified and sequenced by eight DNA universal barcodes:ycf1, matK, ITS2, rpoC1, rbcL, rpoB, trnK, and psbA-trnH. Genetic distance statistics, barcoding gap and phylogenetic tree analysis methods were used to study the phylogenetic relationship and phylogeny of L. chuanxiong. At the same time, the evolutionary tree was constructed to study molecular identification of L. chuanxiong and its common adulterants. Results The results showed that rbcL conserved site was the highest (97.32%) with the highest GC content (44.9%). The rbcL+rpoB fragment had the smallest average intraspecific genetic distance (0.002 5). The psbA-trnH sequence fragment had the largest average interspecific genetic distance (0.429 2). The trnK and rbcL+rpoB sequence had the highest interspecific genetic distance. The overlap of the "barcoding gap" region of psbA-trnH was the least. The species of L. chuanxiong and other adulterated species were not accurately identified by the eight pairs of DNA barcodes. The cluster analysis of 24 InDel markers could accurately identify genuine L. chuanxiong and classify the species of L. chuanxiong and its adulterants into four categories, one of which was genuine L. chuanxiong collected from Sichuan. Conclusion The ability of InDel markers to identify authentic L. chuanxiong and its common adulterants was higher than that of common barcode. According to the above studies, it is found that it is impossible to distinguish L. chuanxiong and its common adulterants by the traditional DNA barcodes because of the large difference in genetic components. The newly developed InDel molecular markers can effectively identify L. chuanxiong and its commonly used adulterants, and provide an effective method for the genuineness of L. chuanxiong at molecular level.