风毛菊属（菊科）药用植物的DNA条形码鉴定

The genus Saussurea, consisting of about 400 species, is one of the largest genera of the family Asteraceae. Moreover, it's one of the most species-rich genera in traditional Chinese medicines, with more than 70 species, such as S. involucrata, being employed in traditional Chinese herbal therapy. However, extreme diversity and prevalent convergent phenotypes combine to complicate the authentication of most medicinal plants. At the same time, some species are endangered or hard to be accessed and species with similar curative effect should be explored. In responce to these calls, here we propose to launch a study of DNA barcoding, an exciting new tool promises to not only provide powerful tool for species identification, but also assist to clarify the systematic relationship among species. Firstly, we plan to conduct a detailed field investigation including more than 60 species of medicinal species and representatives covering ca. 75% of the species diversity of Saussurea. Secondly, we intend to sequence multiple candidate barcode fragments of the representatives sampled from more than 2 localities for each of the sampled species, and evaluate the identification efficiency of each DNA fragments and their combination in different ways employing barcoding gap, Wilcoxon signed-rank tests, fuzzy membership, Blast search, the nearest distance and some other methods. Thirdly, we will use the sequenced data to explore the systematic relationship among species with phylogenetic reconstruction such as maximum parsimony in order to discern sister species or closely related taxa of the medicinal plants. Finally, we propose to build a library of reference sequences of the medical herbs, including a part of genuine medicines, of Saussurea by sequencing the optimal multilocus combination of more than 3 populations of each of the medicinal plants and submitting them, along with the information about its voucher specimen, to a public database. The study is promised to provide an objective and rapid method to identify the herbal medicines of Saussurea and moreover, provide fresh insights into the application of DNA barcoding in taxa experienced explosive speciation.

菊科风毛菊属植物约400种，其中天山雪莲等70余种用于中药或民族药，是我国天然药物品种最多的属之一。但是，罕见的物种多样性与众多趋同特征使属内很多药材的真伪、优劣鉴定非常困难，随着中药现代化与世界化进程的推进，亟待快速、客观的鉴定方法；同时，部分品种药源有限，需要寻找疗效近似的植物。本项目拟对绝大部分已知药用品种在内，涵盖风毛菊属75%以上物种多样性的种群进行野外调查，分析DNA条形码候选片段在种群内、种群间以及种间的变异，以barcoding gap等方法探讨风毛菊属的DNA条形码鉴定方案，并以系统发育方法分析种间关系；在此基础上，建立60个以上药用品种及一些地道药材的DNA条形码鉴定体系，确认各药用品种的近缘类群。研究结果对于探讨DNA条形码在近期爆发成种类群的应用、风毛菊属药用植物的鉴定、新药源的寻找与扩大等问题具有重要意义。

项目执行期间，先后前往青藏高原、黄土高原、内蒙古高原、阿尔泰山脉、天山山脉、昆仑山脉、横断山脉、秦岭山脉、长白山脉、太行山脉、大巴山脉、吕梁山、贺兰山、黄山等地进行野外调查，共采集植物风毛菊属及其近缘类群研究材料共4500余份，经鉴定，确定采集到FOC收录的风毛菊属约65%的物种（189种），以及FOC出版后发表的新种或新纪录14种，另有50个以上的疑似新种或形态过渡类群，这些材料基本涵盖了风毛菊属的形态多样性，其中雪莲亚属、附片亚属、风毛菊亚属羽裂组、风毛菊亚属栗缘组等类群已采集到80%以上物种。我们对每个采到的种选取了3-9个个体，累计1400多个个体进行了7个DNA片段的测序以及ＤＮＡ条形码和系统发育分析。主要结果有：1）风毛菊亚属栗缘组与其他类群亲缘关系较远，应独立成属，已命名为尚武菊属Shangwua Yu J. Wang, Raab-Straube, Susanna & J. Quan Liu；2）风毛菊亚属羽裂组植物尽管叶片形状差异较小，但分支分析发现他们并不组成一个单系分支，且种间关系难以分辨，这个组70多种几乎全部分布在青藏高原，可能是这一地区剧烈环境变迁驱动植物发生辐射分化的结果；3）附片亚属与风毛菊亚属纤维组和无茎组的部分类群亲缘关系较近，相关类群应合并，新界定的附片亚属含2个分支，分别由总苞片具明显膜质附属物的类群和附属物不明显的类群组成，ITS序列能鉴定70%左右的物种，但matK等叶绿体序列单独或联合分析只能鉴定不到35%的物种；4）雪莲亚属成员与风毛菊亚属部分类群聚成一支，因此这个亚属也应重新界定，ITS能鉴定这个亚属约65%的物种，但叶绿体即使多套序列联合分析也只能鉴定不到30%的物种，著名药用植物雪莲花与其他物种能以ITS鉴定，阿尔泰雪莲可能是雪莲花亲缘关系最近的类群，苞叶雪莲尽管形态相似，但亲缘关系较远。目前，部分结果已经发表。这些研究为风毛菊属系统发育历史与属下分类系统的建立，客观准确的物种界定与鉴定，以及风毛菊属药用植物与抗逆基因等资源的开发利用奠定了基础。