目的 评价DNA条形码通用序列对地黄属植物Rehmannia Libosch. ex Fisch. et Mey.的鉴定作用,探讨中药地黄R. glutinosa的栽培起源。方法 对地黄属物种的ITS、psbA-trnH、rbcL和matK序列扩增和测序,比较各序列在种内和种间变异。采用最近距离法、相似性搜索法、构建Neighbor-Joining(NJ)系统聚类树等方法进行鉴定分析。结果 对比地黄属各种的rbcL和matK序列,结果显示rbcL序列完全一致,matK序列同源性为99.9%~100%。ITS和psbA-trnH序列的平均种间变异均大于平均种内变异,且2条序列的种间最小变异均大于种内最大变异。在ITS系统树上,地黄的所有样品与茄叶地黄聚在一支,支持率为96%;psbA-trnH和联合树上,地黄的所有样品聚为一单系分支(支持率为55%和58%)。地黄与茄叶地黄聚在的这一分支分别与裂叶地黄和高地黄聚在一支(在ITS和联合树上支持率为55%和68%),与裂叶地黄和天目地黄聚在一支(在psbA-trnH树上支持率为80%)。ITS和联合树支持栽培地黄的3个品种与来源于河南温县、郑州、南阳和北京野生地黄居群聚在一支,支持率为51%和69%。结论 叶绿体基因rbcL和matK不适合作为条形码对地黄属进行鉴定,ITS和psbA-trnH序列可以作为中药材地黄与同属近缘种进行鉴定的条形码序列。裂叶地黄和天目地黄可能是四倍体地黄的2个亲本来源,栽培的地黄品种可能起源于河南温县区域的野生群体。

Objective To evaluate the suitable candidate DNA barcoding of plants in Rehmannia Libosch. ex Fisch. et Mey., and unravel the origin of cultivated R. glutinosa. Methods Nuclear DNA ITS and chloroplast gene psbA-trnH, rbcL, and matK sequences of species in Rehmannia Libosch. ex Fisch. et Mey. were amplified and sequenced. The Kimura 2-Parameter (K2P) distances were calculated. Identification analyses were performed using Nearest Distance, BLAST1, and Neighbor-Joining (NJ) methods. Results A comparison on the sequences within species in Rehmannia Libosch. ex Fisch. et Mey. indicated that the rbcL sequences were identical, the matK sequences were similar by 99.9%-100%. All inter-specific distances (ITS and psbA-trnH data) were far higher than all intra-specific genetic distances. Minimum interspecific distance (ITS and psbA-trnH data) were higher than coalescent depth. The NJ trees (ITS, psbA-trnH, and combined data) indicated that all population of R. glutinosa formed a monophyletic clade [Bootstrap (BS)=96%, 55%, and 58%]. The clades including R. glutinosa and R. solanifolia were clustered with R. piasezkii and R. elata in ITS and combined trees (BS=55% and 68%), and clustered with R. piasezkii and R.chingii in psbA-trnH tree (BS=80%). The NJ trees (ITS, psbA-trnH, and combined data) supported that three cultivated varieties of R. glutinosa were clustered with wild populations from Wenxian, Zhengzhou, Nanyang, and Beijing (BS=51% and 69%). Conclusion Chloroplast genes rbcL and matK can not be used to identify the medicinal plants of Rehmannia Libosch. ex Fisch. et Mey. ITS and psbA-trnH are two efficient barcodes for authentication of R. glutinosa and its relative species. R. piasezkii and R. chingii may be as both parental species of tetraploid R. glutinosa. Furthermore, it appears that native wild populations are involved in the origin of cultivated R. glutinosa in Wenxian county.

国家自然科学基金河南联合基金项目(U1404302);河南省基础与前沿计划研究项目(152300410198)