Chen Fu-Rong, Wang Tao, Guo Qiao-Sheng, Zhu Zai-Biao, Yang Feng, Zou Qin-Jun, Zhang Ya-Jing
Institute of Chinese Medicinal Materials,Nanjing Agricultural University Nanjing 210095,China.
Zhongguo Zhong Yao Za Zhi. 2019 Feb;44(4):660-665. doi: 10.19540/j.cnki.cjcmm.2019.0015.
DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum and Ch. morifolium based on psbA-trn H,mat K and trn L sequences. The total DNA was extracted from 21 samples collected,and the psbA-trn H,mat K,trn L sequences were amplified by PCR and sequenced. The information of these sequences were obtained. We aligned all 63 sequences,calculated the intraspecific and interspecific distances,analysed the SNPs distribution of psbA-trn H+mat K+trn L combination sequences and constructed the Neighbor-joining( NJ) Tree,using MEGA 7. 0. The results showed that the genetic distances of Ch. indicum,Ch. indicum( Juhuanao)and Ch. morifolium were overlapped. The SNPs analysis of psbA-trn H+mat K+trn L combination sequences showed that there were 19 nucleotide polymorphism loci( SNPs) and nine parsim-informative sites in the combination sequences. In addition,Ch. indicum showed more obvious sequence polymorphism than those of Ch. indicum( Juhuanao) and Ch. morifolium. The psbA-trn H sequences showed obvious length variation.The NJ Tree showed that Ch. morifolium numbered C2-C5 were clustered into a single subbranch with a bootstrap value of 62%,and Ch.morifolium could be distinguished from Ch. indicum and Ch. indicum( Juhuanao). Moreover,Ch. indicum numbered Z9 and Z10 collected from Gansu province were singly clustered into one branch with a bootstrap value of 77%. It was also found that the changes of psbA-trn H and trn L sequences information of Ch. indicum samples from the northwest were obviously related to the geography and environment. Moreover,Ch.indicum and Ch. indicum( Juhuanao) had obvious differentiation,were also regarded as the evolutionary sources of Ch. morifolium. Therefore,psbA-trn H+mat K+trn L combination sequences as DNA barcode can identify Ch. indicum and Ch. morifolium accurately and rapidly,which provides an important basis for germplasm resources identification and species identification.
采用DNA条形码技术，基于psbA-trnH、matK和trnL序列，建立了野菊花和菊花的快速鉴定方法。从采集的21个样本中提取总DNA，通过PCR扩增psbA-trnH、matK、trnL序列并进行测序，获取这些序列的信息。利用MEGA 7.0对所有63条序列进行比对，计算种内和种间距离，分析psbA-trnH+matK+trnL组合序列的单核苷酸多态性（SNP）分布并构建邻接（NJ）树。结果表明，野菊花、野菊花（巨花脑）和菊花的遗传距离存在重叠。psbA-trnH+matK+trnL组合序列的SNP分析显示，组合序列中有19个核苷酸多态性位点（SNP）和9个简约信息位点。此外，野菊花的序列多态性比野菊花（巨花脑）和菊花更明显。psbA-trnH序列表现出明显的长度变异。NJ树显示，编号为C2-C5的菊花聚为一个亚分支，自展值为62%，菊花可与野菊花和野菊花（巨花脑）区分开来。此外，从甘肃采集的编号为Z9和Z10的野菊花单独聚为一个分支，自展值为77%。还发现，西北地区野菊花样本的psbA-trnH和trnL序列信息变化与地理和环境明显相关。而且，野菊花和野菊花（巨花脑）存在明显分化，也被认为是菊花的进化来源。因此，psbA-trnH+matK+trnL组合序列作为DNA条形码可准确快速地鉴定野菊花和菊花，为种质资源鉴定和物种鉴定提供了重要依据。
