Identification Of Chrysanthemum Indicum L.and Its Easily Mixed Species By DNA Barcode

Chrysanthemum indicum L.is a perennial herb of Compositae.It is a Chinese medicinal herb with dried flower heads.With the functions of clearing heat,detoxifying fire and calming liver in the Chinese Pharmacopoeia(2015),it is the main raw material of many kinds of Chinese patent medicines for wind-heat and cold.C.indicum has a wide range of geographical adaptations,and is distributed in Northeast,North,Central,South and Southwest China.During actual harvesting,it was found that the head-like inflorescence was very similar to that of some medicinal plants from the same family and different genera,which caused that the market medicinal materials are likely to be mixed and difficult to distinguish,seriously affecting their quality.In addition,the traditional identification methods of C.indicum and its related species mainly focus on plant morphology and composition,which has certain limitations.At present,the molecular identification of C.indicum mainly focuses on IS SR,RAPD and other aspects,while the molecular identification of DNA barcode about C.indicum has rarely been reported.Consequently,it is of great significance to establish a rapid and effective molecular identification method for C.indicum and its adulterants or related species.In this study,DNA barcode technology was used to explore and establish a rapid identification method for C.indicum and its adulterants or related species through ITS2 sequence analysis,ITS2 secondary structure analysis and psbA-trnH,matK,trnL sequences analysis.The chief results and conclusions in this study are as below:(1)With ITS2 sequences used as DNA barcode to study Chrysanthemum morifolium,Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens,it was found that both intraspecific and interspecific genetic distances and NJ trees constructed from ITS2 sequences confirmed that ITS2 sequences could accurately identify C.indicum and its adulterants.The genetic distances of C.indicum and C.morifolium were overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance among C.indicum,I.lineariifolia,S.oldhamianus and S.scandens,showing an obvious barcoding gap.The NJ tree indicated that C.indicum and C.morifolium shared a clade,while most of C.morifolium and part of C.indicum were shared a subclade.At the same time,I.lineariifolia,S.oldhamianus and S.scandens belonged to one clade separately.ITS2 secondary structures of I.lineariifolia,S.oldhamianus and S.scandens were clearly different enough to identificate completely but C.indicum and C.morifolium shared two secondary structures of A and B.It was proved that C.indicum was one of the evolutionary sources of C.morifolium.Besides,ITS2 sequence analysis made known that there was no visible correlation between samples of C.indicum from different regions and their geographical distribution areas.(2)In order to establish a new method for rapid identification of C.indicum and its related species,we explored additional phylogenetic information hidden in ITS2 secondary structure using DNA barcode.The results show that the secondary structure of ITS2 in all samples shared the same secondary structure model-a four-fingered hand.It not only has the common characteristics of ITS2 secondary structure in plants,but also has many other conservative sequences,with high overall conservativeness.Among all species used in this study,their ITS2 secondary structures has obvious difference.Moreover,the number of mutation sites in the joint matrix increased by nearly 90%compared with the nucleic acid sequences,which greatly enriched the number of mutant site information.Furthermore,the support rate of evolutionary trees and the identification rate of species were obviously improved.Although there was no distinction between C.zawadskii and C.morifolium,it effectively identificated three species of C.hypargyrum,C.oreastrum,and C.dichrum.(3)For the sake of establishing a rapid identification method of C.indicum and C.morifolium,sequences of psbA-trnH,matK and trnL were used as DNA barcode.The genetic distances of C.indicum,C.indicum(Juhuanao)and C.morifolium were overlapped.The SNPs analysis of psbA-trnH+matK+trnL combination sequences showed that there were 19 nucleotide polymorphism loci(SNPs)and nine parsim-informative sites in the combination sequences.Also,C.indicum showed more obvious sequence polymorphism than that of C.indicum(Juhuanao)and C.morifolium.The psbA-trnH sequences demonstrated obvious length variation.What’s more,the NJ tree revealed that C.morifolium could be distinguished from C.indicum and C.indicum(Juhuanao),and C.morifolium numbered C2-C5 clustered into a single subbranch with a bootstrap value of 62%.Moreover,C.indicum numbered Z9 and Z10 collected from Gansu Province were singly clustered into one branch with a bootstrap value of 77%.It turned out that the changes of psbA-trnH and trnL sequences information of C.indicum samples from the northwest were evidently related to the geography and environment in the northwest of China.Besides,C.indicum and C.indicum(Juhuanao)had obvious differentiation,so they were the evolutionary sources of C.morifolium.