Molecular Identification Of Herbal Species In Genus Corydalis Dc. And Its Application

Objective: The tuber of genus Corydalis herbs is widely used to relieve pain,because of the particular alkoloids.C.yanhusuo W.T.Wang ex Z.Y.Su et C.Y.Wu and C.decumbens(Thunb.)Pers.has been listed in the Chinese pharmacopoeia for ages,which belongs to Sect.Corydalis and Sect.Duplotuber separately.The rest herts are uesd as substitutes in different regions of China.Among the rest herbs,C.turtschaninovii Bess.is not only the substitutes in north-east China,but also listed in the Japanese pharmacopoeia as a common medicinal materials.At present,it is difficult to identify the herts between Pharmacopoeia herbs and its substitutes.Moreover,there has long been a debate on taxonomy of some species in Sect.Corydalis and Sect.Duplotuber.In this study,we are aimed to clarify which species are used as medicinal materials,which group are they come from,what classification status of the controversial species,and how to distinguish them in an effective way.Methods:(1)Referring to the records in the books since ancient time to determine the species as medicinal materials.(2)According to the documents data,we summarized the controversial in species naming process by different taxonomist.(3)Using DNA barcoding methods to identify all the tuberous herts of Corydalis based on 4 chloroplast gene(mat K,trn G,trn H-psb A,rbc L)and ITS fragment.Choosing the most suitable DNA barcoding sequences for Corydalis.(4)Using ABGD and PTP,the single-gene species dilimination methods,to divide species and assess the precent taxonomy based on 4 chloroplast gene.(5)Using AS-PCR methods to distinguish C.yanhusuo,C.Decumbens and C.turtschaninovii from each other and they substitutes.Results:(1)We have made clear of the species used for medicinal meterials,which mostly(86%)come from Sect.Corydalis and Sect.Duplotuber.In addition,the source of yanhuuo has been changed in about 400 years ago,since emerging cultivated C.yanhusuo instead of such species with yellow tuber as C.turtschaninovii which had been used for more than 700 years.(2)Classification dispute has great influence on the identification of medicinal plants.The dispute appears seriously in particular on species in Sect.Corydalis: the controversial species pairs,C.fumariifolia and C.ambigua,C.yanhusuo and C.turtschaninovii,C.humosa and C.caudata,have caused great confusions in the identification of medicinal herbs such as yanhusuo until now on.(3)DNA barcoding method has not only realized the identification of C.yanhusuo,C.decumbens and their closely related species in the same section,but also showed certain phylogenetic relationship.ITS/ITS2 are not suitable for the identification of species in Corydalis due to their multi-copy and inner-genomic variation nature.With the most informative positions(13.62%)and the maximum identification efficiency,Cp gene mat K can be accepted as the optimal DNA barcoding of Corydalis.Remarkably,when combined with other three cp genes(rbcl,trn G and trn L),this gene achieved the identification of all species in Sect.Corydalis and Sect.Duplotuber.In addition,phylogenetic researchs of this taxon has not only indicated the relationships between the new species C.huangshanensis and other controversial species,but also revealed the importance of the character “ nectary” in the evolution and identification.(4)Species diliminaion methods provide some new view on taxonomy of Sect.Corydalis and Sect.Duplotuber.First of all,our results suggested that several species that we used to believe: C.watanabei,C.ambigua,C.humilis,C.kiautschouensis、C.linjiangensis and C.fumariifolia should be one.Secondly,samples of C.schanginii were divided into two species.The results also proved the species status of C.huangshanensis.(5)Based on 14 species in Sect.Corydalis and Sect.Duplotuber,the AS-PCR technology realized the rapid identification for specific species.The template quantity(0.6～1200ng)and annealing temperature(42～60℃)have minor affects on the amplification product.The number of cycles had a great influence on the results of amplification.Under the conditions of 20 cycles,with the primers we designed in this study,we got specific products of C.yanhusuo,C.turtschaninovii and C.decumbens,which result in 300 bp(mat K),350 bp(trn G)and 550 bp(mat K)sequences,respectively.Closely related species of them were failed to amplified.The minimum amount of DNA that can be detected is 60 ng,0.6ng,and 60 ng,respectively.Innovations:(1)Applying DNA barcoding methods,we first identified C.yanhusuo,C.decumbens and their closely related species successfully,and revealed the incomplete ITS concerted evolution in the genus Corydalis.So we suggest that ITS sequences should be used cautiously in related phylogenetic researches.(2)Based on the traditional classification and the current popular DNA barcodes for the identification of medicinal herbs,the more advanced methods for determining the species of ABGD and PTP were introduced into the identification of medicinal plants,which enriched the analysis methods and made the research results more reliable.(3)We have built a standard method to rapidly identify C.yanhusuo,C.decumbens and C.turtschaninovii,and develop the identification researchs for other medicinal herts.