Survey And Identification Of Cultivated Bupleurum Spp.germplasm Resources In China

Bupleuri Radix(BR),the dried root of several Bupleurum spp.,is one of the frequently used Chinese herbal medicines.It has been artificially cultivated since the 1980s,but cultivated Bupleurum has not become the main commercial BR source until the 21st century.The botanical origin of commercial BR has been chaotic for a long time,and Chinese pharmacopoeia included only two species,namely Bupleurum chinense and B.scorzonerifolium.Several related species which were used as BR adulterations were introduced and cultivated during early artificial cultivation stages,and these resources were hardly eliminated because:(1)it is difficult to identify Bupleurum spp.morphologically;(2)the distribution areas of wild Bupleurum are extensive and the distribution of different species overlap;(3)different production areas have different usage habits constantly.However,the current situation of cultivated Bupleurum in China has not been comprehensively collected and identified.Moreover,identifying cultivated Bupleurum spp.is confusing because several morphologic characters are modified during cultivation,and the presence of intra-specific diversity also affects species identification.Therefore,establishing a reliable identification system is necessary to remove the limits on cultivated BR identification and usage.In this study,literature research,market investigation,and production area investigation were applied to thoroughly understand the main productive districts,varieties,and challenges of cultivated Bupleurum in China.Cultivated samples were collected from the main production areas in China and planted in experimental plots.Morphologic and microscopic identification,as well as traditional DNA barcoding technology and whole chloroplast genome analysis,were successively conducted to identify the cultivated Bupleurum species,and then the multi-dimensional identification methods were verified with each other,thereby confirming the results.The main results are as follows:1.Resource investigation of BR cultivation.Market investigation revealed that cultivated Bupleurum was the main commercial BR resource,mainly come from the Gansu,Shanxi,and Shaanxi areas,whereas wild resources are mainly from Inner Mongolia.Commercial species are usually unclear and named after their origin locations.The aerial parts of Bupleurum are sold in the market as well,although most of them are roots.Based on our investigation on several BR-producing areas,the main germplasms in each area were different.There are six types of mainly cultivated germplasms,and more than one planting type or variety in some areas,whereas exchanging germplasms also occasionally transpired among the different regions.Literature research showed that the identification results may vary for the same germplasm but show consistent sequences for different species.2.Accurate identification of the main cultivated Bupleurum in China.For the first time,the mainly cultivated Bupleurum spp.in China were systematically sorted out and accurately identified.This was achieved using multiple approaches,including morphologic and microscopic identification,ITS barcode identification,and whole chloroplast genome analysis.The disputed and easily misconstrued materials were also effectively identified and clarified.The main cultivated Bupleurum spp.from Gansu,Shanxi,and Shaanxi were identified as B.chinense,although there were morphologic differences among them.The main cultivated Bupleurum spp.from Heilongjiang was identified as B.scorzonerifolium.In addition to the large-scale cultivation of the aforementioned two species in Chinese pharmacopeia,B.falcatum is also cultivated in Heilongjiang and Hebei,and B.marginatum var.stenophyllum is cultivated in Gansu.3.Establishment of identification methods for cultivated Bupleurum.Through carefully observing,comparing,and recording the traits of the identified germplasms,the main characteristic variations of the cultivated Bupleurum were compared with wild Bupleurum spp.,and the distinguishing characteristics,including seven newly discovered features among all cultivated Bupleurum spp.,were proposed for establishing an identification system for the cultivated species.The nuclear DNA sequence ITS was identified from a screen of four traditional DNA barcodes(ITS,psbA-trnH,rbcL,and matK)to confirm the botanical origin of the cultivated materials.The whole chloroplast genome analysis was subsequently conducted to identify the cultivated Bupleurum spp.and confirm the results of the aforementioned methods.SSR molecular markers and chloroplast DNA barcodes with distinguishing features were simultaneously screened as supplementary barcodes or molecular markers for identifying cultivated Bupleurum spp.Combining practical identification keys,carefully calibrated ITS barcode sequences,chloroplast genome sequences,and new barcode sequences from the perspective of morphology,microscopy,nuclear gene inheritance,and chloroplast gene inheritance,respectively,the multi-dimensional methods provided mutually verified results,which greatly improved the efficiency and accuracy of cultivated Bupleurum species identification.Establishing this identification method is beneficial to the quality control and evaluation of cultivated B.chinense.Moreover,it has a great significance for further developing cultivated BR resources and for safe and effective BR utilization.Moreover,the methods and protocols established in this study could be a reference for future taxonomic analysis of other plant species with similar identification challenges.