Molecular Identification Of Herbal Medicines:From Barcodes To Genomes

There are many differents kinds of medicinal plants with complicated sources.Chinese Materia Medica(CMM)is the raw material of traditional Chinese medicine(TCM)decoction pieces,TCM extract and Chinese patent medicine.Accurate identification of CMM is a basic insurance of the development of TCM.DNA barcoding is an effective supplement of traditional morphological identification method and it has been the research hotspot of biological classification and species identification in recent years.Now,DAN barcoding is widely used in many fields,such as TCM identification,taxonomy,evolution and biodiversity conservation.In this study,DNA barcoding was used to identify the crude drugs of dietary supplements which recorded in the United States pharmacopoeia,the species of traditional She Medicine Shi-Liang Tea,Vaccariae Semen and their adulterants.And the chloroplast genomes of the Dioscorea opposite and D.collettii were sequenced using the high throughput sequencing platforms in order to screen highly variable loci as novel molecular markers for the identification of Dioscorea.The study came to the following results:(1)In this study,we obtained 228 sequences belonging to 128 samples of 49 species from 28 families which covered 98%of the crud drugs in the dietary supplements of the United States Pharmacopoeia(USP).The internal transcribed spacer 2(ITS2)of 125 authenticated samples were amplified and 94 were successfully amplified and sequenced,with success rates of 75.8%and 100%,respectively.35 samples of which originated from several species were amplified using the psbA-trnH and 31 were successfully amplified with success rates of 88.6%.The rate of identification efficiciency of ITS2 and psbA-trnH for these dietary supplements samples were 77.9%and 40.0%at the species,respectively.The NJ tree demonstrated that the ITS2 sequences of each crude drugs formed into one clade,which can be successfully distinguished from each other.a USP dietary supplement reference DNA barcode library was constructed based on the ITS2 and psbA-trnH sequences as a supervisory method to which constituted by samples database,sequences database and bibliographic database.(2)71 ITS2 sequences belonging to seven species of original sources of Shi-Liang Tea and closely related species were analyzed in this study.The interspecific genetic distances of Chimonanthus salicifolius and C.zhejiangensis were ranged from 0 to 0.078,and the average GC content was 71.23%and 71.07%,respectively.The NJ tree showed that the original plants of Shi-Liang Tea can be easily differentiated from closely related species according to their monophyly.ITS2 could be used as an effective marker to identify the original plants of Shi-Liang Tea and their closely related species,thereby establishing a foundation for the clinical safety of TCM.(3)The ITS2 sequences of 85 samples from six species of the seeds of Vaccaria segetalis(Neck.)and its adulterants were amplified and sequenced successfully.The results showed that the sequence lengths of ITS2 regions of V.segetalis were ranged from 219 to 221 bp and the average GC content was 56.6%.The largest intraspecific K2P genetic distance was 0.009,which was smaller than the interspecific K2P genetic distance.There were many variation sites of ITS2 sequences between V.segetalis and its adulterants.The NJ tree showed that V.segetalis and its adulterants can be easily differentiated according to their monophyly.It was concluded that the ITS2 sequences could be used to identify V.segetalis and its adulterants,which not only provided a new method for the identification of germplasm resources of CMM,but also supplied some guidelines for the establishment of the quality standards of Chinese herbal seeds and seedlings.(4)The complete chloroplast genomes of D.opposite and D.collettii were sequenced using Illumina MiSeq.Genome structure,gene order,GC content and codon usage were analyzed.The complete chloroplast genome length of D.opposite and D.collettii was 152,963 bp and 153,870 bp,which including four parts:two inverted repeats(IRs),one large single copy(LSC)and one small single copy(SSC).Both of D.opposite and D.collettii encoded 125 genes,including 87 protein coding genes,30 tRNA genes and 8 rRNA genes.The GC contents of D.opposite and D.collettii were 37.04%and 37.17%,respectively.The result showed that the variation of non-coding regions is higher than that of the protein coding region and the variation of in LSC and SSC is higher than that of the IRs.10 genes molecular markers of Dioscorea were screened to be used as the potential DNA barcodes for Dioscorea identification which including 5 protein coding regions and 5 non-coding regions.Phylogenetic analyses showed the chloroplast genome can be used as an ultra-barcode to distinguish Dioscorea species from each other.Furthermore,this study laid the foundation of super barcode utilization in the Dioscorea and provided us a molecular base for next investigation on this important medicinal species.