Identification,molecular Systematics,and Fungal Community Analysis Of Chinese Herbs Using DNA Barcoding Technique

Traditional Chinese medicine(TCM),an integral part of Chinese culture,plays a predominant role in the healthcare system of China.In the clinical treatment,prevention,and control of COVID-19(corona virus disease 2019),TCM has contributed an important force and further enhanced its influence in the world.However,adulteration and quality problems of Chinese herbal medicines occur from time to time,which not only affect the safety and effectiveness of clinical medications,but also weaken consumers' trust in TCM.In recent years,DNA barcoding,as an emerging molecular identification technology,has been widely used in the identification of Chinese herbal medicines.With the continuous development of sequencing technology and the deepening of scientific research,the introduction of new concepts such as DNA mini-barcode,super barcode/ultrabarcoding,and DNA metabarcoding have further broadened the application of DNA barcoding technique,which not only provides a strong guarantee for monitoring the authenticity and quality of processed Chinese medicinal materials,but also provides new ideas and methods for the systematic evolution analysis of complex groups and the study of microbial diversity in Chinese herbal medicines.In this study,DNA barcoding technology was taken as the core to study its application in the identification,molecular systematics,and fungal diversity of Chinese herbal medicine.The main research contents and conclusions are as follows:1.The ITS2 barcode was used to discriminate the medicinal species of Cynanchum.The identifying capability of ITS2 for 17 medicinal plants of Cynanchum was assessed using the specific genetic divergence,BLAST1,neighbor-joining(NJ)tree,maximum-likelihood(ML)tree,and single-nucleotide polymorphism(SNP)methods.Results indicated that the intra-specific genetic divergences of Cynanchum species were lower than their inter-specific genetic divergences.ITS2 showed a high identification efficiency of 90.8%and 87.4%at the species level through BLAST1 and the nearest distance methods.NJ tree and ML tree also demonstrated the suitability of ITS2 to differentiate Cynanchum species.Meanwhile,a stable SNP was found,and it could accurately authenticate C.paniculatum and C.atratum.Furthermore,we collected 64 commercial samples from three commonly used herbal medicines and evaluated the capability of ITS2 to survey their authentication.Of these samples,Cynanchi Atrati Radix et Rhizoma(Baiwei)showed a potential safety problem,and all the 11 test samples were adulterants.ITS2 can distinguish medicinal species in Cynanchum effectively,greatly improving the identification efficiency and accuracy of herbal medicines in this genus.2.A nucleotide signature of Ophiopogonis Radix(Maidong)was developed using DNA mini-barcode technique to investigate the authenticity of commercial Ophiopogonis Radix and its Chinese patent medicines.A total of 255 ITS2 sequences representing 39 species and 4 varieties from Ophiopogon and Liriope were used to develop a nucleotide signature of Ophiopogonis Radix.A 69bp nucleotide signature unique to Ophiopogonis Radix was found,which could effectively distinguish Ophiopogonis Radix from other species.The nucleotide signature was used to investigate 17 commercial crude drugs of Ophiopogonis Radix and eight batches of Chinese patent medicines containing Ophiopogonis Radix.The survey revealed that 2 of 17 crude drug samples were adulterants identified as Liriopes Radix(Shanmaidong).No adulterants were detected in the eight batches of Chinese patent medicines.The newly developed nucleotide signature could be efficiently applied to identify Ophiopogonis Radix and its Chinese patent medicines,aiding in the authentication,quality control,and supervision of processed products in herbal markets.3.The chloroplast genome is used as a super barcode to study the molecular phylogeny and evolution of Epimedium.We explored the molecular phylogeny,assessed the infrageneric classification,estimated the divergence times,and inferred the ancestral states of Epimedium based on 45 chloroplast genomes from 32 species.The Epimedium plastome length ranged from 156,635bp to 159,956bp.Four types of plastome organization with different inverted repeat boundary changes were identified.Phylogenetic analysis revealed a strong support for the sister relationship of sect.Macroceras and sect.Diphyllon,but did not provide resolution within sect.Diphyllon.Disharmony between molecular phylogenetic relationships and traditional classification of sect.Diphyllon was observed.Results from divergence time analysis showed that Epimedium diverged in the early Pleistocene(?2.11Ma,95%HPD=1.88-2.35Ma).Ancestral character state reconstructions indicated transitions from long spur(large-flowered group)to other petal types(small-flowered group)in Epimedium.These findings provide new insights into the phylogenetic relationships among Epimedium species and pave the way for better elucidation of the classification and evolution of this genus.4.The DNA matabarcoding technique was used to analyze the fungal community in Ziziphi Spinosae Semen,Coicis Semen,Cassiae Semen,and Armeniacae Semen Amarum samples.The total DNA was extracted,and the ITS2 sequences were amplified.Then,the amplicons were sequenced using the Illumina MiSeq PE250/300 platform.Results showed that all the samples tested were contaminated by fungi.Ascomycota was the prevailing fungi at the phylum level in these four medicinal seeds,with the relative abundance of 64.36%-99.74%,93.96%-99.58%,66.50%-99.42%,and 68.57%-99.65%,respectively.At the genus level,Aspergillus(13.52%-87.87%),Candida(0.42%-64.56%),and Wallemia(0.06%-34.31%)were the predominant genera in Ziziphi Spinosae Semen samples;Fusarium(3.05%-60.32%),Aspergillus(2.20%-45.44%),and Beauveria(0.07%-63.21%)were the dominant genera in Coicis Semen samples;Aspergillus(0.66%-85.51%),Cladosporium(0.20%-29.11%),and Penicillium(0.11%-32.92%)were the predominant genera in Cassiae Semen samples;and Aspergillus(25.86%?93.86%)was the most dominant genus in Armeniacae Semen Amarum samples.In addition,potential toxigenic fungi and human pathogens from Aspergillus,Penicillium,Candida,Fusarium,Schizophyllum,Wallemia,and Rhizopus genera were also detected in the samples of the four Chinese herbal medicines.DNA metabarcoding technique is feasible for analyzing fungal communities in medicinal seeds,which provides a new approach to investigate the fungal contamination in herbal materials,thereby ensuring drug efficacy and safety.