Resource Investigation And Identification Of DNA Barcode Of Dalbergia Odorifera And Its Adulterants

Dalbergia Odorifera T.Chen is derived from dry heartwood of the trunk and root of the Leguminous plant Dalbergia odorifera T.Chen.It is mainly produced in Guangxi,Hainan,Yunnan and other places in my country.It has the effects of removing blood stasis,stopping bleeding,regulating qi and relieving pain.In addition to its medicinal value,it is one of the precious mahogany varieties in my country.Due to the high medicinal value and wood value of Dalbergia odorifera,the market demand is huge,resources are scarce,the medicinal materials are more mixed in the market,and the morphological characteristics of the medicinal materials are limited.Traditional identification methods are difficult to accurately identify,which seriously affects quality and clinical drug safety of Dalbergia odorifera.Therefore,clarifying the relationship between the origin of the medicinal material Dalbergia odorifera included in the Pharmacopoeia and its contaminants will provide a favorable scientific basis for the accuracy of the use of Dalbergia odorifera in the Chinese herbal medicine market and the safety of clinical medications in the future,and Lay a scientific foundation for expanding the search for alternative sources of Dalbergia odorifera.Objective:The market for Dalbergia odorifera medicinal materials is complex,and there are many adulternats,which lead to confusion in the medication situation and seriously affect the safety and effectiveness of clinical medication.This study found out the amount and distribution of Dalbergia L.f medicinal resources through the textual research of Materia Medica and the collection of CVH specimens;collected experimental samples of Dalbergia odorifera and its adulterants through field investigation;amplified by DNA barcode technology based on PCR The principle is to realize the identification of Dalbergia odorifera and its genus plants,and the accurate identification of Dalbergia odorifera and its adulterants.Methods:Find out the medicinal resources,efficacy and national medicines of the Dalbergia odoriferaby referring to domestic and foreign references and herbal books,and through the Chinese Virtual Herbarium(CVH)(http: // www.cvh.ac.cn/)collection specimens summarized the Dalbergia L.f distribution of medicinal plants of the Dalbergia odorifera.It provides a reference for the full development and utilization of experiments and provides an experimental basis for subsequent research.This study collected Dalbergia L.f.samples from Yunnan,Hainan,Guangxi,Guangdong,Chongqing,Shanxi,Guang zhou and other places.During the collection process,the information such as the date,location,latitude,longitude,and altitude of the samples were recorded,and accurately identified in combination with the "Chinese Flora" search form.The collected samples are selected from fresh young plant leaves and placed in silica gel for dry storage in time.Collect 289 samples of Dalbergia odorifera and its genus plants,extract the genomic DNA of the sample,use ITS,ITS2,rbc L,psb A-trn H,mat K as primers to amplify and sequence the sequence,calculate the success rate of amplification and sequencing of each sequence,and use MEGA7.0 analyzes the characteristics of the aligned sequence;analyzes the sequence saturation of 5 kinds of sequences,calculates the genetic distance between species within and between species based on the Kimura-2-Parameter(K2P)two-parameter model,and uses MEGA7.0 software to construct NJ tree and Phylosuite software build a BI phylogenetic tree.The total DNA was extracted from 63 leaves and 11 heartwood samples of Dalbergiae Odoriferae Lignum and its same genegric plants.36 sequences originated from Gen Bank.and the rbc L sequences of Dalbergiae Odoriferae Lignum.and same genegric plants were amplified and sequenced bi-directional,and the information of rbc L sequences were obtained.PCR amplified 42 ITS2 sequences.The length and GC content and the variation loci of rbc Land ITS2 sequences were analyzed through MEGA7.0 software,the genetic distances were computed by Kimura-2-parameter(K2P)model.Dalbergiae Odoriferae Lignum and its genegric plants have been identified through Blast identification methods and the construction of phylogenetic trees.Result:From the specimens collected in the CVH library,specimens of 16 types and 8096 Medicinal plants of the Dalbergia odorifera were sorted out.Among the collection specimens,many specimens were collected from Guang xi,Yunnan,Sichuan,Tibet,Guang zhou and other regions of China.A total of 289 samples were collected from 20 species including Dalbergia odorifera,Dalbergia cochinchinensis,Dalbergia mimosoidesand Dalbergia yunnanensis,etc.The 5 DNA barcode sequences of ITS,ITS2,rbc L,psb A-trn H,and mat K were evaluated.Among them,psb A-trn H had the highest amplification success rate of 100%,and the success rate of ITS,rbc L,and matk was more than 90%.The success rate of rbc L and ITS2 sequencing is higher,respectively,93% and 89%.A total of 269 rbc L sequences,252 ITS2 sequences,179 psb A-trn H sequences and 169 mat K sequences were obtained.The ITS,ITS2,rbc L,psb A-trn H,and ITS,ITS2,rbc L,psb A-trn H,and ITS,ITS2,rbc L,psb A-trn H,Variation site information of mat K DNA barcode sequence.Compared with other barcode sequences,ITS2 has the highest resolution,obvious Barcoding gap,and less overlap between species within species;phylogenetic tree shows that ITS2,mat K and psb A-trn H single gene sequences can clearly cluster D.odorifera and its genus plants into differences Branches,and psb A-trn H and rbc L are more difficult to distinguish Dalbergia odorifera and its genus plants.Therefore,ITS,ITS2 and mat K are more suitable for the identification of Dalbergia odorifera and its adulterants.ITS2 has the best effect,and psb A-trn H is the second.The sequence lengths of rbc L of Dalbergiae Odoriferae Lignum were 494 bp,and the GC content was 43.12%,the minimum K2 P interspecific genetic distances of Dalbergiae Odoriferae Lignum and its same generic plantswere 0.002,apart from Dalbergia retusa Baillthe sequences lengths of ITS2 of Dalbergiae Odoriferae Lignumwere 216 bp,and the GC content was 68.52%,the minimum K2 P interspecific genetic distances of Dalbergiae Odoriferae Lignum and its same generic plantswere 0.024,apart from Dalbergia tonkinensis Prain.and which was larger than that of the intraspecific genetic distance of Dalbergiae Odoriferae Lignum.Conclusion:Dalbergiae odorifera is a traditional Chinese medicine used for thousands of years,but its original is complex,the kinship is not clear,mixed with a variety of adulterants,leading to drug confusion,seriously affecting the safety and effectiveness of clinical medication.according to the herb,most species of the Dalbergia odorifera which is from the Lignum can be used as the original of In this experiment,the efficacy,national medicinal status,reserves and distribution of the medicinal plants were investigated.The molecular identification technology of DNA barcode was used to identify Dalbergia odorifera and its adulterants,Dalbergiae Odoriferae Lignum and same generic plants successfully,and an identification method based on ITS2 and supplemented by the psb A-trn H sequence was obtained for fast and accurate identification of them.The study provides an important basis for Dalbergiae Odoriferae Lignum for the identification of germplasm resources and the safety of clinical medication.