DNA Barcoding Chinese Medicinal Bupleurum

Chaihu(Bupleuri radix),the dried roots of Bupleurum chinense DC.and B.scorzonerifolium Willd.,is a famous Traditional Chinese medicinal herb with a history of medical use for more than 2000 years.The taste of Bupleurum is bitter and cool,and has the effect of evacuating the fever,dispersing stagnated liver qi for relieving qi stagnation,lifting yang;which commonly used in cold fever,headache dazzle,chills and fever embolism.Chaihu are distributed throughout the whole of China,according to the statistics recorded in the past,there are 42 species,17 varieties,7 variant bupleurum plants in our country.Most of Bupleurum medicinal plants have been medical use since ancient.In some areas,even 5 or 6 different species of local Bupleurum are used under the same name of Chaihu.This confusion(intentional or not)results in rather variable qualities of the raw materials for Chaihu in Chinese market.Futhermore,it will cause deadly situation,seriously affecting our reputation in the international market of Chaihu.Therefore,the identification of bupleurum is a critical state.Traditional Chinese medicine,which has a wide range of different varieties,has been used in clinic commonly.Drugs of little difference in appearance,the effect will be very different.The same kind of medicine has been affected by the growth of the local environment,climatic conditions and present different effects;and it will have different effects on different conditions of use and treatment.Therefore,medicines management and resolution of complexity to counterfeit on the market to create the conditions so that a portion of poor quality Chinese medicine into the market.With the fast development of science and technology,the classic source,character,identified by physicochemical methods can not meet the authenticity of traditional Chinese medicine,the pros and cons need identification,especially not meet such as animal ingredients,authentic herbs,medicinal herbs and Chinese medicine processing processing compound preparation prescriptions identified needs.In recent years,with emerging biotechnology,medicine identification method has also undergone a rapid development.Recently proposed by the DNA markers can recover and overcome some of the pitfalls of traditional identification methods and problems.Rapid,accurate and simple identification of species by DNA bar code technology through standardized small fragment DNA sequences.The basic operation process of barcoding DNA includes the following steps:designing universal primers,extracting DNA,using universal primers for PCR amplification,purification of PCR products,sequencing and sequence analysis.DNA sequence contains rich biological and chemical information,DNA sequences of different species are significantly different.DNA barcode is not only a powerful supplement to the traditional identification of species,but also makes the process of identification of specimens to achieve automation and standardization,can be established in a relatively short period of time to form an easy to use application system.At present,a single sequence or combination of sequences can not be very accurate for species identification.The conservation of chloroplast genome sequence is very high,the average length is 110-160kb,far more than the length of the DNA bar code 300-700bp.Because the chloroplast genome contains a wealth of information on the evolution of species,it is possible to reflect the difference between species.Therefore,based on the chloroplast genome sequence,the high throughput sequencing of the Bupleurum was carried out to study the phylogeny of the genus.In this study,we collected from all over the country 19 species Bupleurum,51 samples.Four candidate barcode sequence,namely psbA-trnH,matK,rbcL,ITS2 of PCR amplification,sequencing and sequence analysis,and were assessed in terms of the success rate of PCR amplification and sequencing,DNA barcoding gap,the rate of identification and the ability to discriminate species.The chloroplast genome DNA of Bupleurum chinense and Bupleurum scorzonerifolium was extracted and purified.Complete chloroplast genome sequences were geted by high-throughput sequencing technology,then assembled and annotated.The physical map of the chloroplast genome was obtained,which provided a reference for the development of the system of the medicinal plants of Bupleurum medicinal plants.The establishment of DNA barcode identification method for Chaihu1 PurposeWe tested 4 markers,namely nuclear internal transcribed spacer 2(ITS2),psbA-trnH,matK,and rbcL,to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix(Chaihu)from its adulterants througout the success rate of PCR amplification and sequencing,DNA barcoding gap,the rate of identification.2 Method2.1 Samples collectionB.angustissimum,B.candollei,B.chaishoui,B.chinense,B.chinense f.Chiliosciadium,B.chinense f.octoradiatum,B.chinense f.vanheurckii,B.Falcatum,B.hamiltoni,B.longicaule var.giraldii,B.longiradiatum,B.longiradiatum f.australe,B.malconense,B.marginatum,B.marginatum var.Stenophyllum,B.petiolulatum var.tenerum,B.polyclonum,B.Scorzonerifolium,B.sichuanense,B.smithii,B.smithii var.Parvifolium,B.Yinchowense,total 19 species 51 samples of Bupleurum(collected from 2008 to 2015)from different areas of China including Yunnan,Hebei,Shanxi,and Gansu Provinces,etc.All the plant species were identified by one of the author,Chao Zhi,and Professor Pan Shengli of the School of Pharmacy,Fudan University.The voucher specimens were deposited in School of Traditional Chinese Medicine,Southern Medical University.2.2 Species-specific primer designBased on primer designing principles,species-specific primer of B.chinense was designed for ITS2;and the primers used in the amplification of rbcL,psbA t and matK fragments were all references.2.3 DNA extractionThe total DNA was extracted using DNA kit following the manufacturer’s instruction(made by TI AN GEN company).2.4 ITS2 Barcode amplificationThe ITS2(5’-CGTAG CGAAA TGCGA TACTT GGTG-3’)and ITS4(5‘-TCCTC CGCTT ATTGA TATGC-3’)primers were used to amplify the ITS2 barcode region.PCR was amplified in a total reaction volume of 25μA containing lμl genomic DNA,1pl each primer,2xTaq Plus PCR MasterMix12.5μ1,with 9.5μ1 ddH2O.Thermal cycling was performed with an initial denaturing at 93 ℃ for 5min and annealing at 50℃ for 2min,followed by 30 cycles of 93℃ for 30s,50℃ for 45s,and 70 ℃for 45s,with a final extension at 70℃ for 5min and chilling to 4℃.After PCR amplification,PCR products were detected by agarose gel electrophoresis.A blank control group with no DNA template was set up in the experiment.If amplification fails,the primers are required to design and change the PCR condition until the amplified bands are obtained.2.5 rbcL Barcode amplificationThe 1F(5’-ATGTC ACCAC AAACAGAAAC-3’)and 724R(5’-TCGCATGTAC CTGCA GTAGC-3’)primers were used to amplify the rbcLbarcode region.PCR was amplified in a total reaction volume of 25,μ1 containing1μ1 genomic DNA,1μ1 each primer,2×Taq Plus PCR MasterMix12.5μ1,with 9.5,μ1 ddH2O.Thermal cycling was performed with an initial denaturing at 93 ℃ for 5min and annealing at 50℃ for 2min,followed by 30 cycles of 93 ℃ for 30s,50℃ for 45s,and 70℃ for 45s,with a final extension at 70℃ for 5min and chilling to 4℃.After PCR amplification,PCR products were detected by agarose gel electrophoresis.A blank control group with no DNA template was set up in the experiment.If amplification fails,the primers are required to design and change the PCR condition until the amplified bands.are obtained.2.6 matK Barcode amplificationThe 390(5’-CGATC TATTC ATTCA ATTTC-3’)and 1326R(5’-TCTAG CACAC GAAAG TCGAA GT-3’)primers were used to amplify the matK barcode region.PCR was amplified in a total reaction volume of 25,μ1 containing 1μl genomic DNA,1μ1 each primer,2xTaq Plus PCR MasterMix12.5μl,with 9.5,μ1 ddH2O.Thermal cycling was performed with an initial denaturing at 93℃ for 5min and annealing at 50℃ for 2min,followed by 30 cycles of 93℃ for 30s,50℃ for 45s,and 70℃ for 45s,with a final extension at 70℃ for 5min and chilling to 4℃.After PCR amplification,PCR products were detected by agarose gel electrophoresis.A blank control group with no DNA template was set up in the experiment.If amplification fails,the primers are required to design and change the PCR condition until the amplified bands are obtained.2.7 psbA-trnH Barcode amplificationThe trnHF(5’-CGCGC ATGGT GGATT CACAA TGG-3’)and psbA3’f(5’-GTTAT GCATG AACGT AATGC TC-3’)primers were used to amplify the psbA-trnH barcode region.PCR was amplified in a total reaction volume of 25,1 containing 1μl genomic DNA,1μl each primer,2×Taq Plus PCR MasterMixl2.5μ1,with 9.5,μ1 ddH2O.Thermal cycling was performed with an initial denaturing at 93 ℃ for 5min and annealing at 50℃ for 2min,followed by 30 cycles of 93℃for 30s,50℃for 45s,and 70℃ for 45s,with a final extension at 70℃for 5min and chilling to 4℃.After PCR amplification,PCR products were detected by agarose gel electrophoresis.A blank control group with no DNA template was set up in the experiment.If amplification fails,the primers are required to design and change the PCR condition until the amplified bands are obtained.2.8 the sequencing of four barcodesThe PCR products were detected by 1.2%agarose gel electrophoresis,and visualized under ultraviolet light.After purification,the products were sequenced in both directions by invitrogen Biotechnology(Shanghai)Co.,Ltd.2.9 sequence analysisDNA sequences were assembled with Chromas.The advantages and disadvantages of the PCR amplification efficiency of each candidate sequence were discussed and compared with the different sequences.Compare the amplification and sequencing efficiency,intra-and interspecific variation were barcoding gap analysis and the ability to identify.Finally,the similarity search algorithm(BLAST1)and the nearest distance method(Distance Nearest)were used to investigate the success rate of the sequences.3 Results3.1 The ITS2 and rbcL regions exhibited an amplification and sequencing rate of 100%for the test species.The PCR success rate for the psbA-trnH and matK was 96.08%and 66.67%,with a sequencing efficiency of 87.76%and 94.12%,respectively.3.2 The amplicon size was 226-227 bp for ITS2,328-367 bp for psbA-trnH,(923 □)932-933 bp for rbcL,and(823 □)882-883 bp for matK.3.3 intra-and inter-specific analysis:PsbA-trnH had the highest average intra-specific distance,followed by ITS2 and matK,and that of rbcL was the lowest.ITS2 had the highest inter-specific divergence in all the 3 dimensions,followed by psbA-trnH and matK,while that of rbcL was the lowest.3.4 In this study,we found that ITS2 had obvious advantages over the other loci as a barcode for Bupleurum.ITS2 alone showed a success rate of 73.68%in Bupleurum discrimination;while combined with psbA-trnH sequences,the success rate was increased to 84.21%.In addition,ITS2 successfully discriminated 64.13%of the samples at the species level in a large database from GenBank,which included 173 samples from 73 species.3.5 ITS2 had the highest authentication capability and correctly identified 73.68%of the test samples at the species level.PsbA-trnH,following ITS2,exhibited an identification efficiency of 72.22%.When ITS2 and psbA-trnH were combined,the identification efficiency increased to 84.21%.In contrast,the rates of successful species identification using matK and rbcL were lower than 50%.The chloroplast genome of Bupleurum1 PurposeThe research on phylogenetic genomics of the Bupleurum was carried out by means of high-throughput sequencing technology.2 Method2.1 Samples collectionThe same as"DNA barcoding Chinese medicinal Bupleurum,"above.2.2 Chloroplast genome extractionAccording to the "chloroplast genome extraction" method to extract the genome.2.3 High-Throughput SequencingUsing HiSeq 2500 high throughput sequencing platform developed by Illumina company.2.4 The chloroplast genome sequence assemblyThe original data returned by sequencing company was loaded in Fasta QC software to analyze the overall quality of the sequence;FastQ file input to the CLC genomics workbench software;reads filtered based on de Bruijn algorithm,the value of the parameter K-mer de novo assembly.2.5 The annotation and analysis of GenomeChloroplast genome annotation with DOGMA(Dual Organellar Genome Annotator)program,manual adjustments when necessary.And then the chloroplast genome sequence registered in Genbank.Construction of the physical map,and bioinformatics analysis.3 Results3.1 The assemble result of Bupleurum chinese chloroplast genomeThe total length of the chloroplast genome was 156763 bp,and the GC content was 37.65%.It displayed a typical quadripartite structure including two inverted repeat regions(IRA and IRB,26293 bp),one small singlecopy region(SSC,17546 bp)and one large single-copy region(LSC,8663 1bp).It contained 127 genes.3.2 The assemble result of Bupleurum scorzonerifolium chloroplast genome The total length of the chloroplast genome was 156766 bp,and the GC content was 37.65%.It displayed a typical quadripartite structure including two inverted repeat regions(IRA and IRB,26315 bp),one small singlecopy region(SSC,17530 bp)and one large single-copy region(LSC,85607bp).It contained 127 genes.3.3 Phylogenetic tree of gene systemThe construction of the NJ tree analysis showed that the chloroplast genome sequence could be distinguished from Bupleurum Chinese,Bupleurum scorzonerifolium andB.falcatum.ConclusionsAn ideal DNA barcode must have high PCR amplification and sequencing efficiency,a significant inter-species divergences and a minimal intra-species variation.1.In the present study,rbcL exhibited an amplification and sequencing efficiency of 100%,but its identification efficiency was only 26.32%.As to matK,the amplification efficiency was only 66.67%with the recommended universal primer 390F-1326R,and the identification efficiency was 46.15%.Therefore,rbcL and matK fragments at the species level to identify the root of the root had not an advantage.2.Using the primers designed for ITS2,we achieved PCR amplification and sequencing efficiency both of 100%.As to the identification efficiency,ITS2 alone showed a success rate of 73.68%in Bupleurum discrimination;while combined with psbA-trnH sequences,the success rate was increased to 84.21%.In addition,ITS2 successfully discriminated 64.13%of the samples at the species level in a large database from GenBank,which included 173 samples from 73 species.3.The PCR success rate for the psbA-trnH was 96.08%,and the sequence rate was 87.76%;while the identification rate was 72.22%,inter-specific variation was relatively large.4.Bupleurum chinense and Bupleurum scorzonerifolium chloroplast genome DNA were extracted and purified using high-throughput sequencing technologies for sequencing and sequence assembly,complete chloroplast genome sequence;and the successful annotation,access to the respective chloroplast genome physical map.5.The total length of Bupleurum Chinese chloroplast genome was 156763 bp,and the GC content was 37.65%.It displayed a typical quadripartite structure including two inverted repeat regions(IRA and IRB,26293 bp),one small singlecopy region(SSC,17546 bp)and one large single-copy region(LSC,86631 bp).It contained 127 genes.The total length of Bupleurum scorzonerifolium chloroplast genome was 156766 bp,and the GC content was 37.65%.It displayed a typical quadripartite structure including two inverted repeat regions(IRA and IRB,26315 bp),one small singlecopy region(SSC,17530 bp)and one large single-copy region(LSC,85607bp).It contained 127 genes.Based on the amplification and sequencing efficiency,barcoding gaps,and success rate of identification,we found that ITS2 had obvious advantages.When ITS2 and psbA-trnH were combined,the identification efficiency increased to 84.21%.partial ITS2 sequences can be covered and complemented by PsbA-tmH sequences which cannot be analyzed and successfully identified.Therefor,we recommend that ITS2 region be used as a barcode for differentiating Bupleurum species,with psbA-trnH as a complementary locus.The chloroplast genome sequencing and splicing of Bupleurum medicinal plants,will provide reference for the high throughput sequencing of the medicinal plants and their external groups,and lay a theoretical foundation for the next level of the species.