DNA Barcoding Identification Research On Medicinal Plants And Herbs Of Angelica

The identification of herbs is a major issue in Traditional Chinese Medical (TCM) research, the purpose of which is to identify the genuine and high-quality of herbs, eliminate the false and retain the truth. The traditional methods for herbs identification include character identification, microscopic identification, physical and chemical identification have the shortcomings in objectivity, stability and repeatability. DNA barcoding is the latest technology in the molecular identification methods used in Chinese medicine molecular identification, can identify the species rapidly and accurately, the samples can be easily identified just by DNA extraction, PCR amplification, electrophoresis detection, gene sequencing and data-base alignment, thus received more and more attention.There are two limitations of the recent researches that use DNA barcodes to identify the herbs,1) the previous studies on the technology of DNA barcoding for the identification of herbs ignore an important issue, that is the premise of barcode-based identification of herbs is the species of the herbs and their adulterants have DNA differences, that is to be monophyletic with each other. This is the premise of herb identification, only based on this, can give the molecular identification of medicinal plants and herbs a objective and comprehensive molecular scale.2) the previous DNA barcoding studies often use fresh leaves collected from the original plants as the research materials, hence, the DNA of herb-pieces may be degraded and fractured after particularity processing, the aim for DNA barcoding is whether DNA barcode technology can overcome the problem and achieve the purpose of the authenticity Chinese herbs, eliminate the false and retain the truth or not, which is also worth to try and verify.In this study, we use the original plants of Angelica and herbs of Danggui, Duhuo and Baizhi as our research objects. Based on molecular systematic and DNA barcoding technology, we select one nuclear and three chloroplast DNA regions (ITS, rbcL, matK and trnH-psbA) to do DNA extraction, PCR amplification and sequencing, then based on the K2P model using the neighbor joining (NJ) to establish the phylogenetic tree, to achieve the purposes,①To select the most suitable DNA region for DNA barcoding research of Angelica;②To evaluate the ability of DNA barcoding to identify the medicinal plants of Angelica;③To discuss the accessibility of using DNA barcoding to identify the misuse situation between Danggui and Duhuo;④Molecular identification used by DNA barcoding research of Baizhi in different degrees of processing;⑤Locality identification of Baizhi(Chuan, Hang, Qi, Yu);⑥Research on the cultivated origin of Baizhi..The results are as follows:1. ITS is the most suitable DNA region for DNA barcoding research of AngelicaThere are25species original plants of Angelica (including four subspecies), after the analysis of the experimental results of the original plant, we get a total of384sequences, including96sequences of ITS,96sequences of matK,96sequences of rbcL, and96sequences of trnH-psbA. The success rate for the selected four regions of DNA extraction, PCR amplification and sequencing was100%, showing no difference in the versatility, but the species identification, ITS shows significantly better than the other three fragments. All the four fragments of interspecific genetic distance greater than the intraspecific genetic distance, and the decreasing order is ITS> trnH-psbA> matK> rbcL. Especially, For ITS, the interspecific genetic distance is50times more than intraspecific genetic distance, the limits of interspecific/intraspecific genetic distance is obviously, ITS is well for the identification of species.Build support≥50%threshold as the successful identification of species boundaries NJ tree, ITS can successfully identify19species original plant from25species (76%), while the ability to identify the remaining three fragments are trnH-psbA8species (32%), matK11species (44%), rbcL5species (20%). Therefore, this study recommended ITS as the standard regions of the DNA barcoding research of Angelica plants.2. DNA barcodes are able to identify16species of medicinal plants of Angelica effectively25species of original plants are selected in this experiment, and19species have medicinal effect. Based on the NJ tree of ITS sequences,19species of medicinal plants except the A. laxifoliata, A. decursiva and A.cartilaginomarginata var. foliata cannot be separated, the remaining16species of medicinal plants can be well separated from one and another. This is a new approach for the identification of national and local drug can be used as a reliable identification based on medicine quality control and authenticity of the identification.3. DNA barcoding can successfully applied to the identification of Danggui and Duhuo piecesThe previous results show that the original plants of Danggui and Duhuo are both monophyletic in the NJ tree established by ITS, so we can use molecular identification to identify Danggui and Duhuo pieces. There are59samples of Danggui and58samples of Duhuo involved in this study. We get92ITS sequences,103trnH-psbA sequences,89matK sequences and103rbcL sequences. The PCR efficiency/sequencing efficiency is ITS(92.3%/85.2%), trnH-psbA (96.6%/91.2%), matk(88.9%/85.6%),rbcL(92.3%/95.4%). Four regions in the Danggui and Duhuo pieces show a higher efficiency of PCR amplification and sequencing technical feasibility of DNA barcodes in the identification of Danggui and Duhuo pieces.Using the ITS sequences of25species of Angelica original plants and Danggui and Duhuo pieces together to establish the NJ tree, the results show that, in the Danggui pieces, the samples from Dezhou(DZ) GDZ-2GDZ-4、GDZ-5、GDZ-7、 GDZ-8are the alternative uses of Lerisiicum officimale, and the other samples come from9different regions are the same with the orginal plant of A. sinensis, which we can define it as genuine. In Duohuo pieces, the samples from Binzhou(HBZ-4、 HBZ-6) and Dezhou(HDZ-4、 HDZ-6、HDZ-8), are all Leristicum officimale too. We have done traditional characteristic identification between the substitutions and the fake to determine the feasibility and reliability of DNA barcoding.4. The investigation of identification of Baizhi in different progressing methods when using DNA barcoding.In this study, we select60samples from10different regions and14samples from4traditonal production areas, including Suining, Hefei, Anguo, Yuzhou. We have got7ITS sequences,15trnH-psbA sequences,29rbcL sequences and10matK sequences, the PCR efficiency/sequencing efficiency is, ITS (46.7%/25.0%), trnH-psbA (40.0%/62.5%).rbcL (51.7%/93.5%), matK (30.0%/55.6%), and we can conclude that4regions of the PCR efficiency in Baizhi crud drugs is higher than in Baizhi pieces. The sequencing efficiency in Baizhi curd drugs is also higher except rbcL. The reasons is that there is more starchy in Baizhi pieces after purification and we should establish a new method to identification of herbs using a standard system to make a breakthrough in herbs identification.5.The ability of DNA barcoding that applied to locality identification of Baizhi is inadequateFor locality identification of4commercial species of Baizhi, We collect Chuan (Hang), Qi (Yu)Baizhi from their country of orgin, each place collect2-4samples of the fresh root(crud drug), dry naturally before the experiment. We get12sequences of ITS,11sequences of trnH-psbA,12sequences of matK,12sequences of rbcL. After the four regions obtained sequences are compared, we find that each of the four DNA regions have the same sequence, they can not be identified from each other. The essence of locality identification is the genetic differentiation problem of populations. It can be based on the theory of population genetics and molecular phylogeography, which should choose the DNA regions with relative faster rate of evolution than in species level, so that to analyze the degree of genetic differentiation and find the molecular identification of medicinal origin of geographical indications. 6. Reaserch on the cultivated orgin of Baizhi.For the question of Baizhi cultivated origin, we use14samples from the4commercial species of Baizhi, and get2ITS sequences of Chuan Baizhi,3ITS sequences of Hang Baizhi and Yu Baizhi,4ITS sequences of Qi Baizhi, we use all the12sequences of ITS together with the ITS sequences of the original plants of Angelica to establish the NJ tree, the results show that the4species of Baizhi have the closest relationship with XingAn Baizhi(A. dahurica), the conclusion is inconsistent with the think of the original plant of Baizhi is Taiwan Baizhi (A. dahurica var. formosana). Therefore, the question of Baizhi cultivated origin need further discussion with the new theories and methods.The conclusion of our experiments are as following,①ITS is the appropriate sequence in distinguishing the right from wrong in Angelica when using DNA barcoding,②DNA barcoding is the most suitable method to separate Danggui and Duhuo when using ITS.③This study find that when we use DNA barcoding to identify herbs, the fresh plant cannot completely replace the crud drugs and piece to verify the ability of DNA barcoding to solve practical problems, that is because some herbs after concocted processing will affect the efficiency of the experiment.The innovation in this study is that, we first establish the scientific method and guiding principles of DNA barcoding for the identification of herbs, that is the "two-step method" for the identification of medicinal materials. The first step is, we should firstly distinguish the appropriate DNA barcoding sequences to establish the phylogenetic tree based on the traditional molecular systematic, to determine the identification of the original medicinal plant of the herbs that we want to identify in the entire system location is a monophyletic. The second step is, after the species boundaries to be clear, we can use DNA barcoding to make accurate identification of the herbs. For the herbs that in doubt, according to the evolution of the position in the system of the orginal plants of the genus, to provide a scientific explanation and identification credentials of their confusion and substitution.