DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China

DNA barcoding, an increasingly popular mean of species identification, has been widely used for global species identification despite a consensus not being reached regarding which DNA sequences can be used as the best plant barcodes. In this study, we tested the feasibility of five candidate DNA barcodes (nrITS, nrITS2, matk, rbcL and trnH-psbA) for identifying Uncaria species. We collected a total of 54 specimens of 10 Uncaria species across its distributional range. BLAST, barcoding gaps, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capability of the candidate DNA barcodes. The results showed that the ITS2 is most suitable as a candidate DNA barcode for identification of medicinal plants of the genus Uncaria.     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

真菌DNA条形码技术研究进展

DNA条形码(DNA barcoding)技术作为一门新兴的物种鉴定方法以其灵敏、精确、方便和客观的优势,在动植物和微生物的分类鉴定中已经得到广泛应用.真菌鉴定中常用作标准条形码的是核核糖体DNA内转录间隔区(Internal transcribed spacer,ITS),如今也有一些新型条形码被发现和应用到实际操作中,如微条形码、ND6、EF3.本文对DNA条形码技术的产生和发展做出了总结,通过研究其在真菌中应用的实际案例分析了DNA条形码技术的优缺点及发展趋势,并指出DNA条形码技术将以全新的视角来弥补传统分类学的不足,最终实现生物自身的序列变异信息与现有形态分类学的结合.     

药用植物DNA条形码鉴定研究进展

DNA条形码是利用相对较短的标准DNA片段对物种进行快速准确鉴定的一门技术。DNA条形码技术可以从分子水平弥补传统鉴定方法的一些不足。该技术具有良好的通用性,使得物种鉴定过程更加快速,已经广泛应用于动物物种的鉴定研究中。近年来,随着药用植物DNA条形码鉴定研究的快速发展,逐渐形成了药用植物和植物源中药材鉴定的完善体系。本文综述了DNA条形码技术鉴定药用植物的原理,介绍了中草药传统鉴定方法及其缺陷、使用DNA条形码技术鉴定植物源药材的意义以及DNA条形码在药用植物鉴定中的应用,对其应用前景进行了展望。     

ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China

Radix Bupleuri (Chaihu), a famous Traditional Chinese Medicine, is derived from the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd. But nowadays, other nine species and varieties from genus Bupleurum are also used as Radix Bupleuri in northwestern China. The authentic identification of dried roots of B. chinense and B. scorzonerifolium, however, is difficult to just base on the appearance and morphology. A molecular genetic method was developed to help discriminate the original species of Radix Bupleuri. The ITS sequences ( approximately 600 bp) were amplified by the PCR technique from genomic DNA isolated from all the collected samples. According to analyzing the information given by ITS sequences, the conclusion can be made that ITS sequence could serve as markers for authentication of Radix Bupleuri.     

中国蜘蛛抱蛋属植物DNA条形码研究

利用植物DNA条形码候选序列mat K、psb A-trn H、psb K-psb I和rbc L对蜘蛛抱蛋属(Aspidistra)植物的19种104批样品进行扩增和测序,并采用相似性搜索算法(BLAST)对各序列的鉴定效率进行评价,得出蜘蛛抱蛋属物种鉴定的最佳序列。结果显示,psb K-psb I的物种鉴定成功率为88.7%,在单一序列中成功率最高。通过多序列组合鉴定效率的比较,发现组合序列的鉴定成功率明显高于单一序列,其中mat K+(psb K-psb I)组合的鉴定成功率高达100%,基于该序列组合构建蜘蛛抱蛋属植物的系统发育树,结果显示同一物种的样品聚集度较好,多表现为单系。研究结果表明mat K+(psb K-psb I)序列组合可作为蜘蛛抱蛋植物种鉴定的最佳条形码序列。     

基于DNA条形码鉴定植物药常山及其混伪品

虎耳草科（Saxifragaceae）常山属（Dichroa）植物常山（Dichroa febrifuga Lour.）是我国著名的抗疟药物。但目前全国各地使用较为混乱,代用品、混淆品多,鉴定困难。本研究收集常山及其混伪品6种,共64份样品,利用DNA条形码和二维DNA条形码技术进行物种鉴定研究。研究结果表明,DNA条形码技术可准确鉴定常山及其混伪品,20%的常山样品存在混伪现象,混伪品主要为阔叶十大功劳（Mahonia bealei（Fort.） Carr.）。常山ITS2序列长度为230 bp,种内K2P遗传距离为0~0.0253,常山及其混伪品种间K2P遗传距离为0.152~0.748。基于ITS2序列构建的NJ树显示,常山及其混伪品能够明显区分,可为临床安全用药提供科学依据。成功构建常山的二维DNA条形码鉴定体系,实现“互联网+”的跨平台信息交换,可为二维DNA条形码技术在流通监管领域的实践应用提供理论基础。     

Comparison of four DNA barcodes in identifying certain medicinal plants of Lamiaceae

Many species in the family Lamiaceae have been widely used for the treatment of coronary heart disease,stroke,and other conditions,and authenticating each of these species has become an important topic...     

Molecular identification and phylogenetic analysis of Aloe shadensis from Saudi Arabia based on matK,rbcL and ITS DNA barcode sequence

The Kingdom of Saudi Arabia thrives with great plant diversity, including rare plants of the family Asphodelaceae that have multiple benefits and are still being studied. Aloe shadensis is one of these plants that must be preserved and documented in its natural environment. The most appropriate molecular approach currently approved for documentation is the sequencing of some genomic markers. The current study is the first to use genomic markers to record this rare plant. In this study, the plastid genes matK (Maturase K), rbcL (Ribulose-bisphosphate carboxylase/oxygenase large subunit), and the nuclear region ITS (Internal transcribed spacer) were used to reveal their efficiency in identifying the plant under study. This study is the first to deal with this plant and document it using these genetic markers. The study showed a promising result concerning identifying the sequence of the matK gene and ITS region, while the rbcL gene did not give a good indicator through the used primers. The obtained sequences of the matK gene and the ITS region were determined through two different sets of primers in each case then deposited in GenBank. The evolutionary relatedness of Aloe shadensis was established with the different species of Aloe. The study showed that the closest species is Aloe vera with a similarity of more than 99 %. The study concludes with the possibility of using these genes to correctly identify, distinguish and document the species of Aloe shadensis.     

DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs

DNA barcoding has greatly facilitated studies of taxonomy, biodiversity, biological conservation, and ecology. Here, we establish a reliable DNA barcoding library for Chinese snakes, unveiling hidden diversity with implications for taxonomy, and provide a standardized tool for conservation management. Our comprehensive study includes 1638 cytochrome c oxidase subunit I (COI) sequences from Chinese snakes that correspond to 17 families, 65 genera, 228 named species (80.6% of named species) and 36 candidate species. A barcode gap analysis reveals gaps, where all nearest neighbour distances exceed maximum intraspecific distances, in 217 named species and all candidate species. Three species-delimitation methods (ABGD, sGMYC, and sPTP) recover 320 operational taxonomic units (OTUs), of which 192 OTUs correspond to named and candidate species. Twenty-eight other named species share OTUs, such as Azemiops feae and A. kharini, Gloydius halys, G. shedaoensis, and G. intermedius, and Bungarus multicinctus and B. candidus, representing inconsistencies most probably caused by imperfect taxonomy, recent and rapid speciation, weak taxonomic signal, introgressive hybridization, and/or inadequate phylogenetic signal. In contrast, 43 species and candidate species assign to two or more OTUs due to having large intraspecific distances. If most OTUs detected in this study reflect valid species, including the 36 candidate species, then 30% more species would exist than are currently recognized. Several OTU divergences associate with known biogeographic barriers, such as the Taiwan Strait. In addition to facilitating future studies, this reliable and relatively comprehensive reference database will play an important role in the future monitoring, conservation, and management of Chinese snakes.     

DNA barcoding of Canada's skates

DNA-based identifications have been employed across broad taxonomic ranges and provide an especially useful tool in cases where external identification may be problematic. This study explored the utility of DNA barcoding in resolving skate species found in Atlantic Canadian waters. Most species were clearly resolved, expanding the utility for such identification on a taxonomically problematic group. Notably, one genus (Amblyraja) contained three of four species whose distributions do not overlap that could not be readily identified with this method. On the other hand, two common and partially sympatric species (Little and Winter skates) were readily identifiable. There were several instances of inconsistency between the voucher identification and the DNA sequence data. In some cases, these were at the intrageneric level among species acknowledged to be prone to misidentification. However, several instances of intergeneric discrepancies were also identified, suggesting either evidence of past introgressive hybridization or misidentification of vouchered specimens across broader taxonomic ranges. Such occurrences highlight the importance of retaining vouchered specimens for subsequent re-examination in the light of conflicting DNA evidence.     

DNA barcoding of invasive plants in China: A resource for identifying invasive plants

Invasive plants have aroused attention globally for causing ecological damage and having a negative impact on the economy and human health. However, it can be extremely challenging to rapidly and accurately identify invasive plants based on morphology because they are an assemblage of many different families and many plant materials lack sufficient diagnostic characteristics during border inspections. It is therefore urgent to evaluate candidate loci and build a reliable genetic library to prevent invasive plants from entering China. In this study, five common single markers (ITS, ITS2, matK, rbcL and trnH‐psbA) were evaluated using 634 species (including 469 invasive plant species in China, 10 new records to China, 16 potentially invasive plant species around the world but not introduced into China yet and 139 plant species native to China) based on three different methods. Our results indicated that ITS2 displayed largest intra‐ and interspecific divergence (1.72% and 91.46%). Based on NJ tree method, ITS2, ITS, matK, rbcL and trnH‐psbA provided 76.84%, 76.5%, 63.21%, 52.86% and 50.68% discrimination rates, respectively. The combination of ITS + matK performed best and provided 91.03% discriminatory power, followed by ITS2 + matK (85.78%). For identifying unknown individuals, ITS + matK had 100% correct identification rate based on our database, followed by ITS/ITS2 (both 93.33%) and ITS2 + matK (91.67%). Thus, we propose ITS/ITS2 + matK as the most suitable barcode for invasive plants in China. This study also demonstrated that DNA barcoding is an efficient tool for identifying invasive species.     

DNA条形码技术在毒品原植物大麻鉴定中的应用

准确鉴定毒品原植物大麻的种属及品种具有重要的理论和实践意义。为了探讨DNA条形码技术用于毒品原植物大麻种属鉴定及品种鉴定的可行性,该研究以60份大麻原植物(分别采自内蒙、黑龙江、陕西延安、陕西榆林4个地区的栽培大麻雌雄各6株及新疆玛纳斯地区的野生大麻雌雄各6株)为材料,通过从其叶片中提取的DNA为模版,利用核糖体DNA基因间隔区的通用引物ITS2和叶绿体DNA的通用引物psbAtrnH进行PCR扩增,对扩增片段进行双向测序,将测序结果进行人工矫正和比对。结果显示:所有大麻样本的ITS2扩增片段序列没有变异完全一致,但psbA-trnH扩增片段变异较大共检测出8种cpDNA单倍型,用MEGE5.1软件计算种间遗传距离,并构建NJ系统聚类树可以有效把这五个地区的大麻样本区别开来,因此证明DNA条形码技术在毒品原植物大麻的种属鉴定方面具有可行性,但其用于大麻的种属鉴定的准确性、可靠性及在其来源地鉴定及品种鉴定中的可能性还有待进一步深入地研究。     

Chemical investigation of an antimalarial Chinese medicinal herb Picrorhiza scrophulariiflora

An antimalarial medicinal plant Picrorhiza scrophulariiflora was chemically investigated as part of our ongoing research in traditional chinese medicines (TCM). Mass directed fractionation of the active part of the crude extract led to the isolation of ten main components, three new compounds (1–3) and seven known compounds (4–10). Compound 10 inhibited the growth of the Plasmodium falciparum 3D7 malarial parasite line, with an IC₅₀ value of 8.3 μM. This compound accounted for ～95% of P. falciparum growth inhibitory activity in the crude extract confirming, for this TCM, that a single compound was responsible for the antimalarial activity.     

DNA barcoding of the recently evolved genus Holcoglossum (Orchidaceae: Aeridinae): a test of DNA barcode candidates

Orchidaceae is one of the largest families of flowering plants. Many species of orchid are endangered, and all species are included in Conventions on International Trade of Endangered Species of Fauna and Flora (CITES) I and II, but it is very difficult to identify orchid species, even those with fertile parts. The genus Holcoglossum (Orchidaceae: Aeridinae) has long been problematic in taxonomy. It consists of both long-evolved and radiated species and is an excellent case to use for testing DNA barcodes for Orchidaceae. We investigated the power of a subset of proposed plant barcoding loci [rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA and internal transcribed spacer (ITS)] to discriminate between species in this genus. Our results showed that all these DNA regions, except psbK-psbI and atpF-atpH, can be amplified easily from Holcoglossum and sequenced with established primers. The DNA regions matK and ITS had the highest variability. Among the six loci, matK resolved eight of the 12 Holcoglossum species and had the highest discriminatory ability. However, the combination of matK and ITS showed a greater ability to identify species than matK alone. Single or combined DNA markers discriminated between Holcoglossum species distributed in tropical areas effectively, but had less ability to identify radiated species from the temperate Hengduan Mountains of China. In the study, matK proved to be a useful DNA barcode for the genus Holcoglossum; however, complementary DNA regions are still required to accelerate the investigation and preservation of radiated species of orchid.     

DNA条形码在鳞翅目昆虫中的应用

2003年,Hebert等提出DNA条形码后,快速而精确的特点使它在物种鉴定中得到了广泛的应用。鳞翅目是昆虫纲中第二大目,其物种鉴定任务复杂而艰巨,因此DNA条形码具有广阔的应用前景。该文主要针对DNA条形码概况以及近年来它在鳞翅目昆虫中的研究情况予以综述。     

Assessment of candidate plant DNA barcodes using the Rutaceae family

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention.Here,seven regions (psbA-trnH,matK,ycf5,rpoC1,rbcL,ITS2,and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae.To evaluate each barcode’s utility for species authentication,PCR amplification efficiency,genetic divergence,and barcoding gaps were assessed.We found that the ITS2 region exhibited the highest inter-specific divergence,and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests.The ITS2 locus had the highest identification efficiency among all tested regions.In a previous study,we found that ITS2 was able to discriminate a wide range of plant taxa,and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore,we propose that ITS2 is a promising candidate barcode for plant species identification.     

Phylogenetic relationships and DNA barcoding of nine endangered medicinal plant species endemic to Saint Katherine protectorate

A high degree of endemism has been recorded for several plant groups collectively in Saint Katherine Protectorate (SKP) in the Sinai Peninsula. Nine endangered endemic plant species in SKP were selected to test the variable abilities of three different DNA barcodes; Riboluse-1,5- Biphosphate Carboxylase/Oxygenase Large subunit (rbcL), Internal Transcribed Spacer (ITS), and the two regions of the plastid gene (ycf1) as well as Start Codon Targeted (SCoT) Polymorphism to find the phylogenetic relationships among them. The three barcodes were generally more capable of finding the genetic relationships among the plant species under study, new barcodes were introduced to the National Centre for Biotechnology Information (NCBI) for the first time through our work. The barcode sequences were efficient in finding the genetic relationships between the nine species. However, SCoT polymorphism could only cluster plant species belonging to the same genus together in one group, but it could not cluster plant species belonging to the same families except for some primers solely. RbcL was the most easily amplified and identified barcode in eight out of the nine species at the species level and the ninth barcode to the genus level. ITS identified all the species to the genus level. Finally, ycf1 identified six out of the eight species, but it could not identify two of the eight species to the genus level.     

检疫性疫霉DNA条形码标准分子构建

质粒标准分子是指含有外源基因和内源标准基因特异性片段的重组质粒分子.DNA条形码技术是通过对标准目的基因的DNA序列进行分析从而进行物种鉴定的技术.构建基于DNA条形码的质粒标准分子是DNA条形码技术应用于检测实践的要求.本研究将这两种检测鉴定技术相结合应用于检疫性疫霉的检测,构建了11种检疫性疫霉的DNA条形码标准分子,进行了测序验证,均匀性,稳定性和特异性验证.结果表明,构建的质粒标准分子准确度,均匀性,稳定性和特异性均良好,对实际口岸检验检疫工作具有实践应用价值.     

New universal matK primers for DNA barcoding angiosperms

The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and has been suggested to be a "barcode" for land plants. However, matK exhibits low amplification and sequencing rates due to low universality of currently available primers and mononucleotide repeats. To resolve these technical problems, we evaluated the entire matK region to find a region of 600-800 bp that is highly variable, represents the best of all matK regions with priming sites conservative enough to design universal primers, and avoids the mononucleotide repeats. After careful evaluation, a region in the middle was chosen and a pair of primers named natK472F and matK1248R was designed to amplify and sequence the matK fragment of approximately 776 bp. This region encompasses the most variable sites, represents the entire matK region best, and also exhibits high amplification rates and quality of sequences. The universality of this primer pair was tested using 58 species from 47 families of angiosperm plants. The primers showed a strong amplification (93.1%) and sequencing (92.6%)successes in the species tested. We propose that the new primers will solve, in part, the problems encountered when using matK and promote the adoption of matK as a DNA barcode for angiosperms.