基于条形码ITS2序列的青天葵及其混伪品分子鉴别

通过分析青天葵及其常见混伪品的ITS2序列,建立青天葵新型真伪鉴别方法.采用植物基因组DNA提取试剂盒提取青天葵及其混伪品的叶片DNA,一对通用引物PCR扩增ITS2基因片段并直接双向测序.采用DNAMAN、ClustalX软件拼接比对序列,MEGA4.0软件构建NJ树,Schultz等建立的数据库和网站预测ITS2序列的二级结构.结果显示,获得的12条ITS2序列的长度范围为203-242 bp,GC含量范围为53.1％-71.8％.所有样品ITS2序列比对后的长度为249 bp,其中存在226个变异位点.青天葵种间K2P遗传距离(1.125)远大于种内K2P遗传距离(0.004).基于ITS2的序列的NJ树和二级结构均能直观地区分青天葵及其混伪品.     

丹皮药材及其伪品、混淆品核糖体rDNA ITS2的序列分析

分析安徽道地药材丹皮（Paeonia suffruticosa Andr）及其伪品、混淆品核糖体DNA第二内转录间隔区（ITS2）的序列。分析丹皮的分子遗传多样性,为丹皮的真品药材基原鉴定提供分子依据。采用DNA克隆测序技术对从不同市场购得的丹皮样品及其伪品、混淆品的ITS2基因进行测序分析研究。丹皮与其伪品川赤芍、亳州芍药的rDNA-ITs2序列长度均为412bp。排序比较发现不同市场上的丹皮样品rDNA-ITs2序列变异位点丰富。鉴定出与丹皮真品（黄沙与白沙）及伪品的rDNA-ITS2序列完全相同的样品。其他样品的rDNA-ITs2序列与丹皮真品的差异明显。通过丹皮ITS2序列差异比较分析。ITS2序列可以作为鉴别丹皮药材真伪的一种手段。     

基于DNA metabarcoding技术的如意金黄散处方成分鉴定研究

中药的有效性和安全性是国内外关注的热点,本研究以如意金黄散为例,基于DNA metabarcoding技术,尝试从投料药材真伪角度评价中药的有效性和安全性.首先,收集如意金黄散处方成分药材及其混伪品样品161份,从已发表文献中下载密切相关物种序列154条,构建"如意金黄散DNA条形码鉴定数据库",该数据库包含10属119个物种.收集不同批次的如意金黄散样品3份,以ITS2序列为条形码,使用Illumina HiSeq平台进行PE250深度测序,利用本研究构建的"如意金黄散DNA条形码鉴定数据库"进行投料药材基原物种识别,结果表明,基于DNA metabarcoding技术可检测到除厚朴外的全部处方成分,不同药材的测序量存在显著差异;检测到苍术药材的混伪品朝鲜苍术和天南星药材的混伪品虎掌南星,提示如意金黄散临床使用的有效性和安全性存在潜在风险;不同批次鉴定结果具有稳定性和可重复性.本研究表明,基于DNA metabarcoding技术的如意金黄散处方成分鉴定方法符合中药复杂体系特点,有望成为中成药鉴定的新方法,并为中成药的有效性和安全性评价提供参考.     

基于DNA条形码的半夏属及其伪品分子鉴别

利用DNA条形码技术对半夏属及其伪品进行分子鉴定, 研究半夏属药用植物鉴定的新方法。该实验使用matK序列对半夏(Pinellia ternata)及其伪品进行扩增测序, 结合GenBank数据库数据, 分析ITS、ITS2、psbA-trnH、rbcL和matK各序列的种内与种间变异及barcoding gap, 并采用最近距离法(nearest distance)和相似性搜索算法(BLAST1)评价不同序列的鉴定能力。结果显示, matK序列的种间变异最大, rbcL序列的种内变异最小; rbcL序列的种内和种间遗传变异重叠比例最小, 其次为matK序列; 各序列的Neighbor Joining树均可明显地将不同种分开。实验结果表明, 利用DNA条形码能够准确地鉴别半夏属药用植物及其伪品, matK和rbcL序列为鉴别半夏属及其伪品的较理想条形码组合。该研究为半夏属植物的分子鉴别提供了科学依据与新的思路。     

山葡萄种质资源DNA条形码通用序列的筛选

对山葡萄(Vitis amurensis)种质资源样品的ITS、ITS2、psb A-trn H、rbc L和mat K序列进行PCR扩增及测序,优化PCR反应的退火温度,比较各序列的扩增效率、测序成功率、品种间和品种内的差异及barcoding gap图,使用BLAST和NJ树法比较不同序列的鉴定能力,最终从5条DNA片段中筛选出可用于山葡萄种质资源鉴定的DNA条形码通用序列。结果表明,在采集的11份33个山葡萄样品中,psb A-trn H和ITS2序列的扩增与测序成功率较高,其品种间、品种内差异及barcoding gap较ITS、rbc L和mat K序列具有明显的优势,且ITS2序列能够鉴别psb A-trn H序列无法鉴别的品种。实验证明,ITS2和psb A-trn H序列是较适合鉴别山葡萄资源的DNA条形码序列组合。DNA条形码弥补了形态学鉴定的不足,可为山葡萄种质资源的准确鉴定提供科学依据。     

应用matK基因鉴别青天葵及其常见混伪品

分析了青天葵及其混伪品matK序列,以期在分子水平建立青天葵及其常见混伪品的鉴别方法。采用一对通用引物对matK基因进行PCR扩增并测序,所得序列用DNAMAN、MEGA等软件进行分析。获得青天葵及其混伪品的matK基因序列长度为587 bp,平均GC含量为32.8%。青天葵的种内遗传距离为0,与混伪品种间遗传距离范围为0.016～0.375。基于matK基因序列构建的NJ聚类树能明显地区别青天葵及其混伪品。因此,应用matK基因序列可以有效鉴别青天葵及其混伪品。     

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。     

莪术基原植物DNA条形码序列的筛选与鉴定

为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件对获得的高质量的序列通过变异位点分析、遗传距离计算和系统树分析等进一步进行评估,最后将筛选到的DNA条形码序列对未知基原的待测样品进行基原鉴定。结果表明:(1) ITS、ITS2和matK等条形码序列在莪术基原植物中的扩增或测序成功率较低,难以应用于实际鉴定;而psbA-trnH、trnL-trnF和rpoB条形码序列变异位点信息过少,不足于区分莪术的三种不同基原植物;只有atpB-rbcL条形码序列的扩增和测序成功率较高,容易获得高质量的序列,同时序列长度(642～645 bp)理想,变异位点多(11个),可实现莪术的三种不同基原的区分鉴别。(2)待测样品经基于atpB-rbcL序列构建的系统发育树鉴别为温郁金。综上所述,叶绿体atpB-rbcL序列能够准确鉴定莪术不同基原植物,可以作为中药材莪术基原植物鉴定的条形码序列。     

滇重楼的psbA-trnH条形码分子鉴定研究北大核心CSCD

为探讨滇重楼药材鉴定新方法,本研究通过对重楼样品提取基因组DNA,PCR扩增叶绿体psb A-trn H序列并进行双向测序,所得序列经Codon Code Aligner拼接后,采用MEGA5.0软件进行序列比对,计算种内和种间遗传距离(K2P),构建邻接数(neighbor-joining tree)进行结果分析。研究表明,叶绿体psb A-trn H片段在滇重楼种内变异较小,平均K2P遗传距离为0.007,而滇重楼与其它重楼种间平均K2P遗传距离为0.025,滇重楼种间最大K2P遗传距离明显小于滇重楼与重楼属其它种内的最小K2P遗传距离。中药材DNA条形码鉴定系统比对和NJ树鉴定结果均表明psb A-trn H序列可区分滇重楼及其同属物种,且具有较好的稳定性和准确性,为保障临床安全用药提供了新的技术手段。     

滇重楼的psbA-trnH条形码分子鉴定研究

为探讨滇重楼药材鉴定新方法,本研究通过对重楼样品提取基因组DNA,PCR扩增叶绿体psb A-trn H序列并进行双向测序,所得序列经Codon Code Aligner拼接后,采用MEGA5.0软件进行序列比对,计算种内和种间遗传距离(K2P),构建邻接数(neighbor-joining tree)进行结果分析。研究表明,叶绿体psb A-trn H片段在滇重楼种内变异较小,平均K2P遗传距离为0.007,而滇重楼与其它重楼种间平均K2P遗传距离为0.025,滇重楼种间最大K2P遗传距离明显小于滇重楼与重楼属其它种内的最小K2P遗传距离。中药材DNA条形码鉴定系统比对和NJ树鉴定结果均表明psb A-trn H序列可区分滇重楼及其同属物种,且具有较好的稳定性和准确性,为保障临床安全用药提供了新的技术手段。     

ITS2 barcode for identifying the officinal rhubarb source plants from its adulterants

Officinal rhubarb, the dried root and rhizome of Rheum officinale Baill., Rh. palmatum L., and Rh. tanguticum (Maxim. ex Regel) Maxim. ex Balf. of Polygonaceae family, is one of the most well-known and important traditional Chinese medicines. The growing herbal market has led to the introduction of adulterants, and difficulties in morphological based discrimination has also resulted in concerns over consumer safety. ITS2 as a potential barcode was employed to discriminate officinal rhubarb sampled from the entire distribution range and its adulterants. The minimum K2P interspecific distance between officinal rhubarb source plants and the adulterants were higher than the maximum K2P intraspecific distance within the officinal rhubarb source plants. When the phylogenetic trees were constructed by neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian methods, all Rheum species were grouped into one clade, while Polygonum and Rumex species were clustered in another clade. In the Rheum clade, all the officinal rhubarb source plants constituted a distinct subclade, while other Rheum species formed other subclades. Our results suggested that the ITS2 region is a more suitable and accurate DNA barcode for distinguishing officinal rhubarb source plants from their adulterants.     

药用和观赏植物常山产地适宜性分析和定量评价

为了解常山(Dichroa febrifuga Lour.)现有产区分布并预测新的产区,本文利用中药材产地适宜性分析地理信息系统TCMGIS(Geographic Information System for Traditional Chinese Medicine)对426个常山样品信息进行了适宜生态因子生长范围确认,分析了常山适宜产地空间分布状况。结果显示,常山主要分布于我国温暖湿润的东部季风区,生态相似系数100%区域分布较为集中,总面积达760 640.1 km2,共829个县区,其中广西、云南、广东、湖南分布较多。本研究结果对常山的野生资源保护、人工规范化种植和资源可持续利用具有重要意义。     

Identification of species within Tetrastigma (Miq.) Planch. (Vitaceae) based on DNA Barcoding Techniques

Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA, and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. hemsleyanum, could be identified precisely using DNA barcoding.     

Identification of species within Tetrastigma (Miq.) Planch.(Vitaceae) based on DNA barcoding techniques

Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. Hemsleyanum, could be identified precisely using DNA barcoding.     

A DNA mini‐barcode for land plants

Small portions of the barcode region – mini‐barcodes – may be used in place of full‐length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini‐barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini‐barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30 472)]. PCR amplification for all mini‐barcodes, as estimated by validated electronic simulation, was successful for 90.2–99.8% of species. Overall Sanger sequence quality for mini‐barcodes was very low – the best mini‐barcode tested produced sequences of adequate quality (B₂₀ ≥ 0.5) for 74.5% of samples. The majority of mini‐barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini‐barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini‐barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini‐barcode D (F52/R193).     

New DNA barcodes for identification of Korean birds

DNA barcode is a short sequence of standardized genomic region that is specific to a species and therefore, helps in species identification. According to studies of animal species, the 648-bp sequence of the mitochondrial gene encoding cytochrome c oxidase 1 (CO1) is extremely useful for species identification. Several studies of birds have already ascertained the reliability of CO1 barcodes. In this study, we investigated the validity of DNA barcoding in Korean bird species by using additional barcode records. We analyzed the CO1 barcodes of 154 species of Korean birds, and discovered that the average genetic distance between congeneric species was 25 times higher than the average genetic distance within species. Most (98.7 %) bird species possessed a barcode distinct from that of other bird species. However, among the remaining 1.3 %, species had overlapping barcode clusters. Thus, we reemphasize that CO1 barcodes are an effective identification tool for Korean bird species.     

DNA barcodes for discriminating the medicinal plant Isatis indigotica Fort. (Cruciferae) and its adulterants

Isatis indigotica Fort. (Cruciferae) is a biennial medicinal plant. In order to protect the decreasing natural genetic resources of I. indigotica, three candidate DNA barcodes (ITS2, trnL-F and rbcL) were employed to establish an accurate and effective identification system for I. indigotica. The results demonstrated that all three candidate DNA barcodes have performed very well in I. indigotica. The interspecific genetic distances were obviously greater than the intraspecific distance among I. indigotica as indicated by ITS2, trnL-F and rbcL. Sequence alignment analysis of I. indigotica genotypes revealed that four SNPs (54, 108, 146 and 181 bp) located in ITS2, three (2, 30, 709 bp) in trnL-F and one (531 bp) in rbcL, respectively. UPGMA phylogenetic tree constructed from trnL-F and rbcL could allote I. indigotica to the correct corresponding genus, whereas rbcL could not distinguish I. indigotica from its adulterants. Meanwhile, UPGMA tree of ITS2 could accurately identify I. indigotica from its adulterants according to the corresponding species. Consequently, it can be concluded that ITS2 is a more suitable and accurate DNA barcode for identifying I. Indigotica and its adulterants than trnL-F and rbcL.     

Species identification of the medicinal plant Tulipa edulis (Liliaceae) by DNA barcode marker

Tulipa edulis (Liliaceae) is the botanical origin of the traditional Chinese medicine (TCM) “Guangcigu”. Due to overexploitation that induced a decline in natural sources, many dried bulbs from other species of Tulipa have been used, adulterating the medicine in recent years. This practice may cause a series of inconsistent therapeutic effects and quality control problems in the herbal medicine industry. Hence, three DNA regions (matK, psbA-trnH and rbcL) were evaluated as barcodes for identifying T. edulis and its adulterants. All candidate DNA barcodes were successfully amplified from leaf samples. Based on the sequence divergences, rbcL and psbA-trnH can assign T. edulis and its adulterants to the correct genus, while matK can accurately differentiate T. edulis and its adulterants. Thus, at the DNA level, the matK intergenic region is a more suitable, accurate and applicable identification of T. edulis and its adulterants than rbcL and psbA-trnH.     

青海省6种裂腹鱼COI基因作为DNA条形码的适用性初探

青海省裂腹鱼鱼类至少有20种,占土著鱼类的40%以上,具有重要的生态价值。由于生态环境的恶化和人为因素的干扰,很多物种群已濒临灭绝。快速和准确的物种鉴定对于这些物种的保护至关重要,而基于形态学的传统分类法很难满足这一需求。因此,本研究通过DNA条形码技术初步探讨了在青海省裂腹鱼物种鉴定中的适用性。本研究中,测序获得了青海26尾裂腹鱼(6个物种)的细胞色素c氧化酶亚基I (COI)基因,并经GenBank数据库进行比对;基于K2P模型分析COI序列变异;运用贝叶斯法(BI)和最大似然法(ML)构建系统发育树。结果显示,6种裂腹鱼COI序列检测得到15个单倍型,且各物种间无共享的单倍型。基于K2P模型,最大种内遗传距离和最小种间遗传距离分别为(0.621±0.297)%和(2.792±0.644)%。种间平均遗传距离为(12.205±1.307)%,约为种内平均遗传距离((0.327±0.162)%)的37倍,表明各物种的COI序列间已经形成明显"条形码间隙"。AMOVA分析显示,遗传变异主要来自种间,约占97.05%;FST=0.970 54,p<0.01,说明各物种间分化程度极高。此外,基于BI和ML方法构建的系统树具有一致的拓扑结构,分辨率较高;各物种均单独聚为一个发育枝,拓扑结构合理。以上结果表明,COI基因作为DNA条形码在青海裂腹鱼物种鉴定中具有较高的适用性。