锦葵科植物DNA条形码通用序列的筛选

对锦葵科植物样品的ITS、ITS2、rbcL、matK和psbA-trnH序列进行PCR扩增和测序,比较各序列的扩增效率、测序成功率、种内和种间变异的差异以及barcoding gap图,使用BLAST1和Nearest Distance方法评价不同序列的鉴定能力,进而从这些候选序列中筛选出较适合锦葵科植物鉴别的DNA条形码序列。结果表明,ITS序列在采集的锦葵科植物11个种26个样品中的扩增成功率较高,其种内、种间变异差异和barcoding gap较ITS2、psbA-trnH及rbcL序列具有更明显的优势,且纳入60个属316个种共1228个样品的网上数据后,其鉴定成功率可达89.9%。psbA-trnH序列的扩增和测序成功率最高,其鉴定成功率为63.2%,并能鉴别一些ITS序列无法鉴别的种。实验结果表明,ITS和psbA-trnH是较适合鉴别锦葵科植物的DNA条形码序列组合。     

锦葵科植物DNA条形码通用序列的筛选

对锦葵科植物样品的ITS、ITS2、rbcL、matK和psbA-trnH序列进行PCR扩增和测序, 比较各序列的扩增效率、测序成功率、种内和种间变异的差异以及barcoding gap图, 使用BLAST1和Nearest Distance方法评价不同序列的鉴定能力, 进而从这些候选序列中筛选出较适合锦葵科植物鉴别的DNA条形码序列。结果表明, ITS序列在采集的锦葵科植物11个种26个样品中的扩增成功率较高, 其种内、种间变异差异和barcoding gap较ITS2、psbA-trnH及rbcL序列具有更明显的优势, 且纳入60个属316个种共1 228个样品的网上数据后, 其鉴定成功率可达89.9%。psbA-trnH序列的扩增和测序成功率最高, 其鉴定成功率为63.2%, 并能鉴别一些ITS序列无法鉴别的种。实验结果表明, ITS和psbA-trnH是较适合鉴别锦葵科植物的DNA条形码序列组合。     

云南常见药用石斛DNA条形码筛选

选择云南省内常见的12种药用石斛，采用分子生物学鉴定技术筛选其适用的DNA条形码序列。以12种药用石斛共36个样品为材料，提取样品总DNA，对核基因片段ITS和ITS2、叶绿体基因片段psbA-trnH和matK序列进行扩增、测序，结合GenBank下载部分石斛序列；利用生物信息学软件进行序列分析和系统发育分析。结果表明，4条序列扩增和测序成功率均为100%；4条序列没有明显的Barcoding Gap，但ITS序列与ITS2序列的种内和种间重叠部分较少，有偏向两端的趋势；系统发育树显示，ITS和psbA-trnH序列能成功区分12种云南常见的药用石斛，ITS2序列未能区分长距石斛(Dendrobium longicornu)和矮石斛(D. bellatulum)，matK序列仅区分6种石斛。建议以ITS和psbA-trnH序列作为云南药用石斛鉴定序列，为药用石斛的种源鉴定提供理论依据。     

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。     

基于芸香科的植物通用DNA条形码研究

植物DNA条形码研究是近10年来进展最迅速的学科之一,其通用序列的筛选一直是该领域研究的热点问题.2009年,生命条形码联盟植物工作组推荐rbcL+matK组成复合序列作为植物通用条形码,但其研究对象中近缘属、种较少,且物种水平鉴定成功率仅为72%,所以仍在进行验证和新序列的研究工作.本研究选取nrDNAITS2序列,利用其具有Ⅱ级结构的特性、通过不同物种类型模型判定全长,将其和目前热点候选序列(matK,rbcL,psbA-trnH,rpoC1,ycf5)及nrDNAITS序列针对芸香科72属192种300个样本进行比较,试图在同一科属下更多近缘种存在时,真实判定候选序列的鉴定能力.结果表明,自行设计引物的ITS2序列具有较好的PCR扩增和测序成功率,在所考察的候选序列中具有最大的种间变异和较小的种内变异,且两者存在极显著差异,同时物种鉴定成功率最高,各评价指标均优于其他候选序列.证实ITS2序列在单一科属内的"高效性",故推荐其作为植物DNA条形码通用序列之一.     

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。     

秦巴山区漆树nrDNAITS和cpDNA序列分子进化特点的分析

应用PCR产物直接测序法分析漆树种群nrDNA （核糖体DNA） ITS序列和cpDNA序列（matK、rbcL、psbA-trnH）的碱基差异,初步研究两套植物基因组的变异速率。结果表明：nrDNA ITS序列共有518 bp,有变异位点15处,变异位点百分率为2.9%,（G+C）含量为61.8%。cpDNA序列合并后长度1 907 bp,有变异位点20处,变异位点百分率为1.05%,（G+C）含量为36.1%。通过对ITS序列核糖型（Ribotype）和叶绿体序列单倍型（Haploype）进行分析发现,秦巴山区漆树区域性分布明显,不同区域拥有自己独特的单倍型,漆树居群历史近期没有扩张。nrDNA ITS序列较叶绿体序列进化较快,变异速率较快。nrDNA ITS序列及叶绿体matK、psbA-trnH适合漆树的亲缘地理学研究。     

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。     

含有小檗碱的药用植物DNA条形码鉴定研究

[目的]评价ITS2与psbA-trnH条形码候选序列对含有小檗碱的药用植物的鉴定作用。[方法]对含有小檗碱与不含有小檗碱的药用植物的ITS2与psbA-trnH序列进行PCR扩增和测序,进行种内和种间变异分析,构建NJ进化树评价不同序列的鉴定能力。[结果]ITS2与psbA-trnH序列均能够成功地区分含有小檗碱与不含有小檗碱的药用植物。然而,ITS2在区分含有小檗碱的不同科药用植物的效率优于psbA-trnH序列。[结论]建立了以ITS2序列为主,psbA-trnH序列为辅的鉴定含有小檗碱的药用植物的方法,这为含有小檗碱的药用植物种质资源的整理与标准化,甚至育种都提供了有效的基础数据。     

利用DNA条形码技术识别植物物种

DNA条形码技术能够快速、准确地识别物种,对于开展基础性的分类学研究和应用性的生物多样性研究极为重要.本文对鼎湖山20 hm²大样地183个植物物种进行DNA条形码测序.结果表明： 单个条形码片段时, psbA-trnH的综合成功率最高(75%),其次是matK(70%)和rbcL(56%);片段组合时,matK+rbcL+psbA-trnH三片段组合的物种水平识别率在87%以上,随后是matK+psbA-trnH(85%)、rbcL+psbA-trnH(83%)和matK+rbcL(81%).综合了亚热带波多黎各的LFDP样地(143个种)和热带巴拿马的BCI样地(296个种)以及圭亚那的Nouragues样地(254个种)3个森林类型的研究结果,评价DNA条形码各片段在4个森林样地的通用性.在热带和亚热带地区的森林样地中,各片段测序成功率分别为rbcL(93%,95.1%)、psbA-trnH(91.5%,94.6%)和matK(68.5%,79.7%).在植物类群水平上,核心条形码片段matK+rbcL组合的物种准确识别率不高,只在局部群落中表现较为理想;而三位点DNA条形码片段组合在热带和亚热带森林样地中综合成功率可达84%和90%.     

Applying DNA barcodes for identification of plant species in the family Araliaceae

An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Molecular phylogeny of genus Vigna subgenus Ceratotropis based on rDNA ITS and atpB-rbcL intergenic spacer of cpDNA sequences

The genetic diversity and phylogenetic relationships among species in the genus Vigna subgenus Ceratotropis were investigated using sequence data from the ribosomal DNA ITS and atpB-rbcL intergenic spacer of chloroplast DNA regions. While both sets of sequences were of similar lengths about 700bp the rDNA-ITS was more informative than atpB-rbcL having 170% more polymorphic sites and five times as many parsimony-informative sites. The atpB-rbcL spacer may be appropriate for analysis of taxa above the species level in the genus Vigna. Results of analyzing rDNA-ITS revealed, with low level of statistical bias, separation of the subgenus into three groups that correspond to the three sections Aconitifoliae, Angulares, and Ceratotropis. The ancestral section is Aconitifoliae based on comparison with the outgroup species cowpea, Vigna unguiculata. The V. minima complex, V. minima, V. riukiuensis, and V. nakashimae, has a distinct evolutionary path within section Angulares. Other species in section Angulares are very closely related except V. trinervia. Vigna trinervia has an intermediate position between sections. Sequence data suggests one genome donor to V. reflexo-pilosa came from a lineage within section Angulares close to V. exilis, V. hirtella, and V. umbellata. Data presented supports the view that section Angulares is the most recently diversified section in the subgenus, as inferred by short terminal branch lengths among the species of this section.     

DNA barcoding of Pedicularis L. (Orobanchaceae): Evaluating four universal barcode loci in a large and hemiparasitic genus

Abstract One application of DNA barcoding is species identification based on sequences of a short and standardized DNA region. In plants, various DNA regions, alone or in combination, have been proposed and investigated, but consensus on a universal plant barcode remains elusive. In this study, we tested the utility of four candidate barcoding regions (rbcL, matK, trnH‐psbA, and internal transcribed spacer (ITS)) as DNA barcodes for discriminating species in a large and hemiparasitic genus Pedicularis (Orobanchaceae). Amplification and sequencing was successful using single primer pairs for rbcL, trnH‐psbA, and ITS, whereas two primer pairs were required for matK. Patterns of sequence divergence commonly showed a “barcoding gap”, that is, a bimodal frequency distribution of pairwise distances representing genetic diversity within and between species, respectively. Considering primer universality, ease of amplification and sequencing, and performance in discriminating species, we found the most effective single‐region barcode for Pedicularis to be ITS, and the most effective two‐region barcode to be rbcL + ITS. Both discriminated at least 78% of the 88 species and correctly identified at least 89% of the sequences in our sample, and were effective in placing unidentified samples in known species groups. Our results suggest that DNA barcoding has the potential to aid taxonomic research in Pedicularis, a species‐rich cosmopolitan clade much in need of revision, as well as ecological studies in its center of diversity, the Hengduan Mountains region of China.     

Phylogenetic relationship of China Dendrobium species based on the sequence of the internal transcribed spacer of ribosomal DNA

The genetic relationship of 36 Dendrobium species in China was determined based on sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA. Aligned sequences of the complete ITS region obtained from the 36 Dendrobium species and 2 outgroup species (Epigeneium amplum and Epigeneium nakaharaei) by using PCR amplification and direct DNA sequencing. The nrDNA ITS1 of Dendrobium was 225–234 bp and ITS2 was 239–248 bp. Phylogenetic tree was constructed, and seven main clusters were generated among the 36 Dendrobium species. From the results, D. moulmeinense was not grouped in the classification of Dendrobium and E. amplum and E. nakaharaei were shown to be divergent from Dendrobium species. The phylogenetic relationships revealed by ITS DNA analysis partially supported previously published morphological data.     

基于rbcL和matK序列探讨马鞭草科部分植物的系统学位置

为探究适用于马鞭草科植物的DNA条形码及该类群的系统分类关系,对豆腐柴(Premna microphylla)的叶绿体基因ycf6-psbM、trnV-atpE、rbcL、trnL-F、psbM-trnD、atpB-rbcL、trnC-ycf6、trnH-psbA、rpl36-infA-rps8和核基因ITS序列进行了PCR扩增和测序,结果表明仅rbcL、trnl-F、trnH-psbA序列的PCR扩增以及测序效果较好,而ITS不能得到明显的扩增条带,ycf6-psbM不能成功测序,其它序列存在有部分双峰或噪值高等问题。根据DNA条形码标准,rbcL序列是所有测试条码中相对最适合的。应用rbcL和matK序列对马鞭草科(Verbenaceae)豆腐柴属、牡荆属(Vitex L.)、马鞭草属(Verbena L.)和大青属(Clerodendrum L.)等4属与唇形科宝盖草属(Lamium L.)、水苏属(Stachys L.)、鼠尾草属(Salvia L.)和香科科属(Teucrium L.)等4属的分类和系统发育关系进行分析,以紫草科Lithospermum multiflorum L.为外群,最大简约法对2个片段的单独和联合矩阵分别构建系统发育树。豆腐柴属和大青属应从马鞭草科划入唇形科,马鞭草属仍归于马鞭草科,而牡荆属的系统学位置还需更多的证据。     

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

Background

The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over.

Methodology/Principal Findings

Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level.

Conclusions

The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.     

ITS1: a DNA barcode better than ITS2 in eukaryotes?

A DNA barcode is a short piece of DNA sequence used for species determination and discovery. The internal transcribed spacer (ITS/ITS2) region has been proposed as the standard DNA barcode for fungi and seed plants and has been widely used in DNA barcoding analyses for other biological groups, for example algae, protists and animals. The ITS region consists of both ITS1 and ITS2 regions. Here, a large‐scale meta‐analysis was carried out to compare ITS1 and ITS2 from three aspects: PCR amplification, DNA sequencing and species discrimination, in terms of the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality. In total, 85 345 sequence pairs in 10 major groups of eukaryotes, including ascomycetes, basidiomycetes, liverworts, mosses, ferns, gymnosperms, monocotyledons, eudicotyledons, insects and fishes, covering 611 families, 3694 genera, and 19 060 species, were analysed. Using similarity‐based methods, we calculated species discrimination efficiencies for ITS1 and ITS2 in all major groups, families and genera. Using Fisher's exact test, we found that ITS1 has significantly higher efficiencies than ITS2 in 17 of the 47 families and 20 of the 49 genera, which are sample‐rich. By in silico PCR amplification evaluation, primer universality of the extensively applied ITS1 primers was found superior to that of ITS2 primers. Additionally, shorter length of amplification product and lower GC content was discovered to be two other advantages of ITS1 for sequencing. In summary, ITS1 represents a better DNA barcode than ITS2 for eukaryotic species.     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.