DNA barcoding of the main cultivated yams and selected wild species in the genus Dioscorea

Distinguishing yam species based on morphological traits is extremely difficult and unreliable, posing a challenge to breeders and genebank curators. Development of a molecular assay based on DNA barcoding can facilitate rapid and accurate identification of important Dioscorea species. To develop a DNA barcoding system forDioscorea species identification, the rbcL and matK loci (in unison and in combination), the non-coding intergenic spacer trnH-psbA of the chloroplast genome, and the nuclear ITS regions were investigated using criteria for developing candidate DNA barcodes. All DNA barcoding sequences were assessed for ease of PCR amplification, sequence quality and species discriminatory power. Amongst the markers investigated, the matK locus performed well in terms of species identification (63.2%), in addition to detecting high interspecific variation with mean divergence of 0.0196 (SD=0.0209). The combination of the two coding regions (rbcL + matK) was determined to be the optimal (76.2%) DNA barcoding approach as 16 out of 21 species could be defined. While the rbcL exhibited good PCR amplification efficiency and sequence quality, its species discriminatory power was relatively poor with 47.6% identification. Similarly, the trnH-psbA region had a weak discrimination efficiency of only 36.8%. While the development of more robust DNA barcoding systems is an ongoing challenge, our results indicate that therbcL + matK combination can be utilized as multi-locus DNA barcode regions for Dioscorea species identification.     

Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals

Considerable debate remains as to which DNA region should be used to barcode plants. Several different chloroplast (cp) DNA regions (rbcL, matK, and trnH-psbA) and nuclear ribosomal internal transcribed regions (ITS) have been suggested as suitable barcodes in plants. Recently, low-copy nuclear loci were also suggested to be potentially ideal barcode regions. The aim of the present study was to test the effectiveness of these proposed DNA fragments and five additional low-copy loci (CHS, DETl, COPl, PGICl, and RPS2; comprising both coding and non-coding regions) in barcoding closely related species. We examined the divergences within and between two species of Pugioniun (Brassicaceae). We failed to find any interspecific variation from three cpDNA fragments with which to discriminate the two species. However, a single base mutation in the internal transcribed spacer (ITS) could discriminate between the two species consistently. We found more variations among all individuals of the two species using each of the other five low-copy nuclear loci. However, only alleles from one locus (DET1) of the five low-copy loci related to flowering regulations was able to distinguish the sampled individuals into two species. We failed to amplify the corresponding fragments out of Brassicaceae using the designed DETl primers. We further discussed the discrimination power of different loci due to incomplete lineage sorting, gene flow, and species-specific evolution. Our results highlight the possibility of using the nuclear ITS as a core or complementary fragment to barcode recent diverged species.     

藓类植物DNA条码的初步研究——以蔓藓科部分属为例

选取7个叶绿体基因片段,其中3个编码区基因片段（matK,rps4 and rbcL-a）和4个非编码区基因片段（atpB-rbcL,atpF-H,psbK-Iandtrn H-psbA）,一个线粒体基因片段（nad5）和一个核基因片段（ITS2）,材料包括5个属14个种的74份样品。分析比较了6个基因片段的多样性、种内和种间的遗传距离、鉴别率等。结果显示,在使用单个片段时ITS2的鉴定效果最好,而atpB-rbcL则是叶绿体基因片段中鉴别能力最高的。使用组合片段的结果发现,两个基因片段组合的鉴别效果最好。通过NJ树的方法检验,atpB-rbcL＋trn H-psbA和rbcL-a＋＋trn H-psbA的鉴定成功率是64%,当再增加一个或两个基因片段时其鉴定效果并没有提高。本研究表明在植物中应用DNA条码需要用质体基因和核基因联合。     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Testing candidate plant barcode regions in the Myristicaceae

The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon - UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.     

Plant DNA barcoding: from gene to genome

DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single‐locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole‐chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource‐effective and does not yet offer the speed of analysis provided by single‐locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super‐barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single‐locus barcodes and super‐barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.     

DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH-psbA, trnL-F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear-cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty-seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH-psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH-psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.     

植物物种的快速鉴定与iFlora：DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比,DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（PedicularisL．）植物具对生（轮生）叶的种类70％以上分布在中国．近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH-psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81．40％和89．57％,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。     

Testing four barcoding markers for species identification of Potamogetonaceae

The pondweeds (Potamogetonaceae) are among the most important plant groups in the aquatic environment. Owing to their high morphological and ecological diversity, species identification of this aquatic family remains problematic. DNA barcoding involves sequencing a standard DNA region and has been shown to be a powerful tool for species identification. In the present study, we tested four barcoding markers (rbcL, matK, internal transcribed spacer (ITS), and trnH-psbA) in 15 Potamogeton species and two Stuckenia species, representing most species of the Potamogetonaceae in China. The results show that all four regions can distinguish and support the newly proposed genera of Stuckenia from Potamogeton. Using ITS and trnH-psbA, significant interspecific genetic variability was shown. However, intraspecific genetic variability of trnH-psbA is high and so it is not suitable for barcoding in Potamogetonaceae. The ITS and matK regions showed good discrimination. However, matK was not easy to sequence using universal primers. The best performing single locus was ITS, making it a potentially useful DNA barcode in Potamogetonaceae.     

Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF-atpH, and psbK-psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85-100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK-psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69-71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.     

悬钩子属DNA条形码通用序列的初步筛选

为了建立悬钩子属（Rubus）植物的DNA条形码分子鉴定技术,筛选获得适用于悬钩子属植物的通用条形码序列。该研究基于GenBank数据对ITS、ITS2、matK、rbcL、trnH-psbA、trnL-trnF 6个DNA条形码序列进行了遗传变异、barcoding gap、建树等评估分析。结果显示,trnH-psbA、matK、rbcL、rtnL-trnF的种内变异与种间变异差异较大,变异分辨率分别为97.32%、83.33%、79.07%、64.95%,存在较大的barcoding gap;NJ一致树分析显示,matK的单系性比例最高（67%）,其次为trnH-psbA（64%）,rtnL-trnF（43%）,rbcL（30%）。结果表明,悬钩子属植物的matK与trnH-psbA序列种内变异与种间变异差异较大,能较好地区分不同物种,具有较大的鉴定潜力。建议将matK和trnH-psbA作为悬钩子属植物鉴定的核心条形码序列,rtnL-trnF、rbcL作为辅助条形码序列。     

Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals

Considerable debate remains as to which DNA region should be used to barcode plants. Several different chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA) and nuclear ribosomal internal transcribed regions (ITS) have been suggested as suitable barcodes in plants. Recently, low-copy nuclear loci were also suggested to be potentially ideal barcode regions. The aim of the present study was to test the effectiveness of these proposed DNA fragments and five additional low-copy loci (CHS, DET1, COP1, PGIC1, and RPS2; comprising both coding and non-coding regions) in barcoding closely related species. We examined the divergences within and between two species of Pugionium (Brassicaceae). We failed to find any interspecific variation from three cpDNA fragments with which to discriminate the two species. However, a single base mutation in the internal transcribed spacer (ITS) could discriminate between the two species consistently. We found more variations among all individuals of the two species using each of the other five low-copy nuclear loci. However, only alleles from one locus (DET1) of the five low-copy loci related to flowering regulations was able to distinguish the sampled individuals into two species. We failed to amplify the corresponding fragments out of Brassicaceae using the designed DET1 primers. We further discussed the discrimination power of different loci due to incomplete lineage sorting, gene flow, and species-specific evolution. Our results highlight the possibility of using the nuclear ITS as a core or complementary fragment to barcode recent diverged species.     

中国秋海棠属（秋海棠科）植物的DNA条形码评价

秋海棠属植物种类繁多,形态变异多样,导致种类的系统放置混乱,近缘种类鉴定困难。利用DNA条形码实现物种快速准确的鉴定技术具有不受形态特征约束的优势,为秋海棠属植物的分类鉴定提供了新的方法。本研究选择4个DNA条形码候选片段（rbcL,matK,trnH-psbA,ITS）对中国秋海棠属26种136个个体进行了分析。结果显示：叶绿体基因rbcL,matK和trnH-psbA种内和种间变异小,对秋海棠属植物的鉴别能力有限：ITS／ITS2种内和种间变异大,在本研究中物种正确鉴定率达到100％／96％,可考虑作为秋海棠属DNA条形码鉴定的候选片段。研究结果支持中国植物条形码研究组建议将核基因ITS／ITS2纳人种子植物DNA条形码核心片段中的观点。     

DNA barcodes identify Chinese medicinal plants and detect geographical patterns of Sinosenecio (Asteraceae)

DNA barcodes have proved to be efficient for plants species discrimination and identification using short and standardized genomic regions. The genus Sinosenecio(Asteraceae) is used for traditional medicinal purposes in China. Most species of the genus occur in restricted geographical regions and exhibit a wide range of morphological variations within species, making them difficult to differentiate in the field. Previously, taxonomic revisions have been made based on morphological and cytological evidence. In the present study, barcoding analysis was performed on 107 individuals representing 38 species in this genus to evaluate the performance of four candidate barcoding loci (matK, rbcL, trnH-psbA and internal transcribed spacer [ITS]) and detect geographical patterns. Three different methods based on genetic distance, sequence similarity, and the phylogenetic tree were used. Comparably high species discrimination power was detected in species-level taxonomic process by the ITS dataset alone or combined with other loci, which was suggested to be the most suitable barcode for Sinosenecio. Our results are congruent with previous taxonomic studies concerning the monophyly of the S. oldhamianus group. The present study provides an empirical paradigm for the identification of medicinal plant species and their geographical patterns, ascertaining the congruence between taxonomical studies and barcoding analysis inSinosenecio.     

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost‐effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non‐trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost‐effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single‐locus DNA barcode for plants.     

DNA barcoding in closely related species: A case study of Primula L.sect.Proliferae Pax (Primulaceae) in China

DNA barcoding is a method of identifying species by analyzing one or a few short standardized DNA sequences. There are particular challenges in barcoding plants, especially for distinguishing closely related species. Hence, there is an urgent need to evaluate the performance of candidate loci for distinguishing between species, especially closely related species, to complement the rbcL + matK combination suggested as the core barcode for land plants. We sampled 48 individuals representing 12 species in Primula sect. Proliferae Pax in China to evaluate the performance of eight leading candidate barcode loci (matK, rbcL, rpoB, rpoCl, trnH-psbA, psbK-psbI, atpFatpH, and internal transcribed spacer (ITS)). The core combination rbcL + matK gave only 50% species resolution in sect. Proliferae. In terms of intraspecies and interspecies divergence, degree of monophyly, and sequence similarity, ITS, trnH-psbA, and psbK-psbI showed good performance as single-locus barcodes. Internal transcribed spacer displayed the highest genetic divergence and best discriminatory power, both alone and in combination with rbcL +matK (83.3% species resolution). We recommend evaluating the use of ITS for barcoding in other species. Low or single copy nuclear regions would provide more sophisticated barcoding tools in the long term, even though further research is required to find suitable loci.     

Identification of seagrasses in the gut of a marine herbivorous fish using DNA barcoding and visual inspection techniques

Traditional visual diet analysis techniques were compared with DNA barcoding in juvenile herbivorous rabbitfish Siganus fuscescens collected in Moreton Bay, Australia, where at least six species of seagrass occur. The intergenic spacer trnH-psbA, suggested as the optimal gene for barcoding angiosperms, was used for the first time to identify the seagrass in fish guts. Four seagrass species and one alga were identified visually from gut contents; however, there was considerable uncertainty in visual identification with 38 of 40 fish having unidentifiable plant fragments in their gut. PCR and single-strand conformational polymorphism (SSCP) were able to discriminate three seagrass families from visually cryptic gut contents. While effective in identifying cryptic gut content to family level, this novel method is likely to be most efficient when paired with visual identification techniques.     

DNA barcoding in closely related species: A case study of Primula L. sect. Proliferae Pax (Primulaceae) in China

DNA barcoding is a method of identifying species by analyzing one or a few short standardized DNA sequences. There are particular challenges in barcoding plants, especially for distinguishing closely related species. Hence, there is an urgent need to evaluate the performance of candidate loci for distinguishing between species, especially closely related species, to complement the rbcL + matK combination suggested as the core barcode for land plants. We sampled 48 individuals representing 12 species in Primula sect. Proliferae Pax in China to evaluate the performance of eight leading candidate barcode loci (matK, rbcL, rpoB, rpoC1, trnH-psbA, psbK-psbI, atpF-atpH, and internal transcribed spacer (ITS)). The core combination rbcL+matK gave only 50% species resolution in sect. Proliferae. In terms of intraspecies and interspecies divergence, degree of monophyly, and sequence similarity, ITS, trnH-psbA, and psbK-psbI showed good performance as single-locus barcodes. Internal transcribed spacer displayed the highest genetic divergence and best discriminatory power, both alone and in combination with rbcL+matK (83.3% species resolution). We recommend evaluating the use of ITS for barcoding in other species. Low or single copy nuclear regions would provide more sophisticated barcoding tools in the long term, even though further research is required to find suitable loci.     

植物物种的快速鉴定与iFlora: DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比, DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（Pedicularis L.）植物具对生（轮生）叶的种类70%以上分布在中国,近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81.40%和89.57%,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。