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.     

Biodiversity studies in Phaseolus species by DNA barcoding

The potential of DNA barcoding was tested as a system for studying genetic diversity and genetic traceability in bean germplasm. This technique was applied to several pure lines of Phaseolus vulgaris L. belonging to wild, domesticated, and cultivated common beans, along with some accessions of Phaseolus coccineus L., Phaseolus lunatus L., and Vigna unguiculata (L.) Walp. A multilocus approach was exploited using three chloroplast genic regions (rbcL, trnL, and matK), four intergenic spacers (rpoB-trnC, atpBrbcL, trnT-trnL, and psbA-trnH), and nuclear ITS1 and ITS2 rDNA sequences. Our main goals were to identify the markers and SNPs that show the best discriminant power at the variety level in common bean germplasm, to examine two methods (tree based versus character based) for biodiversity analysis and traceability assays, and to evaluate the overall utility of chloroplast DNA barcodes for reconstructing the origins of modern Italian varieties. Our results indicate that the neighbor-joining method is a powerful approach for comparing genetic diversity within plant species, but it is relatively uninformative for the genetic traceability of plant varieties. In contrast, the character-based method was able to identify several distinct haplotypes over all target regions corresponding to Mesoamerican or Andean accessions; Italian accessions originated from both gene pools. On the whole, our findings raise some concerns about the use of DNA barcoding for intraspecific genetic diversity studies in common beans and highlights its limitations for resolving genetic relationships between landraces and varieties.     

Evaluation of 11 single‐locus and seven multilocus DNA barcodes in Lamium L. (Lamiaceae)

The aim of this work was to evaluate the suitability of selected DNA regions in the barcoding of plants, based on the species belonging to the genus Lamium (Lamiaceae). For this purpose, nine chloroplast barcodes, that is, accD, matK, rbcL, rpoA, rpoB, rpoC1, rpoC2, trnH‐psbA, trnL‐trnF, as well as ITS nuclear region, and intron of mitochondrial nad5 gene were tested. Among the single‐locus barcodes, most effective in the identification of Lamium species was the trnH‐psbA spacer and matK gene. The high level of variability and resolving power was also observed in the case of rpoA and rpoC2 genes. Despite the high interspecies variability of ITS region, it turned out to be inapplicable in Lamium identification. An important disadvantage of ITS as a barcode is a limitation of its use in polyploid plants, samples contaminated with fungal material or samples with partially degraded DNA. We have also evaluated five‐two‐locus and two‐three‐locus barcode regions created from a combination of most effective single loci. The best‐performing barcode combinations were matK + trnH‐psbA and matK + rpoA. Both of them had equally high discriminative power to identify Lamium 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.     

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.     

A character‐based approach in the Mexican cycads supports diverse multigene combinations for DNA barcoding

A DNA barcoding study was conducted to determine the optimal combination of loci needed for successful species‐level molecular identification in three extant cycad genera—Ceratozamia, Dioon, and Zamia—that occur in Mexico. Based on conclusions of a previous multigene study in representative species of all genera in the Cycadales, we tested the DNA barcoding performance of seven chloroplast coding (matK, rpoB, rpoC1, and rbcL) and non‐coding (atpF/H, psbK/I, and trnH‐psbA) regions, plus sequences of the nuclear internal transcribed spacer. We analysed data under the assumptions of the “character attributes organization system” (CAOS), a character‐based approach in which species are identified through the presence of ‘DNA diagnostics’. In Ceratozamia, four chloroplast regions and one nuclear region were needed to achieve > 70% unique species identification. In contrast, the two‐gene combination atpF/H + psbK/I and the four‐gene combination atpF/H + psbK/I + rpoC1 + ITS2 were needed to reach 79% and 75% unique species identification in Dioon and Zamia, respectively. The combinations atpF/H + psbK/I and atpF/H + psbK/I + rpoC1 + ITS2 include loci previously considered by the international DNA barcoding community. However, none of the three combinations of potential DNA barcoding loci found to be optimal with a character‐based approach in the Mexican cycads coincides with the ‘core barcode’ of chloroplast markers (matK + rbcL) recently proposed for universal use in the plant kingdom.     

Evaluation of candidate DNA barcoding loci for economically important timber species of the mahogany family (Meliaceae)

There has been considerable debate regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from proposed candidate loci on a common set of samples. In this study, we evaluated main candidate plastid regions (rpoC1, rpoB, accD) and additional plastid markers (psbB, psbN, psbT exons and the trnS-trnG spacer) as well as the nuclear ribosomal spacer region (ITS1-5.8S-ITS2) in a group of land plants belonging to the mahogany family, Meliaceae. Across these samples, only ITS showed high levels of resolvability. Interspecific sharing of sequences from individual plastid loci was common. The combination of multiple loci did not improve performance. DNA barcoding with ITS alone revealed cryptic species and proved useful in identifying species listed in Convention on International Trade of Endangered Species appendixes.     

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。     

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。     

Evaluation of 10 plant barcodes in Bryophyta (Mosses)

DNA barcoding is a molecular tool that uses a standardized DNA region to identify species. Our preliminary study reported here is the first attempt to specifically focus on universality and attributes of candidate barcodes across a wide systematic range of mosses. We tested eight previously proposed plant barcoding regions (atpF-atpH, ITS2, marK, psbK-psbI, rbcL, rpoB, rpoC1, and trnH-psbA) and two popular phylogenetic markers (rps4 and trnL-trnF of cpDNA) in 49 moss species and 9 liverwort species, representing half of the orders in moss lineages. The ITS2, rbcL, rpoC1, rps4, trnH-psbA and trnL-trnF regions showed good universality, and therefore the efficacy of these loci as DNA barcodes was further evaluated in 36 mosses and 2 liverworts, each of which included two to three individuals per taxa. The five loci, viz. rbcL, rpoC1, rps4, trnH-psbA and trnL-trnF, were easy to amplify and sequence and showed significant inter-specific genetic variability, making them potentially useful DNA barcodes for mosses. The best performing single loci were the rbcL and rpoC1 coding regions. Several loci showed equivalent performance and combinations of them did not greatly increase their discrimination capacity. In addition, phylogenies generated from each of the separate regions and multi-locus combinations by using best-fit and Kimura 2-parameter models were compared, but no significant difference was found.     

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。     

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.     

nrDNA ITS和cpDNA rpl16基因测序法对六种鼠尾草的检测

为从鼠尾草属植物中鉴别丹参品种,采用基因测序方法,用核糖体核酸内转录间隔区基因(nrDNA ITS),编码核蛋白体大亚基多肽L16的基因(rpl16)及叶绿体DNA上包含trnL以及trnL和trnF间隔区的区域基因(trnL-trnF)的序列,检测六种鼠尾草属新鲜植物.由于nrDNA ITS和rpl16突变率较高,可以做为6种鼠尾草的基源鉴定标记,依此设计了两对特异引物,从6种鼠尾草中鉴定出丹参(Salvia miltiorrhiza)和云南鼠尾草(S.yunnanensis).但trnL-trnF突变率太低,未能用于鉴别.商品干燥中药材因加工和储藏的方式致使DNA降解严重,基因测序法难于应用.     

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.     

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.     

Barcoding Poplars (Populus L.) from Western China

Background

Populus is an ecologically and economically important genus of trees, but distinguishing between wild species is relatively difficult due to extensive interspecific hybridization and introgression, and the high level of intraspecific morphological variation. The DNA barcoding approach is a potential solution to this problem.

Methodology/Principal Findings

Here, we tested the discrimination power of five chloroplast barcodes and one nuclear barcode (ITS) among 95 trees that represent 21 Populus species from western China. Among all single barcode candidates, the discrimination power is highest for the nuclear ITS, progressively lower for chloroplast barcodes matK (M), trnG-psbK (G) and psbK-psbI (P), and trnH-psbA (H) and rbcL (R); the discrimination efficiency of the nuclear ITS (I) is also higher than any two-, three-, or even the five-locus combination of chloroplast barcodes. Among the five combinations of a single chloroplast barcode plus the nuclear ITS, H+I and P+I differentiated the highest and lowest portion of species, respectively. The highest discrimination rate for the barcodes or barcode combinations examined here is 55.0% (H+I), and usually discrimination failures occurred among species from sympatric or parapatric areas.

Conclusions/Significance

In this case study, we showed that when discriminating Populus species from western China, the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions. Meanwhile, combining the ITS region with chloroplast regions may improve the barcoding success rate and assist in detecting recent interspecific hybridizations. Failure to discriminate among several groups of Populus species from sympatric or parapatric areas may have been the result of incomplete lineage sorting, frequent interspecific hybridizations and introgressions. We agree with a previous proposal for constructing a tiered barcoding system in plants, especially for taxonomic groups that have complex evolutionary histories (e.g. Populus).     

DNA barcoding is a new approach in comparative genomics of plants

DNA barcoding was proposed as a method for recognition and identification of eukaryotic species through comparison of sequences of a standard short DNA fragment—DNA barcode—from an unknown specimen to a library of reference sequences from known species. This allows identifying an organism at any stage of development from a very small tissue sample, fresh or conserved many years ago. Molecular identification of plant samples can be used in various scientific and applied fields. It would also help to find new species, which is particularly important for cryptogamic plants. An optimal DNA barcode region is a small fragment presented in all species of a major taxonomic group, having invariable nucleotide sequence in all members of the same species, but with sufficient variation to discriminate among the species. This fragment should be flanked by low-variable regions for use of universal primers in PCR for amplification and sequencing. The DNA barcode that is well established in animals is a sequence of a fragment of the mitochondrial cytochrome c oxidase gene CO1. However, searching for DNA barcode in plants proved to be a more challenging task. No DNA region universally suitable for all plants and meeting all of the necessary criteria has been found. Apparently, a multilocus or two-stage approach should be applied for this purpose. Several fragments of the chloroplast genome (trnH-psbA, matK, rpoC, rpoB, rbcL) in combinations of two or three regions were suggested as candidate regions with highest potential, but more representative samples should be examined to choose the best candidate. The possibility is discussed to use as DNA barcode internal transcribed spacers (ITS) of nuclear rRNA genes, which are highly variable, widely employed in molecular phylogenetic studies at the species level, but also have some limitations.     

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.     

蒟蒻薯属(薯蓣科)植物DNA条形码研究

蒟蒻薯属(Tacca)植物种间在形态上差别不大,导致分类上存在一定的困难.DNA条形码是一种利用短的DNA标准片段来鉴别和发现物种的方法.本研究利用核基因ITS片段和叶绿体基因trn H-psbA,rbcL,matK片段对蒟蒻薯属6个种的DNA条形码进行研究,对4个DNA片段可用性,种内种间变异,barcode gap进行了分析,采用Tree-based和BBA两种方法比较不同序列的鉴定能力.结果显示:单片段ITS正确鉴定率最高,片段组合rbcL+matK正确鉴定率最高.支持CBOL植物工作组推荐的条码组合rbcL+matK可作为蒟蒻薯属物种鉴定的标准条码,建议ITS片段作为候选条码.丝须蒟蒻薯Tacca integrifolia采自西藏的居群与马来西亚居群形成了2个不同的遗传分支,且两者在形态上也存在一定的差异,很可能是一个新种.     

地黄属植物的DNA条形码研究

地黄属(Rehmannia)为玄参科(Scrophulariaceae)药用植物,广泛分布于中国中东部及北部地区。由于地黄属植物经历了快速成种,导致其属内物种间形态性状差异较小,运用传统的形态学分类方法已难以准确地鉴定物种,近年来迅速发展起来的DNA条形码技术为快速、准确地鉴别物种提供了新思路。本研究选用3个叶绿体DNA非编码区片段(trn L-trn F、trn M-trn V和trn S-trn G)及核基因ITS片段,运用PWG-distance和TreeBuilding两种方法对地黄属5个物种75个个体进行了DNA条形码分析。结果表明:单个叶绿体DNA片段或核基因ITS片段对地黄属物种的鉴别率较低(0%~20%),组合的叶绿体DNA片段分辨能力虽然高于单个DNA片段,但并不能将地黄属5个物种完全区分开;trn S-trn G+ITS片段组合的分辨率可达100%,能够将地黄属5个物种准确区分,与所有叶绿体DNA片段和核基因ITS片段组合(trn L-trn F+trn M-trn V+trn S-trn G+ITS)的辨别率相同,因此推荐trn S-trn G+ITS作为地黄属植物的标准条形码。此外,利用DNA条形码鉴别物种时,可采用叶绿体DNA片段和核DNA片段组合的方法来提高物种鉴定的成功率。