High universality of matK primers for barcoding gymnosperms

DNA barcoding is a tool to provide rapid and accurate taxonomic identification using a standard DNA region. A two-marker combination of rnatK+rbcL was formally proposed as the core barcode for land plants by the Consortium for the Barcode of Life Plant Working Group. However, there are currently no barcoding primers for matK showing high universality in gymnosperms. We used 57 gymnosperm species representing 40 genera, 11families and four subclasses to evaluate the universality of nine candidate matK primers and one rbcL primer in this study. Primer (1F/724R) of rbcL is proposed here as a universal primer for gymnosperms due to high universality. One of the nine candidate matK primers (Gym_F1A/Gym_R1A) is proposed as the best "universal" matK primer for gynnosperms because of high polymerase chain reaction success and routine generation of high quality bidirectional sequences. A specific matK primer for Ephedra was newly designed in this study, which performed well on the sampled species. The primers proposed here for rbcL and matK can be easily and successfully amplified for most gymnosperms.     

竹亚科叶绿体DNA条形码筛选

竹亚科是一个形态上鉴定困难且易于混淆的类群。DNA条形码技术为这一类群的鉴定提供了一个辅助手段。核心条形码（rbcL＋matK）在竹亚科中的鉴别率很低,因此．有必要针对竹亚科筛选有效的DNA条形码片段。本研究根据已发表的竹亚科叶绿体基因组序列,筛选出16个片段,包括1个基因片段和15个基因间隔区,选取青篱竹族和莉竹族的10属22种30个个体,进行引物通用性、测序成功率和变异位点分析。研究结果表明：（1）引物具有较高的扩增成功率,但部分片段中由于存在poly结构造成测序失败;（2）片段中的插入／缺失可以编码,作为信息位点;（3）trnG—trnT（t）具有最多变异位点,建议可作为竹亚科优先选择的DNA条形码;（4）在开展不同类群的DNA条形码和分子系统学研究时,可以按照“trnG—trnT（t）＋x”的方式选择合适的叶绿体DNA片段。     

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable.     

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。     

Conserved primers for DNA barcoding historical and modern samples from New Zealand and Antarctic birds

Our ability to DNA barcode the birds of the world is based on the effective amplification and sequencing of a 648 base pair (bp) region of the mitochondrial cytochrome c oxidase (COI or cox1) gene. For many geographic regions the large numbers of vouchered specimens necessary for the construction of a DNA barcoding database have already been collected and are available in museums and other institutions. However, many of these specimens are old (>20 years) and are stored as either fixed study skins or dried skeletons. DNA extracted from such historical samples is typically degraded and, generally, only short DNA fragments can be recovered from such specimens making the recovery of the barcoding region as a single fragment difficult. We report two sets of conserved primers that allow the amplification of the entire DNA barcoding region in either three or five overlapping fragments. These primer sets allow the recovery of DNA barcodes from valuable historical specimens that in many cases are unique in that they are unable or unlikely to be collected again. We also report three new primers that in combination allow the effective amplification from modern samples of the entire DNA barcoding region as a single DNA fragment for 17 orders of Southern Hemisphere birds.     

First insights into fern matK phylogeny

MatK, the only maturase gene in the land plant plastid genome, is a very popular phylogenetic marker that has been extensively applied in reconstructing angiosperm phylogeny. However, the use of matK in fern phylogeny is largely unknown, due to difficulties with amplification: ferns have lost the flanking trnK exons, typically the region used for designing stable priming sites. We developed primers that are either universal or lineage-specific that successfully amplify matK across all fern families. To evaluate whether matK is as powerful a phylogenetic marker in ferns as in angiosperms, we compared its sequence characteristics and phylogenetic performance to those of rbcL and atpA. Among these three genes, matK has the highest variability and substitution evenness, yet shows the least homoplasy. Most importantly, applying matK in fern phylogenetics better resolved relationships among families, especially within eupolypods I and II. Here we demonstrate the power of matK for fern phylogenetic reconstruction, as well as provide primers and extensive sequence data that will greatly facilitate future evolutionary studies of ferns.     

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

One application ofDNA 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.     

Developing DNA barcodes for species identification in Podophylloideae (Berberidaceae)

Species of Podophyllum, Dysosma, Sinopodophyllum, and Diphylleia, genera from Podophylloideae of Berberidaceae, have long been used in traditional herbal medicine in East Asia and/or North America. Accurate identification of the species of these four genera is crucial to their medicinal uses. In this study, we tested the utility of nine barcodes (matK, rbcL, atpH-atpI, rpl32-trnL^UAG, rps18-clpp, trnL-trnF, trnL-ndhJ, trnS-trnfM, and internal transcribed spacer (ITS)) to discriminate different species of Podophylloideae. Thirty-six individuals representing 12 species of Podophylloideae were collected from different locations in China, Japan, and North America. We assessed the feasibility of amplification and sequencing of all markers, examined the levels of the barcoding gap based on DNA sequence divergence between ranges of intra- and interspecific variation using pairwise distances, and further evaluated successful identifications using each barcode by similarity-based and tree-based methods. Results showed that nine barcodes, except rps18-clpp, have a high level of primer universality and sequencing success. As a single barcode, ITS has the most variable sites, greater intra- and interspecific divergences, and the highest species discrimination rate (83%), followed by matKwhich has moderate variation and also high species discrimination rates. However, these species can also be discriminated by ITS alone, except Dysosma versipellis (Hance) M. Cheng ex T. S. Ying and D. pleiantha (Hance) Woodson. The combination of ITS + matK did not improve species resolution over ITS alone. Thus, we propose that ITS may be used as a sole region for identification of most species in Podophylloideae. The failure of ITS to distinguish D. versipellis and D. pleiantha is likely attributed to incomplete lineage sorting due to recent divergence of the two species.     

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.     

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。     

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.     

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.     

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.     

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%.     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.     

Panicum spikelets from the Early Holocene Takarkori rockshelter (SW Libya): Archaeo-molecular and -botanical investigations

This paper deals with the extraction, amplification and sequencing of ancient DNA (aDNA) from spikelets of wild cereals dated at ca. 9000 cal yr BP, representing the most ancient plants with preserved genetic material from the Sahara desert. The sub-fossil records were collected from the archaeological excavation carried out at Takarkori, an archaeological site located in south-western Libya. Morphological and genetic analyses were made on 100 well preserved dried spikelets. Ten DNA extraction protocols were performed to evaluate nucleic acid recovery in terms of DNA yield, purity and amplification success of the chloroplast barcode region matK. The extraction protocol that returned the most suitable DNA to be amplified is the Kistler and Shapiro (2011: J Archaeol Sci 38: 3549-3554) modified protocol. In our study, the results from matK amplification suggested that four specimens are the most appropriate number of spikelets for these analyses. DNA was then used for PCR amplifications of four chloroplast barcode genes: rbcL, matK, trnH-psbA and trnL. A phylogenetic analysis shows the strict relation between the archaeological specimens and modern Panicoideae, supporting the morphological identification. The results indicate that spikelets have a close relation to Panicum laetum Kunth, a wild cereal still collected in tropical Africa.     

Design of phylum-specific hybrid primers for DNA barcoding: addressing the need for efficient COI amplification in the Echinodermata

Recent research has shown the usefulness of the Folmer region of the cytochrome oxidase I (COI) as a genetic barcode to assist in species delimitation of echinoderms. However, amplification of COI is often challenging in echinoderms (low success or pseudogenes). We present a method that allows the design of phylum-specific hybrid primers, and use this to develop COI primers for the Echinodermata. We aligned COI sequences from 310 echinoderm species and designed all possible primers along the consensus sequence with two methods (standard degenerate and hybrid). We found much lower degeneracy for hybrid primers (4-fold degeneracy) than for standard degenerate primers (≥48-fold degeneracy). We then designed the most conserved hybrid primers to amplify a >500-bp region within COI. These primers successfully amplified this gene region in all tested taxa (123 species across all echinoderm classes). Sequencing of 30 species among these confirmed both the quality of the sequences (>500 bp, no pseudogenes) and their utility as a DNA barcode. This method should be useful for developing primers for other mitochondrial genes and other phyla. The method will also be of interest for the development of future projects involving both community-based genetic assessments on macroorganisms and biodiversity assessment of environmental samples using high-throughput sequencing.     

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle-scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long-range polymerase chain reaction and using next-generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel-dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel-dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.