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.     

Single nucleotide polymorphism barcoding of cytochrome c oxidase I sequences for discriminating 17 species of Columbidae by decision tree algorithm

DNA barcodes are widely used in taxonomy, systematics, species identification, food safety, and forensic science. Most of the conventional DNA barcode sequences contain the whole information of a given barcoding gene. Most of the sequence information does not vary and is uninformative for a given group of taxa within a monophylum. We suggest here a method that reduces the amount of noninformative nucleotides in a given barcoding sequence of a major taxon, like the prokaryotes, or eukaryotic animals, plants, or fungi. The actual differences in genetic sequences, called single nucleotide polymorphism (SNP) genotyping, provide a tool for developing a rapid, reliable, and high‐throughput assay for the discrimination between known species. Here, we investigated SNPs as robust markers of genetic variation for identifying different pigeon species based on available cytochrome c oxidase I (COI) data. We propose here a decision tree‐based SNP barcoding (DTSB) algorithm where SNP patterns are selected from the DNA barcoding sequence of several evolutionarily related species in order to identify a single species with pigeons as an example. This approach can make use of any established barcoding system. We here firstly used as an example the mitochondrial gene COI information of 17 pigeon species (Columbidae, Aves) using DTSB after sequence trimming and alignment. SNPs were chosen which followed the rule of decision tree and species‐specific SNP barcodes. The shortest barcode of about 11 bp was then generated for discriminating 17 pigeon species using the DTSB method. This method provides a sequence alignment and tree decision approach to parsimoniously assign a unique and shortest SNP barcode for any known species of a chosen monophyletic taxon where a barcoding sequence is available.     

Testing evolutionary hypotheses for DNA barcoding failure in willows

The goal of DNA barcoding is to enable the rapid identification of taxa from short diagnostic DNA sequence profiles. But how feasible is this objective when many evolutionary processes, such as hybridization and selective sweeps, cause alleles to be shared among related taxa? In this issue of Molecular Ecology, Percy et al. (2014) test the full suite of seven candidate plant barcoding loci in a broad geographic sample of willow species. They show exceptional plastid haplotype sharing between species across continents, with most taxa not possessing a unique barcode sequence. Using population genetic and molecular dating analyses, they implicate hybridization and selective sweeps, but not incomplete lineage sorting, as the historical processes causing widespread haplotype sharing among willow taxa. This study represents an exceptional case of how poorly barcoding can perform, and highlights methodological issues using universal organellar regions for species identification.     

DNA条形码分析方法研究进展

DNA条形码是一段短的、标准化的DNA序列,DNA条形码技术通过对DNA条形码序列分析实现物种的有效鉴定.随着生物DNA条形码序列的大量测定,DNA条形码分析方法得到迅速发展,推动了其在生物分子鉴定中的应用.2003年以来,DNA条形码技术已广泛应用于动物、植物和真菌等物种的鉴定,并有力地推动了生物分类学、生物多样性和生态学等学科的发展.本文在综述DNA条形码技术的基础上,总结了5类主要的DNA条形码分析方法,即基于遗传距离的分析、基于遗传相似度的分析、基于系统发育树的分析、基于序列特征的分析和基于统计分类法的分析,并进一步展望了DNA条形码技术的发展与应用.     

DNA barcoding of stylommatophoran land snails: a test of existing sequences

DNA barcoding has attracted attention because it is a potentially simple and universal method for taxonomic assignment. One anticipated problem in applying the method to stylommatophoran land snails is that they frequently exhibit extreme divergence of mitochondrial DNA sequences, sometimes reaching 30% within species. We therefore trialled the utility of barcodes in identifying land snails, by analysing the stylommatophoran cytochrome oxidase subunit I sequences from GenBank. Two alignments of 381 and 228 base pairs were used to determine potential error rates among a test data set of 97 or 127 species, respectively. Identification success rates using neighbour‐joining phylogenies were 92% for the longer sequence and 82% for the shorter sequence, indicating that a high degree of mitochondrial variation may actually be an advantage when using phylogeny‐based methods for barcoding. There was, however, a large overlap between intra‐ and interspecific variation, with assignment failure (per cent of samples not placed with correct species) particularly associated with a low degree of mitochondrial variation (Kimura 2‐parameter distance < 0.05) and a small GenBank sample size (< 25 per species). Thus, while the optimum intra/interspecific threshold value was 4%, this was associated with an overall error of 32% for the longer sequences and 44% for the shorter sequences. The high error rate necessitates that barcoding of land snails is a potentially useful method to discriminate species of land snail, but only when a baseline has first been established using conventional taxonomy and sample DNA sequences. There is no evidence for a barcoding gap, ruling out species discovery based on a threshold value alone.     

Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae)

DNA barcoding is a new technology which can identify species rapidly based on short and standardized DNA sequences. Ligularia, a genus of Asteraceae with about 140 species, exhibits high morphological and ecological diversity, which makes the classification and species delimitation difficult, especially in the cases of closely related taxa. In this study, we tested four DNA core barcoding regions (ITS, matK, psbA trnH and rbcL) in 144 samples representing 35 species of Ligularia. The results revealed that the chloroplast regions (matK, psbA trnH and rbcL) have extremely low species identification rate due to low interspecific variation. Conversely, ITS sequence showed higher species identification rate (60%) and could discriminate the species which are difficult to identify. The combination of these four gene fragments did not improve the ability of species discrimination.     

DNA条形码技术在植物中的研究现状

DNA条形码技术(DNA barcoding)是用短的DNA片段对物种进行识别和鉴定的分子生物学技术。在动物研究中该技术已经成功应用于利用线粒体细胞色素c氧化酶亚基I(COI)进行物种鉴定和发现隐种或新物种。相对于动物, COI基因在高等植物中进化速率较慢, 因此植物条形码研究以叶绿体基因组作为重点, 但目前还处于寻找合适的基因片段阶段。许多学者对此进行了积极的探索, 报道了多种植物条形码的候选片段或组合, 但还没有获得满足所有标准的特征位点片段。该文介绍了DNA条形码的标准、优点、工作流程及数据分析方法, 总结了DNA条形码在植物中的研究现状。     

Bayesian species identification under the multispecies coalescent provides significant improvements to DNA barcoding analyses

DNA barcoding methods use a single locus (usually the mitochondrial COI gene) to assign unidentified specimens to known species in a library based on a genetic distance threshold that distinguishes between‐species divergence from within‐species diversity. Recently developed species delimitation methods based on the multispecies coalescent (MSC) model offer an alternative approach to individual assignment using either single‐locus or multiloci sequence data. Here, we use simulations to demonstrate three features of an MSC method implemented in the program bpp . First, we show that with one locus, MSC can accurately assign individuals to species without the need for arbitrarily determined distance thresholds (as required for barcoding methods). We provide an example in which no single threshold or barcoding gap exists that can be used to assign all specimens without incurring high error rates. Second, we show that bpp can identify cryptic species that may be misidentified as a single species within the library, potentially improving the accuracy of barcoding libraries. Third, we show that taxon rarity does not present any particular problems for species assignments using bpp and that accurate assignments can be achieved even when only one or a few loci are available. Thus, concerns that have been raised that MSC methods may have problems analysing rare taxa (singletons) are unfounded. Currently, barcoding methods enjoy a huge computational advantage over MSC methods and may be the only approach feasible for massively large data sets, but MSC methods may offer a more stringent test for species that are tentatively assigned by barcoding.     

DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae)

Morphological and molecular studies on a tardigrade species have been carried out to verify the possibility of using a DNA barcoding approach for species identification in this phylum. Macrobiotus macrocalix Bertolani & Rebecchi, 1993 was chosen as the test species since it belongs to a group of species in which the taxonomy is quite problematic. Animals and eggs belonging to three Italian and one Swedish populations have been investigated. Both morphological and molecular analyses show that all the populations belong to the same species. The low genetic distances recorded among the studied populations (0.3-1.0%) and the high genetic distance (15.9-16.3%) between these populations and a closely related species confirm the possibility of identifying a specimen of this species by its cytochrome oxidase subunit I sequence. Data from other authors support our results indicating that DNA barcoding can be applied to tardigrades. With our protocols, we have obtained voucher specimens that enable us to show a correspondence between morphology and molecular data.     

The origin of the Tibetan Mastiff and species identification of Canis based on mitochondrial cytochrome c oxidase subunit I (COI) gene and COI barcoding

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n = 115) and Canis latrans (n = 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic dogs had four maternal origins (A to D) and that the Tibetan Mastiff originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the Old English sheepdog, Newfoundland, Rottweiler and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.     

DNA条形码技术在植物中的研究现状

DNA条形码技术（DNA barcoding）是用短的DNA片段对物种进行识别和鉴定的分子生物学技术。在动物研究中该技术已经成功应用于利用线粒体细胞色素c氧化酶亚基I（COI）进行物种鉴定和发现隐种或新物种。相对于动物, COI基因在高等植物中进化速率较慢, 因此植物条形码研究以叶绿体基因组作为重点, 但目前还处于寻找合适的基因片段阶段。许多学者对此进行了积极的探索, 报道了多种植物条形码的候选片段或组合, 但还没有获得满足所有标准的特征位点片段。该文介绍了DNA条形码的标准、优点、工作流程及数据分析方法, 总结了DNA条形码在植物中的研究现状。     

Benchmarking DNA barcodes: An assessment using available primate sequences.

DNA barcoding has been recently promoted as a method for both assigning specimens to known species and for discovering new and cryptic species. Here we test both the potential and the limitations of DNA barcodes by analysing a group of well-studied organisms--the primates. Our results show that DNA barcodes provide enough information to efficiently identify and delineate primate species, but that they cannot reliably uncover many of the deeper phylogenetic relationships. Our conclusion is that these short DNA sequences do not contain enough information to build reliable molecular phylogenies or define new species, but that they can provide efficient sequence tags for assigning unknown specimens to known species. As such, DNA barcoding provides enormous potential for use in global biodiversity studies.     

DNA barcode-based molecular identification system for fish species

In this study, we applied DNA barcoding to identify species using short DNA sequence analysis. We examined the utility of DNA barcoding by identifying 53 Korean freshwater fish species, 233 other freshwater fish species, and 1339 saltwater fish species. We successfully developed a web-based molecular identification system for fish (MISF) using a profile hidden Markov model. MISF facilitates efficient and reliable species identification, overcoming the limitations of conventional taxonomic approaches. MISF is freely accessible at .     

New DNA barcodes for identification of Korean birds

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

Use of DNA barcoding to detect invertebrate invasive species from diapausing eggs

The global transhipment of ballast water and associated flora and fauna by cargo vessels has increased dramatically in recent decades. Invertebrate species are frequently carried in ballast water and sediment, although identification of diapausing eggs can be extremely problematic. Here we test the application of DNA barcoding using mitochondrial cytochrome c oxidase subunit I and 16S rDNA to identify species from diapausing eggs collected in ballast sediment of ships. The accuracy of DNA barcoding identification was tested by comparing results from the molecular markers against each other, and by comparing barcoding results to traditional morphological identification of individuals hatched from diapausing eggs. Further, we explored two public genetic databases to determine the broader applicability of DNA barcodes. Of 289 diapausing eggs surveyed, sufficient DNA for barcoding was obtained from 96 individuals (33%). Unsuccessful DNA extractions from 67% of eggs in our study were most likely due to degraded condition of eggs. Of 96 eggs with successful DNA extraction, 61 (64%) were identified to species level, while 36% were identified to possible family/order level. Species level identifications were always consistent between methodologies. DNA barcoding was suitable for a wide range of taxa, including Branchiopoda, Copepoda, Rotifera, Bryozoa and Ascidia. Branchiopoda and Copepoda were respectively the best and worst represented groups in genetic databases. Though genetic databases remain incomplete, DNA barcoding resolved nearly double the number of species identified by traditional taxonomy (19 vs. 10). Notorious invaders are well represented in existing databases, rendering these NIS detectable using molecular methods. DNA barcoding provides a rapid and accurate approach to identification of invertebrate diapausing eggs that otherwise would be very difficult to identify.     

Identifying Chinese species of Gammarus （Crustacea： Amphipoda） using DNA barcoding

Using a standard cytochrome c oxidase I sequence, DNA barcoding has been shown to be effective to distinguish known species and to discover cryptic species. Here we assessed the efficiency of DNA barcoding for the amphipod genus Gammarus from China. The maximum intraspecific divergence for widespread species, Gammarus lacustris, was 3.5%, and mean interspecific divergence reached 21.9%. We presented a conservative benchmark for determining provisional species using maximum intraspecific divergence of Gammarus lacustris. Thirty-one species possessed distinct barcode clusters. Two species were comprised of highly divergent clades with strong neighbor-joining bootstrap values, and likely indicated the presence of cryptic species. Although DNA barcoding is effective, future identification of species of Gammarus should incorporate DNA barcoding and morphological detection[Current Zoology 55(2):158-164,2009].     

DNA条形码在中国金合欢属中的应用

根据形态特征难以准确地辨别金合欢属植物,DNA条形码技术提供了一种准确地鉴定物种的方法。本文利用条形码技术对中国金合欢属物种的序列(psbA trnH、matK、rbcL和ITS)及其不同组合进行比较,通过计算种内和种间变异进行barcoding gap分析,运用Wilcoxon秩和检验比较不同序列的变异性,构建系统树。结果表明：4个片段均存在barcoding gap,ITS序列种间变异率较psbA trnH、rbcL和matK序列有明显优势,单片段ITS正确鉴定率最高,ITS+rbcL片段联合条码的正确鉴定率最高,因此我们认为ITS片段或条形码组合ITS+rbcL是金合欢属的快速鉴别最理想的条码。     

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.     

DNA barcoding and phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) based on mitochondrial COI and 16S rRNA genes

DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish these species. Sequences from homologous regions of four other species of this family were gathered from GenBank. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species. When using neighbour-joining clustering based on COI and 16S genes, all species fell into reciprocally monophyletic clades with high bootstrap values. These evidenced that these scallop species can be efficiently identified by DNA barcoding. Evolutionary relationships of Pectinidae were also examined using the two mitochondrial genes. The results are almost consistent with Waller’s classification, which was proposed on the basis of shell microstructure and the morphological characteristics of juveniles.