TaxI: a software tool for DNA barcoding using distance methods

PubMed Central

Steinke, Dirk; Vences, Miguel; Salzburger, Walter; Meyer, Axel

2005-01-01

DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding. PMID:16214755

Efficient alignment-free DNA barcode analytics.

PubMed

Kuksa, Pavel; Pavlovic, Vladimir

2009-11-10

In this work we consider barcode DNA analysis problems and address them using alternative, alignment-free methods and representations which model sequences as collections of short sequence fragments (features). The methods use fixed-length representations (spectrum) for barcode sequences to measure similarities or dissimilarities between sequences coming from the same or different species. The spectrum-based representation not only allows for accurate and computationally efficient species classification, but also opens possibility for accurate clustering analysis of putative species barcodes and identification of critical within-barcode loci distinguishing barcodes of different sample groups. New alignment-free methods provide highly accurate and fast DNA barcode-based identification and classification of species with substantial improvements in accuracy and speed over state-of-the-art barcode analysis methods. We evaluate our methods on problems of species classification and identification using barcodes, important and relevant analytical tasks in many practical applications (adverse species movement monitoring, sampling surveys for unknown or pathogenic species identification, biodiversity assessment, etc.) On several benchmark barcode datasets, including ACG, Astraptes, Hesperiidae, Fish larvae, and Birds of North America, proposed alignment-free methods considerably improve prediction accuracy compared to prior results. We also observe significant running time improvements over the state-of-the-art methods. Our results show that newly developed alignment-free methods for DNA barcoding can efficiently and with high accuracy identify specimens by examining only few barcode features, resulting in increased scalability and interpretability of current computational approaches to barcoding.

Decision Tree Algorithm-Generated Single-Nucleotide Polymorphism Barcodes of rbcL Genes for 38 Brassicaceae Species Tagging.

PubMed

Yang, Cheng-Hong; Wu, Kuo-Chuan; Chuang, Li-Yeh; Chang, Hsueh-Wei

2018-01-01

DNA barcode sequences are accumulating in large data sets. A barcode is generally a sequence larger than 1000 base pairs and generates a computational burden. Although the DNA barcode was originally envisioned as straightforward species tags, the identification usage of barcode sequences is rarely emphasized currently. Single-nucleotide polymorphism (SNP) association studies provide us an idea that the SNPs may be the ideal target of feature selection to discriminate between different species. We hypothesize that SNP-based barcodes may be more effective than the full length of DNA barcode sequences for species discrimination. To address this issue, we tested a r ibulose diphosphate carboxylase ( rbcL ) S NP b arcoding (RSB) strategy using a decision tree algorithm. After alignment and trimming, 31 SNPs were discovered in the rbcL sequences from 38 Brassicaceae plant species. In the decision tree construction, these SNPs were computed to set up the decision rule to assign the sequences into 2 groups level by level. After algorithm processing, 37 nodes and 31 loci were required for discriminating 38 species. Finally, the sequence tags consisting of 31 rbcL SNP barcodes were identified for discriminating 38 Brassicaceae species based on the decision tree-selected SNP pattern using RSB method. Taken together, this study provides the rational that the SNP aspect of DNA barcode for rbcL gene is a useful and effective sequence for tagging 38 Brassicaceae species.

DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

PubMed

Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

2012-01-01

DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

Potential for DNA-based identification of Great Lakes fauna: match and mismatch between taxa inventories and DNA barcode libraries.

PubMed

Trebitz, Anett S; Hoffman, Joel C; Grant, George W; Billehus, Tyler M; Pilgrim, Erik M

2015-07-22

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to bioassessment and non-native species monitoring. The ability to assign species identities to DNA sequences found depends on the availability of comprehensive DNA reference libraries. Here, we compile inventories for aquatic metazoans extant in or threatening to invade the Laurentian Great Lakes and examine the availability of reference mitochondrial COI DNA sequences (barcodes) in the Barcode of Life Data System for them. We found barcode libraries largely complete for extant and threatening-to-invade vertebrates (100% of reptile, 99% of fish, and 92% of amphibian species had barcodes). In contrast, barcode libraries remain poorly developed for precisely those organisms where morphological identification is most challenging; 46% of extant invertebrates lacked reference barcodes with rates especially high among rotifers, oligochaetes, and mites. Lack of species-level identification for many aquatic invertebrates also is a barrier to matching DNA sequences with physical specimens. Attaining the potential for DNA-based identification of mixed-organism samples covering the breadth of aquatic fauna requires a concerted effort to build supporting barcode libraries and voucher collections.

Potential for DNA-based identification of Great Lakes fauna: match and mismatch between taxa inventories and DNA barcode libraries

NASA Astrophysics Data System (ADS)

Trebitz, Anett S.; Hoffman, Joel C.; Grant, George W.; Billehus, Tyler M.; Pilgrim, Erik M.

2015-07-01

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to bioassessment and non-native species monitoring. The ability to assign species identities to DNA sequences found depends on the availability of comprehensive DNA reference libraries. Here, we compile inventories for aquatic metazoans extant in or threatening to invade the Laurentian Great Lakes and examine the availability of reference mitochondrial COI DNA sequences (barcodes) in the Barcode of Life Data System for them. We found barcode libraries largely complete for extant and threatening-to-invade vertebrates (100% of reptile, 99% of fish, and 92% of amphibian species had barcodes). In contrast, barcode libraries remain poorly developed for precisely those organisms where morphological identification is most challenging; 46% of extant invertebrates lacked reference barcodes with rates especially high among rotifers, oligochaetes, and mites. Lack of species-level identification for many aquatic invertebrates also is a barrier to matching DNA sequences with physical specimens. Attaining the potential for DNA-based identification of mixed-organism samples covering the breadth of aquatic fauna requires a concerted effort to build supporting barcode libraries and voucher collections.

A laboratory information management system for DNA barcoding workflows.

PubMed

Vu, Thuy Duong; Eberhardt, Ursula; Szöke, Szániszló; Groenewald, Marizeth; Robert, Vincent

2012-07-01

This paper presents a laboratory information management system for DNA sequences (LIMS) created and based on the needs of a DNA barcoding project at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands). DNA barcoding is a global initiative for species identification through simple DNA sequence markers. We aim at generating barcode data for all strains (or specimens) included in the collection (currently ca. 80 k). The LIMS has been developed to better manage large amounts of sequence data and to keep track of the whole experimental procedure. The system has allowed us to classify strains more efficiently as the quality of sequence data has improved, and as a result, up-to-date taxonomic names have been given to strains and more accurate correlation analyses have been carried out.

The campaign to DNA barcode all fishes, FISH-BOL.

PubMed

Ward, R D; Hanner, R; Hebert, P D N

2009-02-01

FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System (http://www.barcodinglife.org). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections.

PubMed

Chambers, E Anne; Hebert, Paul D N

2016-01-01

High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale.

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections

PubMed Central

Chambers, E. Anne; Hebert, Paul D. N.

2016-01-01

Background High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. Methodology/Principal Findings This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. Conclusions/Significance This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale. PMID:27116180

Efficient alignment-free DNA barcode analytics

PubMed Central

Kuksa, Pavel; Pavlovic, Vladimir

2009-01-01

Background In this work we consider barcode DNA analysis problems and address them using alternative, alignment-free methods and representations which model sequences as collections of short sequence fragments (features). The methods use fixed-length representations (spectrum) for barcode sequences to measure similarities or dissimilarities between sequences coming from the same or different species. The spectrum-based representation not only allows for accurate and computationally efficient species classification, but also opens possibility for accurate clustering analysis of putative species barcodes and identification of critical within-barcode loci distinguishing barcodes of different sample groups. Results New alignment-free methods provide highly accurate and fast DNA barcode-based identification and classification of species with substantial improvements in accuracy and speed over state-of-the-art barcode analysis methods. We evaluate our methods on problems of species classification and identification using barcodes, important and relevant analytical tasks in many practical applications (adverse species movement monitoring, sampling surveys for unknown or pathogenic species identification, biodiversity assessment, etc.) On several benchmark barcode datasets, including ACG, Astraptes, Hesperiidae, Fish larvae, and Birds of North America, proposed alignment-free methods considerably improve prediction accuracy compared to prior results. We also observe significant running time improvements over the state-of-the-art methods. Conclusion Our results show that newly developed alignment-free methods for DNA barcoding can efficiently and with high accuracy identify specimens by examining only few barcode features, resulting in increased scalability and interpretability of current computational approaches to barcoding. PMID:19900305

Supervised DNA Barcodes species classification: analysis, comparisons and results

PubMed Central

2014-01-01

Background Specific fragments, coming from short portions of DNA (e.g., mitochondrial, nuclear, and plastid sequences), have been defined as DNA Barcode and can be used as markers for organisms of the main life kingdoms. Species classification with DNA Barcode sequences has been proven effective on different organisms. Indeed, specific gene regions have been identified as Barcode: COI in animals, rbcL and matK in plants, and ITS in fungi. The classification problem assigns an unknown specimen to a known species by analyzing its Barcode. This task has to be supported with reliable methods and algorithms. Methods In this work the efficacy of supervised machine learning methods to classify species with DNA Barcode sequences is shown. The Weka software suite, which includes a collection of supervised classification methods, is adopted to address the task of DNA Barcode analysis. Classifier families are tested on synthetic and empirical datasets belonging to the animal, fungus, and plant kingdoms. In particular, the function-based method Support Vector Machines (SVM), the rule-based RIPPER, the decision tree C4.5, and the Naïve Bayes method are considered. Additionally, the classification results are compared with respect to ad-hoc and well-established DNA Barcode classification methods. Results A software that converts the DNA Barcode FASTA sequences to the Weka format is released, to adapt different input formats and to allow the execution of the classification procedure. The analysis of results on synthetic and real datasets shows that SVM and Naïve Bayes outperform on average the other considered classifiers, although they do not provide a human interpretable classification model. Rule-based methods have slightly inferior classification performances, but deliver the species specific positions and nucleotide assignments. On synthetic data the supervised machine learning methods obtain superior classification performances with respect to the traditional DNA Barcode classification methods. On empirical data their classification performances are at a comparable level to the other methods. Conclusions The classification analysis shows that supervised machine learning methods are promising candidates for handling with success the DNA Barcoding species classification problem, obtaining excellent performances. To conclude, a powerful tool to perform species identification is now available to the DNA Barcoding community. PMID:24721333

Long-range barcode labeling-sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Feng; Zhang, Tao; Singh, Kanwar K.

Methods for sequencing single large DNA molecules by clonal multiple displacement amplification using barcoded primers. Sequences are binned based on barcode sequences and sequenced using a microdroplet-based method for sequencing large polynucleotide templates to enable assembly of haplotype-resolved complex genomes and metagenomes.

Accelerated construction of a regional DNA-barcode reference library: Caddisflies (Trichoptera) in the Great Smoky Mountains National Park

USGS Publications Warehouse

Zhou, X.; Robinson, J.L.; Geraci, C.J.; Parker, C.R.; Flint, O.S.; Etnier, D.A.; Ruiter, D.; DeWalt, R.E.; Jacobus, L.M.; Hebert, P.D.N.

2011-01-01

Deoxyribonucleic acid (DNA) barcoding is an effective tool for species identification and lifestage association in a wide range of animal taxa. We developed a strategy for rapid construction of a regional DNA-barcode reference library and used the caddisflies (Trichoptera) of the Great Smoky Mountains National Park (GSMNP) as a model. Nearly 1000 cytochrome c oxidase subunit I (COI) sequences, representing 209 caddisfly species previously recorded from GSMNP, were obtained from the global Trichoptera Barcode of Life campaign. Most of these sequences were collected from outside the GSMNP area. Another 645 COI sequences, representing 80 species, were obtained from specimens collected in a 3-d bioblitz (short-term, intense sampling program) in GSMNP. The joint collections provided barcode coverage for 212 species, 91% of the GSMNP fauna. Inclusion of samples from other localities greatly expedited construction of the regional DNA-barcode reference library. This strategy increased intraspecific divergence and decreased average distances to nearest neighboring species, but the DNA-barcode library was able to differentiate 93% of the GSMNP Trichoptera species examined. Global barcoding projects will aid construction of regional DNA-barcode libraries, but local surveys make crucial contributions to progress by contributing rare or endemic species and full-length barcodes generated from high-quality DNA. DNA taxonomy is not a goal of our present work, but the investigation of COI divergence patterns in caddisflies is providing new insights into broader biodiversity patterns in this group and has directed attention to various issues, ranging from the need to re-evaluate species taxonomy with integrated morphological and molecular evidence to the necessity of an appropriate interpretation of barcode analyses and its implications in understanding species diversity (in contrast to a simple claim for barcoding failure).

Towards writing the encyclopaedia of life: an introduction to DNA barcoding

PubMed Central

Savolainen, Vincent; Cowan, Robyn S; Vogler, Alfried P; Roderick, George K; Lane, Richard

2005-01-01

An international consortium of major natural history museums, herbaria and other organizations has launched an ambitious project, the ‘Barcode of Life Initiative’, to promote a process enabling the rapid and inexpensive identification of the estimated 10 million species on Earth. DNA barcoding is a diagnostic technique in which short DNA sequence(s) can be used for species identification. The first international scientific conference on Barcoding of Life was held at the Natural History Museum in London in February 2005, and here we review the scientific challenges discussed during this conference and in previous publications. Although still controversial, the scientific benefits of DNA barcoding include: (i) enabling species identification, including any life stage or fragment, (ii) facilitating species discoveries based on cluster analyses of gene sequences (e.g. cox1=CO1, in animals), (iii) promoting development of handheld DNA sequencing technology that can be applied in the field for biodiversity inventories and (iv) providing insight into the diversity of life. PMID:16214739

A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection.

PubMed

Hebert, Paul D N; Dewaard, Jeremy R; Zakharov, Evgeny V; Prosser, Sean W J; Sones, Jayme E; McKeown, Jaclyn T A; Mantle, Beth; La Salle, John

2013-01-01

DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity - insects.

Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos

PubMed Central

Hajibabaei, Mehrdad; Shokralla, Shadi; Zhou, Xin; Singer, Gregory A. C.; Baird, Donald J.

2011-01-01

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs. PMID:21533287

Molecular identification and phylogenetic analysis of important medicinal plant species in genus Paeonia based on rDNA-ITS, matK, and rbcL DNA barcode sequences.

PubMed

Kim, W J; Ji, Y; Choi, G; Kang, Y M; Yang, S; Moon, B C

2016-08-05

This study was performed to identify and analyze the phylogenetic relationship among four herbaceous species of the genus Paeonia, P. lactiflora, P. japonica, P. veitchii, and P. suffruticosa, using DNA barcodes. These four species, which are commonly used in traditional medicine as Paeoniae Radix and Moutan Radicis Cortex, are pharmaceutically defined in different ways in the national pharmacopoeias in Korea, Japan, and China. To authenticate the different species used in these medicines, we evaluated rDNA-internal transcribed spacers (ITS), matK and rbcL regions, which provide information capable of effectively distinguishing each species from one another. Seventeen samples were collected from different geographic regions in Korea and China, and DNA barcode regions were amplified using universal primers. Comparative analyses of these DNA barcode sequences revealed species-specific nucleotide sequences capable of discriminating the four Paeonia species. Among the entire sequences of three barcodes, marker nucleotides were identified at three positions in P. lactiflora, eleven in P. japonica, five in P. veitchii, and 25 in P. suffruticosa. Phylogenetic analyses also revealed four distinct clusters showing homogeneous clades with high resolution at the species level. The results demonstrate that the analysis of these three DNA barcode sequences is a reliable method for identifying the four Paeonia species and can be used to authenticate Paeoniae Radix and Moutan Radicis Cortex at the species level. Furthermore, based on the assessment of amplicon sizes, inter/intra-specific distances, marker nucleotides, and phylogenetic analysis, rDNA-ITS was the most suitable DNA barcode for identification of these species.

DNA barcodes for Nearctic Auchenorrhyncha (Insecta: Hemiptera).

PubMed

Foottit, Robert G; Maw, Eric; Hebert, P D N

2014-01-01

Many studies have shown the suitability of sequence variation in the 5' region of the mitochondrial cytochrome c oxidase I (COI) gene as a DNA barcode for the identification of species in a wide range of animal groups. We examined 471 species in 147 genera of Hemiptera: Auchenorrhyncha drawn from specimens in the Canadian National Collection of Insects to assess the effectiveness of DNA barcoding in this group. Analysis of the COI gene revealed less than 2% intra-specific divergence in 93% of the taxa examined, while minimum interspecific distances exceeded 2% in 70% of congeneric species pairs. Although most species are characterized by a distinct sequence cluster, sequences for members of many groups of closely related species either shared sequences or showed close similarity, with 25% of species separated from their nearest neighbor by less than 1%. This study, although preliminary, provides DNA barcodes for about 8% of the species of this hemipteran suborder found in North America north of Mexico. Barcodes can enable the identification of many species of Auchenorrhyncha, but members of some species groups cannot be discriminated. Future use of DNA barcodes in regulatory, pest management, and environmental applications will be possible as the barcode library for Auchenorrhyncha expands to include more species and broader geographic coverage.

DNA Barcodes for Nearctic Auchenorrhyncha (Insecta: Hemiptera)

PubMed Central

Foottit, Robert G.; Maw, Eric; Hebert, P. D. N.

2014-01-01

Background Many studies have shown the suitability of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene as a DNA barcode for the identification of species in a wide range of animal groups. We examined 471 species in 147 genera of Hemiptera: Auchenorrhyncha drawn from specimens in the Canadian National Collection of Insects to assess the effectiveness of DNA barcoding in this group. Methodology/Principal Findings Analysis of the COI gene revealed less than 2% intra-specific divergence in 93% of the taxa examined, while minimum interspecific distances exceeded 2% in 70% of congeneric species pairs. Although most species are characterized by a distinct sequence cluster, sequences for members of many groups of closely related species either shared sequences or showed close similarity, with 25% of species separated from their nearest neighbor by less than 1%. Conclusions/Significance This study, although preliminary, provides DNA barcodes for about 8% of the species of this hemipteran suborder found in North America north of Mexico. Barcodes can enable the identification of many species of Auchenorrhyncha, but members of some species groups cannot be discriminated. Future use of DNA barcodes in regulatory, pest management, and environmental applications will be possible as the barcode library for Auchenorrhyncha expands to include more species and broader geographic coverage. PMID:25004106

A “Rosetta Stone” for metazoan zooplankton: DNA barcode analysis of species diversity of the Sargasso Sea (Northwest Atlantic Ocean)

NASA Astrophysics Data System (ADS)

Bucklin, Ann; Ortman, Brian D.; Jennings, Robert M.; Nigro, Lisa M.; Sweetman, Christopher J.; Copley, Nancy J.; Sutton, Tracey; Wiebe, Peter H.

2010-12-01

Species diversity of the metazoan holozooplankton assemblage of the Sargasso Sea, Northwest Atlantic Ocean, was examined through coordinated morphological taxonomic identification of species and DNA sequencing of a ˜650 base-pair region of mitochondrial cytochrome oxidase I (mtCOI) as a DNA barcode (i.e., short sequence for species recognition and discrimination). Zooplankton collections were made from the surface to 5,000 meters during April, 2006 on the R/V R.H. Brown. Samples were examined by a ship-board team of morphological taxonomists; DNA barcoding was carried out in both ship-board and land-based DNA sequencing laboratories. DNA barcodes were determined for a total of 297 individuals of 175 holozooplankton species in four phyla, including: Cnidaria (Hydromedusae, 4 species; Siphonophora, 47); Arthropoda (Amphipoda, 10; Copepoda, 34; Decapoda, 9; Euphausiacea, 10; Mysidacea, 1; Ostracoda, 27); and Mollusca (Cephalopoda, 8; Heteropoda, 6; Pteropoda, 15); and Chaetognatha (4). Thirty species of fish (Teleostei) were also barcoded. For all seven zooplankton groups for which sufficient data were available, Kimura-2-Parameter genetic distances were significantly lower between individuals of the same species (mean=0.0114; S.D. 0.0117) than between individuals of different species within the same group (mean=0.3166; S.D. 0.0378). This difference, known as the barcode gap, ensures that mtCOI sequences are reliable characters for species identification for the oceanic holozooplankton assemblage. In addition, DNA barcodes allow recognition of new or undescribed species, reveal cryptic species within known taxa, and inform phylogeographic and population genetic studies of geographic variation. The growing database of "gold standard" DNA barcodes serves as a Rosetta Stone for marine zooplankton, providing the key for decoding species diversity by linking species names, morphology, and DNA sequence variation. In light of the pivotal position of zooplankton in ocean food webs, their usefulness as rapid responders to environmental change, and the increasing scarcity of taxonomists, the use of DNA barcodes is an important and useful approach for rapid analysis of species diversity and distribution in the pelagic community.

DNA barcodes for dragonflies and damselflies (Odonata) of Mindanao, Philippines.

PubMed

Casas, Princess Angelie S; Sing, Kong-Wah; Lee, Ping-Shin; Nuñeza, Olga M; Villanueva, Reagan Joseph T; Wilson, John-James

2018-03-01

Reliable species identification provides a sounder basis for use of species in the order Odonata as biological indicators and for their conservation, an urgent concern as many species are threatened with imminent extinction. We generated 134 COI barcodes from 36 morphologically identified species of Odonata collected from Mindanao Island, representing 10 families and 19 genera. Intraspecific sequence divergences ranged from 0 to 6.7% with four species showing more than 2%, while interspecific sequence divergences ranged from 0.5 to 23.3% with seven species showing less than 2%. Consequently, no distinct gap was observed between intraspecific and interspecific DNA barcode divergences. The numerous islands of the Philippine archipelago may have facilitated rapid speciation in the Odonata and resulted in low interspecific sequence divergences among closely related groups of species. This study contributes DNA barcodes for 36 morphologically identified species of Odonata reported from Mindanao including 31 species with no previous DNA barcode records.

Selection of a DNA barcode for Nectriaceae from fungal whole-genomes.

PubMed

Zeng, Zhaoqing; Zhao, Peng; Luo, Jing; Zhuang, Wenying; Yu, Zhihe

2012-01-01

A DNA barcode is a short segment of sequence that is able to distinguish species. A barcode must ideally contain enough variation to distinguish every individual species and be easily obtained. Fungi of Nectriaceae are economically important and show high species diversity. To establish a standard DNA barcode for this group of fungi, the genomes of Neurospora crassa and 30 other filamentous fungi were compared. The expect value was treated as a criterion to recognize homologous sequences. Four candidate markers, Hsp90, AAC, CDC48, and EF3, were tested for their feasibility as barcodes in the identification of 34 well-established species belonging to 13 genera of Nectriaceae. Two hundred and fifteen sequences were analyzed. Intra- and inter-specific variations and the success rate of PCR amplification and sequencing were considered as important criteria for estimation of the candidate markers. Ultimately, the partial EF3 gene met the requirements for a good DNA barcode: No overlap was found between the intra- and inter-specific pairwise distances. The smallest inter-specific distance of EF3 gene was 3.19%, while the largest intra-specific distance was 1.79%. In addition, there was a high success rate in PCR and sequencing for this gene (96.3%). CDC48 showed sufficiently high sequence variation among species, but the PCR and sequencing success rate was 84% using a single pair of primers. Although the Hsp90 and AAC genes had higher PCR and sequencing success rates (96.3% and 97.5%, respectively), overlapping occurred between the intra- and inter-specific variations, which could lead to misidentification. Therefore, we propose the EF3 gene as a possible DNA barcode for the nectriaceous fungi.

A DNA barcode library for ground beetles (Insecta, Coleoptera, Carabidae) of Germany: The genus Bembidion Latreille, 1802 and allied taxa

PubMed Central

Raupach, Michael J.; Hannig, Karsten; Morinière, Jérome; Hendrich, Lars

2016-01-01

Abstract As molecular identification method, DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has been proven to be a useful tool for species determination in many insect taxa including ground beetles. In this study we tested the effectiveness of DNA barcodes to discriminate species of the ground beetle genus Bembidion and some closely related taxa of Germany. DNA barcodes were obtained from 819 individuals and 78 species, including sequences from previous studies as well as more than 300 new generated DNA barcodes. We found a 1:1 correspondence between BIN and traditionally recognized species for 69 species (89%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for three species pairs, including two species pairs with haplotype sharing (Bembidion atrocaeruleum/Bembidion varicolor and Bembidion guttula/Bembidion mannerheimii). In contrast to this, deep intraspecific sequence divergences with distinct lineages were revealed for two species (Bembidion geniculatum/Ocys harpaloides). Our study emphasizes the use of DNA barcodes for the identification of the analyzed ground beetles species and represents an important step in building-up a comprehensive barcode library for the Carabidae in Germany and Central Europe as well. PMID:27408547

Application of DNA barcodes in wildlife conservation in Tropical East Asia.

PubMed

Wilson, John-James; Sing, Kong-Wah; Lee, Ping-Shin; Wee, Alison K S

2016-10-01

Over the past 50 years, Tropical East Asia has lost more biodiversity than any tropical region. Tropical East Asia is a megadiverse region with an acute taxonomic impediment. DNA barcodes are short standardized DNA sequences used for taxonomic purposes and have the potential to lessen the challenges of biodiversity inventory and assessments in regions where they are most needed. We reviewed DNA barcoding efforts in Tropical East Asia relative to other tropical regions. We suggest DNA barcodes (or metabarcodes from next-generation sequencers) may be especially useful for characterizing and connecting species-level biodiversity units in inventories encompassing taxa lacking formal description (particularly arthropods) and in large-scale, minimal-impact approaches to vertebrate monitoring and population assessments through secondary sources of DNA (invertebrate derived DNA and environmental DNA). We suggest interest and capacity for DNA barcoding are slowly growing in Tropical East Asia, particularly among the younger generation of researchers who can connect with the barcoding analogy and understand the need for new approaches to the conservation challenges being faced. © 2016 Society for Conservation Biology.

Future collaborations between NEON and the U.S. EPA: linking molecular genomics for bioassessment with national ecological data sets

EPA Science Inventory

Molecular taxonomic techniques such as DNA barcoding offer interesting new capabilities for studying community biodiversity for applications like biological monitoring. Beyond DNA barcoding, new DNA sequencing technologies (i.e. Next-Generation Sequencing) present even greater po...

[Hydrophidae identification through analysis on Cyt b gene barcode].

PubMed

Liao, Li-xi; Zeng, Ke-wu; Tu, Peng-fei

2015-08-01

Hydrophidae, one of the precious traditional Chinese medicines, is generally drily preserved to prevent corruption, but it is hard to identify the species of Hydrophidae through the appearance because of the change due to the drying process. The identification through analysis on gene barcode, a new technique in species identification, can avoid the problem. The gene barcodes of the 6 species of Hydrophidae like Lapemis hardwickii were aquired through DNA extraction and gene sequencing. These barcodes were then in sequence alignment and test the identification efficency by BLAST. Our results revealed that the barcode sequences performed high identification efficiency, and had obvious difference between intra- and inter-species. These all indicated that Cyt b DNA barcoding can confirm the Hydrophidae identification.

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

PubMed

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

VIP Barcoding: composition vector-based software for rapid species identification based on DNA barcoding.

PubMed

Fan, Long; Hui, Jerome H L; Yu, Zu Guo; Chu, Ka Hou

2014-07-01

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time-consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user-friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two-stage algorithm. First, an alignment-free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment-based K2P distance nearest-neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment-free methods and (ii) higher scalability than alignment-based distance methods and character-based methods. These results suggest that this platform is able to deal with both large-scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/. © 2014 John Wiley & Sons Ltd.

Two new computational methods for universal DNA barcoding: a benchmark using barcode sequences of bacteria, archaea, animals, fungi, and land plants.

PubMed

Tanabe, Akifumi S; Toju, Hirokazu

2013-01-01

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used "1-nearest-neighbor" (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research.

Two New Computational Methods for Universal DNA Barcoding: A Benchmark Using Barcode Sequences of Bacteria, Archaea, Animals, Fungi, and Land Plants

PubMed Central

Tanabe, Akifumi S.; Toju, Hirokazu

2013-01-01

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used “1-nearest-neighbor” (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research. PMID:24204702

With a little help from DNA barcoding: investigating the diversity of Gastropoda from the Portuguese coast

PubMed Central

Borges, Luísa M. S.; Hollatz, Claudia; Lobo, Jorge; Cunha, Ana M.; Vilela, Ana P.; Calado, Gonçalo; Coelho, Rita; Costa, Ana C.; Ferreira, Maria S. G.; Costa, Maria H.; Costa, Filipe O.

2016-01-01

The Gastropoda is one of the best studied classes of marine invertebrates. Yet, most species have been delimited based on morphology only. The application of DNA barcodes has shown to be greatly useful to help delimiting species. Therefore, sequences of the cytochrome c oxidase I gene from 108 specimens of 34 morpho-species were used to investigate the molecular diversity within the gastropods from the Portuguese coast. To the above dataset, we added available COI-5P sequences of taxonomically close species, in a total of 58 morpho-species examined. There was a good match between ours and sequences from independent studies, in public repositories. We found 32 concordant (91.4%) out of the 35 Barcode Index Numbers (BINs) generated from our sequences. The application of a ranking system to the barcodes yield over 70% with top taxonomic congruence, while 14.2% of the species barcodes had insufficient data. In the majority of the cases, there was a good concordance between morphological identification and DNA barcodes. Nonetheless, the discordance between morphological and molecular data is a reminder that even the comparatively well-known European marine gastropods can benefit from being probed using the DNA barcode approach. Discordant cases should be reviewed with more integrative studies. PMID:26876495

With a little help from DNA barcoding: investigating the diversity of Gastropoda from the Portuguese coast.

PubMed

Borges, Luísa M S; Hollatz, Claudia; Lobo, Jorge; Cunha, Ana M; Vilela, Ana P; Calado, Gonçalo; Coelho, Rita; Costa, Ana C; Ferreira, Maria S G; Costa, Maria H; Costa, Filipe O

2016-02-15

The Gastropoda is one of the best studied classes of marine invertebrates. Yet, most species have been delimited based on morphology only. The application of DNA barcodes has shown to be greatly useful to help delimiting species. Therefore, sequences of the cytochrome c oxidase I gene from 108 specimens of 34 morpho-species were used to investigate the molecular diversity within the gastropods from the Portuguese coast. To the above dataset, we added available COI-5P sequences of taxonomically close species, in a total of 58 morpho-species examined. There was a good match between ours and sequences from independent studies, in public repositories. We found 32 concordant (91.4%) out of the 35 Barcode Index Numbers (BINs) generated from our sequences. The application of a ranking system to the barcodes yield over 70% with top taxonomic congruence, while 14.2% of the species barcodes had insufficient data. In the majority of the cases, there was a good concordance between morphological identification and DNA barcodes. Nonetheless, the discordance between morphological and molecular data is a reminder that even the comparatively well-known European marine gastropods can benefit from being probed using the DNA barcode approach. Discordant cases should be reviewed with more integrative studies.

DNA Barcoding for Species Identification of Insect Skins: A Test on Chironomidae (Diptera) Pupal Exuviae

PubMed Central

Ekrem, Torbjørn; Stur, Elisabeth

2017-01-01

Abstract Chironomidae (Diptera) pupal exuviae samples are commonly used for biological monitoring of aquatic habitats. DNA barcoding has proved useful for species identification of chironomid life stages containing cellular tissue, but the barcoding success of chironomid pupal exuviae is unknown. We assessed whether standard DNA barcoding could be efficiently used for species identification of chironomid pupal exuviae when compared with morphological techniques and if there were differences in performance between temperate and tropical ecosystems, subfamilies, and tribes. PCR, sequence, and identification success differed significantly between geographic regions and taxonomic groups. For Norway, 27 out of 190 (14.2%) of pupal exuviae resulted in high-quality chironomid sequences that match species. For Costa Rica, 69 out of 190 (36.3%) Costa Rican pupal exuviae resulted in high-quality sequences, but none matched known species. Standard DNA barcoding of chironomid pupal exuviae had limited success in species identification of unknown specimens due to contaminations and lack of matching references in available barcode libraries, especially from Costa Rica. Therefore, we recommend future biodiversity studies that focus their efforts on understudied regions, to simultaneously use morphological and molecular identification techniques to identify all life stages of chironomids and populate the barcode reference library with identified sequences.

Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina).

PubMed

Dentinger, Bryn T M; Didukh, Maryna Y; Moncalvo, Jean-Marc

2011-01-01

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (~450 bp) representing ~100 morphospecies from ~650 collections of Agaricomycotina using several sets of new primers. Large introns (~1500 bp) at variable locations were detected in ~5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (~30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.

Comparing COI and ITS as DNA Barcode Markers for Mushrooms and Allies (Agaricomycotina)

PubMed Central

Dentinger, Bryn T. M.; Didukh, Maryna Y.; Moncalvo, Jean-Marc

2011-01-01

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (∼450 bp) representing ∼100 morphospecies from ∼650 collections of Agaricomycotina using several sets of new primers. Large introns (∼1500 bp) at variable locations were detected in ∼5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (∼30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms. PMID:21966418

Choosing and Using a Plant DNA Barcode

PubMed Central

Hollingsworth, Peter M.; Graham, Sean W.; Little, Damon P.

2011-01-01

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance. PMID:21637336

Googling DNA sequences on the World Wide Web.

PubMed

Hajibabaei, Mehrdad; Singer, Gregory A C

2009-11-10

New web-based technologies provide an excellent opportunity for sharing and accessing information and using web as a platform for interaction and collaboration. Although several specialized tools are available for analyzing DNA sequence information, conventional web-based tools have not been utilized for bioinformatics applications. We have developed a novel algorithm and implemented it for searching species-specific genomic sequences, DNA barcodes, by using popular web-based methods such as Google. We developed an alignment independent character based algorithm based on dividing a sequence library (DNA barcodes) and query sequence to words. The actual search is conducted by conventional search tools such as freely available Google Desktop Search. We implemented our algorithm in two exemplar packages. We developed pre and post-processing software to provide customized input and output services, respectively. Our analysis of all publicly available DNA barcode sequences shows a high accuracy as well as rapid results. Our method makes use of conventional web-based technologies for specialized genetic data. It provides a robust and efficient solution for sequence search on the web. The integration of our search method for large-scale sequence libraries such as DNA barcodes provides an excellent web-based tool for accessing this information and linking it to other available categories of information on the web.

On site DNA barcoding by nanopore sequencing

PubMed Central

Menegon, Michele; Cantaloni, Chiara; Rodriguez-Prieto, Ana; Centomo, Cesare; Abdelfattah, Ahmed; Rossato, Marzia; Bernardi, Massimo; Xumerle, Luciano; Loader, Simon; Delledonne, Massimo

2017-01-01

Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet’s biological heritage. The use of genetic markers i.e. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. In situ sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities. PMID:28977016

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description.

PubMed

Goldstein, Paul Z; DeSalle, Rob

2011-02-01

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.

Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus (Digenea): Species Differentiation Based On mtDNA (Barcode) and Partial LSU–rDNA Sequences

USGS Publications Warehouse

Bergmame, Laura; Huffman, Jane; Cole, Rebecca; Dayanandan, Selvadurai; Tkach, Vasyl; McLaughlin, J. Daniel

2011-01-01

Flukes belonging to Sphaeridiotrema are important parasites of waterfowl, and 2 morphologically similar species Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus, have been implicated in waterfowl mortality in North America. Cytochrome oxidase I (barcode region) and partial LSU-rDNA sequences from specimens of S. globulus and S. pseudoglobulus, obtained from naturally and experimentally infected hosts from New Jersey and Quebec, respectively, confirmed that these species were distinct. Barcode sequences of the 2 species differed at 92 of 590 nucleotide positions (15.6%) and the translated sequences differed by 13 amino acid residues. Partial LSU-rDNA sequences differed at 29 of 1,208 nucleotide positions (2.4%). Additional barcode sequences from specimens collected from waterfowl in Wisconsin and Minnesota and morphometric data obtained from specimens acquired along the north shore of Lake Superior revealed the presence of S. pseudoglobulus in these areas. Although morphometric data suggested the presence of S. globulus in the Lake Superior sample, it was not found among the specimens sequenced from Wisconsin or Minnesota.

Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus (Digenea): Species Differentiation Based on mtDNA (Barcode) and Partial LSUrDNA Sequences

USGS Publications Warehouse

Bergmame, L.; Huffman, J.; Cole, R.; Dayanandan, S.; Tkach, V.; McLaughlin, J.D.

2011-01-01

Flukes belonging to Sphaeridiotrema are important parasites of waterfowl, and 2 morphologically similar species Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus, have been implicated in waterfowl mortality in North America. Cytochrome oxidase I (barcode region) and partial LSU-rDNA sequences from specimens of S. globulus and S. pseudoglobulus, obtained from naturally and experimentally infected hosts from New Jersey and Quebec, respectively, confirmed that these species were distinct. Barcode sequences of the 2 species differed at 92 of 590 nucleotide positions (15.6%) and the translated sequences differed by 13 amino acid residues. Partial LSU-rDNA sequences differed at 29 of 1,208 nucleotide positions (2.4%). Additional barcode sequences from specimens collected from waterfowl in Wisconsin and Minnesota and morphometric data obtained from specimens acquired along the north shore of Lake Superior revealed the presence of S. pseudoglobulus in these areas. Although morphometric data suggested the presence of S. globulus in the Lake Superior sample, it was not found among the specimens sequenced from Wisconsin or Minnesota. ?? 2011 American Society of Parasitologists.

The Barcode of Life Data Portal: Bridging the Biodiversity Informatics Divide for DNA Barcoding

PubMed Central

Sarkar, Indra Neil; Trizna, Michael

2011-01-01

With the volume of molecular sequence data that is systematically being generated globally, there is a need for centralized resources for data exploration and analytics. DNA Barcode initiatives are on track to generate a compendium of molecular sequence–based signatures for identifying animals and plants. To date, the range of available data exploration and analytic tools to explore these data have only been available in a boutique form—often representing a frustrating hurdle for many researchers that may not necessarily have resources to install or implement algorithms described by the analytic community. The Barcode of Life Data Portal (BDP) is a first step towards integrating the latest biodiversity informatics innovations with molecular sequence data from DNA barcoding. Through establishment of community driven standards, based on discussion with the Data Analysis Working Group (DAWG) of the Consortium for the Barcode of Life (CBOL), the BDP provides an infrastructure for incorporation of existing and next-generation DNA barcode analytic applications in an open forum. PMID:21818249

DNA barcodes for ecology, evolution, and conservation.

PubMed

Kress, W John; García-Robledo, Carlos; Uriarte, Maria; Erickson, David L

2015-01-01

The use of DNA barcodes, which are short gene sequences taken from a standardized portion of the genome and used to identify species, is entering a new phase of application as more and more investigations employ these genetic markers to address questions relating to the ecology and evolution of natural systems. The suite of DNA barcode markers now applied to specific taxonomic groups of organisms are proving invaluable for understanding species boundaries, community ecology, functional trait evolution, trophic interactions, and the conservation of biodiversity. The application of next-generation sequencing (NGS) technology will greatly expand the versatility of DNA barcodes across the Tree of Life, habitats, and geographies as new methodologies are explored and developed. Published by Elsevier Ltd.

A k-mer-based barcode DNA classification methodology based on spectral representation and a neural gas network.

PubMed

Fiannaca, Antonino; La Rosa, Massimo; Rizzo, Riccardo; Urso, Alfonso

2015-07-01

In this paper, an alignment-free method for DNA barcode classification that is based on both a spectral representation and a neural gas network for unsupervised clustering is proposed. In the proposed methodology, distinctive words are identified from a spectral representation of DNA sequences. A taxonomic classification of the DNA sequence is then performed using the sequence signature, i.e., the smallest set of k-mers that can assign a DNA sequence to its proper taxonomic category. Experiments were then performed to compare our method with other supervised machine learning classification algorithms, such as support vector machine, random forest, ripper, naïve Bayes, ridor, and classification tree, which also consider short DNA sequence fragments of 200 and 300 base pairs (bp). The experimental tests were conducted over 10 real barcode datasets belonging to different animal species, which were provided by the on-line resource "Barcode of Life Database". The experimental results showed that our k-mer-based approach is directly comparable, in terms of accuracy, recall and precision metrics, with the other classifiers when considering full-length sequences. In addition, we demonstrate the robustness of our method when a classification is performed task with a set of short DNA sequences that were randomly extracted from the original data. For example, the proposed method can reach the accuracy of 64.8% at the species level with 200-bp fragments. Under the same conditions, the best other classifier (random forest) reaches the accuracy of 20.9%. Our results indicate that we obtained a clear improvement over the other classifiers for the study of short DNA barcode sequence fragments. Copyright © 2015 Elsevier B.V. All rights reserved.

The effectiveness of three regions in mitochondrial genome for aphid DNA barcoding: a case in Lachininae.

PubMed

Chen, Rui; Jiang, Li-Yun; Qiao, Ge-Xia

2012-01-01

The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding. Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in "best match" and 90.8% in "best close match") and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of "tag barcodes" is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the "barcoding overlap" can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the "best close match" technique. A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of "tag barcodes" can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.

Identification of Dendrobium species by a candidate DNA barcode sequence: the chloroplast psbA-trnH intergenic region.

PubMed

Yao, Hui; Song, Jing-Yuan; Ma, Xin-Ye; Liu, Chang; Li, Ying; Xu, Hong-Xi; Han, Jian-Ping; Duan, Li-Sheng; Chen, Shi-Lin

2009-05-01

DNA barcoding is a novel technology that uses a standard DNA sequence to facilitate species identification. Although a consensus has not been reached regarding which DNA sequences can be used as the best plant barcodes, the psbA-trnH spacer region has been tested extensively in recent years. In this study, we hypothesize that the psbA-trnH spacer regions are also effective barcodes for Dendrobium species. We have sequenced the chloroplast psbA-trnH intergenic spacers of 17 Dendrobium species to test this hypothesis. The sequences were found to be significantly different from those of other species, with percentages of variation ranging from 0.3 % to 2.3 % and an average of 1.2 %. In contrast, the intraspecific variation among the Dendrobium species studied ranged from 0 % to 0.1 %. The sequence difference between the psbA-trnH sequences of 17 Dendrobium species and one Bulbophyllum odoratissimum ranged from 2.0 % to 3.1 %, with an average of 2.5 %. Our results support the notion that the psbA-trnH intergenic spacer region could be used as a barcode to distinguish various Dendrobium species and to differentiate Dendrobium species from other adulterating species. Copyright Georg Thieme Verlag KG Stuttgart. New York.

Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.

PubMed

Xu, Chang; Nezami Ranjbar, Mohammad R; Wu, Zhong; DiCarlo, John; Wang, Yexun

2017-01-03

Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller.

DNA Barcoding in the Cycadales: Testing the Potential of Proposed Barcoding Markers for Species Identification of Cycads

PubMed Central

Sass, Chodon; Little, Damon P.; Stevenson, Dennis Wm.; Specht, Chelsea D.

2007-01-01

Barcodes are short segments of DNA that can be used to uniquely identify an unknown specimen to species, particularly when diagnostic morphological features are absent. These sequences could offer a new forensic tool in plant and animal conservation—especially for endangered species such as members of the Cycadales. Ideally, barcodes could be used to positively identify illegally obtained material even in cases where diagnostic features have been purposefully removed or to release confiscated organisms into the proper breeding population. In order to be useful, a DNA barcode sequence must not only easily PCR amplify with universal or near-universal reaction conditions and primers, but also contain enough variation to generate unique identifiers at either the species or population levels. Chloroplast regions suggested by the Plant Working Group of the Consortium for the Barcode of Life (CBoL), and two alternatives, the chloroplast psbA-trnH intergenic spacer and the nuclear ribosomal internal transcribed spacer (nrITS), were tested for their utility in generating unique identifiers for members of the Cycadales. Ease of amplification and sequence generation with universal primers and reaction conditions was determined for each of the seven proposed markers. While none of the proposed markers provided unique identifiers for all species tested, nrITS showed the most promise in terms of variability, although sequencing difficulties remain a drawback. We suggest a workflow for DNA barcoding, including database generation and management, which will ultimately be necessary if we are to succeed in establishing a universal DNA barcode for plants. PMID:17987130

ITS1: a DNA barcode better than ITS2 in eukaryotes?

PubMed

Wang, Xin-Cun; Liu, Chang; Huang, Liang; Bengtsson-Palme, Johan; Chen, Haimei; Zhang, Jian-Hui; Cai, Dayong; Li, Jian-Qin

2015-05-01

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. © 2014 John Wiley & Sons Ltd.

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

PubMed

Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

2016-06-20

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The problems and promise of DNA barcodes for species diagnosis of primate biomaterials

PubMed Central

Lorenz, Joseph G; Jackson, Whitney E; Beck, Jeanne C; Hanner, Robert

2005-01-01

The Integrated Primate Biomaterials and Information Resource (www.IPBIR.org) provides essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing DNA and RNA derived from primate cell cultures. The IPBIR uses mitochondrial cytochrome c oxidase subunit I sequences to verify the identity of samples for quality control purposes in the accession, cell culture, DNA extraction processes and prior to shipping to end users. As a result, IPBIR is accumulating a database of ‘DNA barcodes’ for many species of primates. However, this quality control process is complicated by taxon specific patterns of ‘universal primer’ failure, as well as the amplification or co-amplification of nuclear pseudogenes of mitochondrial origins. To overcome these difficulties, taxon specific primers have been developed, and reverse transcriptase PCR is utilized to exclude these extraneous sequences from amplification. DNA barcoding of primates has applications to conservation and law enforcement. Depositing barcode sequences in a public database, along with primer sequences, trace files and associated quality scores, makes this species identification technique widely accessible. Reference DNA barcode sequences should be derived from, and linked to, specimens of known provenance in web-accessible collections in order to validate this system of molecular diagnostics. PMID:16214744

Identification of Neoceratitis asiatica (Becker) (Diptera: Tephritidae) based on morphological characteristics and DNA barcode.

PubMed

Guo, Shaokun; He, Jia; Zhao, Zihua; Liu, Lijun; Gao, Liyuan; Wei, Shuhua; Guo, Xiaoyu; Zhang, Rong; Li, Zhihong

2017-12-12

Neoceratitis asiatica (Becker), which especially infests wolfberry (Lycium barbarum L.), could cause serious economic losses every year in China, especially to organic wolfberry production. In some important wolfberry plantings, it is difficult and time-consuming to rear the larvae or pupae to adults for morphological identification. Molecular identification based on DNA barcode is a solution to the problem. In this study, 15 samples were collected from Ningxia, China. Among them, five adults were identified according to their morphological characteristics. The utility of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) gene sequence as DNA barcode in distinguishing N. asiatica was evaluated by analysing Kimura 2-parameter distances and phylogenetic trees. There were significant differences between intra-specific and inter-specific genetic distances according to the barcoding gap analysis. The uncertain larval and pupal samples were within the same cluster as N. asiatica adults and formed sister cluster to N. cyanescens. A combination of morphological and molecular methods enabled accurate identification of N. asiatica. This is the first study using DNA barcode to identify N. asiatica and the obtained DNA sequences will be added to the DNA barcode database.

Ultra-barcoding in cacao (Theobroma spp.; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA.

PubMed

Kane, Nolan; Sveinsson, Saemundur; Dempewolf, Hannes; Yang, Ji Yong; Zhang, Dapeng; Engels, Johannes M M; Cronk, Quentin

2012-02-01

To reliably identify lineages below the species level such as subspecies or varieties, we propose an extension to DNA-barcoding using next-generation sequencing to produce whole organellar genomes and substantial nuclear ribosomal sequence. Because this method uses much longer versions of the traditional DNA-barcoding loci in the plastid and ribosomal DNA, we call our approach ultra-barcoding (UBC). We used high-throughput next-generation sequencing to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an individual of the related species T. grandiflorum, as well as an additional publicly available whole plastid genome of T. cacao. All individuals of T. cacao examined were uniquely distinguished, and evidence of reticulation and gene flow was observed. Sequence variation was observed in some of the canonical barcoding regions between species, but other regions of the chloroplast were more variable both within species and between species, as were ribosomal spacers. Furthermore, no single region provides the level of data available using the complete plastid genome and rDNA. Our data demonstrate that UBC is a viable, increasingly cost-effective approach for reliably distinguishing varieties and even individual genotypes of T. cacao. This approach shows great promise for applications where very closely related or interbreeding taxa must be distinguished.

Pay Attention to the Overlooked Cryptic Diversity in Existing Barcoding Data: the Case of Mollusca with Character-Based DNA Barcoding.

PubMed

Zou, Shanmei; Li, Qi

2016-06-01

With the global biodiversity crisis, DNA barcoding aims for fast species identification and cryptic species diversity revelation. For more than 10 years, large amounts of DNA barcode data have been accumulating in publicly available databases, most of which were conducted by distance or tree-building methods that have often been argued, especially for cryptic species revelation. In this context, overlooked cryptic diversity may exist in the available barcoding data. The character-based DNA barcoding, however, has a good chance for detecting the overlooked cryptic diversity. In this study, marine mollusk was as the ideal case for detecting the overlooked potential cryptic species from existing cytochrome c oxidase I (COI) sequences with character-based DNA barcode. A total of 1081 COI sequences of mollusks, belonging to 176 species of 25 families of Gastropoda, Cephalopoda, and Lamellibranchia, were conducted by character analysis. As a whole, the character-based barcoding results were consistent with previous distance and tree-building analysis for species discrimination. More importantly, quite a number of species analyzed were divided into distinct clades with unique diagnostical characters. Based on the concept of cryptic species revelation of character-based barcoding, these species divided into separate taxonomic groups might be potential cryptic species. The detection of the overlooked potential cryptic diversity proves that the character-based barcoding mode possesses more advantages of revealing cryptic biodiversity. With the development of DNA barcoding, making the best use of barcoding data is worthy of our attention for species conservation.

A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.

PubMed

Kress, W John; Erickson, David L

2007-06-06

A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.

The unholy trinity: taxonomy, species delimitation and DNA barcoding

PubMed Central

DeSalle, Rob; Egan, Mary G; Siddall, Mark

2005-01-01

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this ‘DNA barcoding’ initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the ‘DNA barcoding’ initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the ‘DNA barcoding’ initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings—Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of ‘DNA barcoding’. PMID:16214748

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case.

PubMed

Avin, Farhat A; Subha, Bhassu; Tan, Yee-Shin; Braukmann, Thomas W A; Vikineswary, Sabaratnam; Hebert, Paul D N

2017-09-01

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5' terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus , the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5' region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus . Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

DNA barcoding of five common stored-product pest species of genus Cryptolestes (Coleoptera: Laemophloeidae).

PubMed

Wang, Y J; Li, Z H; Zhang, S F; Varadínová, Z; Jiang, F; Kučerová, Z; Stejskal, V; Opit, G; Cao, Y; Li, F J

2014-10-01

Several species of the genus Cryptolestes Ganglbauer, 1899 (Coleoptera: Laemophloeidae) are commonly found in stored products. In this study, five species of Cryptolestes, with almost worldwide distribution, were obtained from laboratories in China, Czech Republic and the USA: Cryptolestes ferrugineus (Stephens, 1831), Cryptolestes pusillus (Schönherr, 1817), Cryptolestes turcicus (Grouvelle, 1876), Cryptolestes pusilloides (Steel & Howe, 1952) and Cryptolestes capensis (Waltl, 1834). Molecular identification based on a 658 bp fragment from the mitochondrial DNA cytochrome c oxidase subunit I (COI) was adopted to overcome some problems of morphological identification of Cryptolestes species. The utility of COI sequences as DNA barcodes in discriminating the five Cryptolestes species was evaluated on adults and larvae by analysing Kimura 2-parameter distances, phylogenetic tree and haplotype networks. The results showed that molecular approaches based on DNA barcodes were able to accurately identify these species. This is the first study using DNA barcoding to identify Cryptolestes species and the gathered DNA sequences will complement the biological barcode database.

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada.

PubMed

Kuzmina, Maria L; Braukmann, Thomas W A; Fazekas, Aron J; Graham, Sean W; Dewaard, Stephanie L; Rodrigues, Anuar; Bennett, Bruce A; Dickinson, Timothy A; Saarela, Jeffery M; Catling, Paul M; Newmaster, Steven G; Percy, Diana M; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R; Zakharov, Evgeny V; Hebert, Paul D N

2017-12-01

Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa.

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada1

PubMed Central

Kuzmina, Maria L.; Braukmann, Thomas W. A.; Fazekas, Aron J.; Graham, Sean W.; Dewaard, Stephanie L.; Rodrigues, Anuar; Bennett, Bruce A.; Dickinson, Timothy A.; Saarela, Jeffery M.; Catling, Paul M.; Newmaster, Steven G.; Percy, Diana M.; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P.; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

Premise of the study: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Methods: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Results: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Discussion: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa. PMID:29299394

Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies.

PubMed

Thaler, David S; Stoeckle, Mark Y

2016-10-01

DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648-bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein-encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most - possibly all - synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well-curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.

DNA Barcoding the Geometrid Fauna of Bavaria (Lepidoptera): Successes, Surprises, and Questions

PubMed Central

Hausmann, Axel; Haszprunar, Gerhard; Hebert, Paul D. N.

2011-01-01

Background The State of Bavaria is involved in a research program that will lead to the construction of a DNA barcode library for all animal species within its territorial boundaries. The present study provides a comprehensive DNA barcode library for the Geometridae, one of the most diverse of insect families. Methodology/Principal Findings This study reports DNA barcodes for 400 Bavarian geometrid species, 98 per cent of the known fauna, and approximately one per cent of all Bavarian animal species. Although 98.5% of these species possess diagnostic barcode sequences in Bavaria, records from neighbouring countries suggest that species-level resolution may be compromised in up to 3.5% of cases. All taxa which apparently share barcodes are discussed in detail. One case of modest divergence (1.4%) revealed a species overlooked by the current taxonomic system: Eupithecia goossensiata Mabille, 1869 stat.n. is raised from synonymy with Eupithecia absinthiata (Clerck, 1759) to species rank. Deep intraspecific sequence divergences (>2%) were detected in 20 traditionally recognized species. Conclusions/Significance The study emphasizes the effectiveness of DNA barcoding as a tool for monitoring biodiversity. Open access is provided to a data set that includes records for 1,395 geometrid specimens (331 species) from Bavaria, with 69 additional species from neighbouring regions. Taxa with deep intraspecific sequence divergences are undergoing more detailed analysis to ascertain if they represent cases of cryptic diversity. PMID:21423340

Opening the treasure chest: A DNA-barcoding primer set for most higher taxa of Central European birds and mammals from museum collections

PubMed Central

Schäffer, Sylvia; Zachos, Frank E.

2017-01-01

DNA-barcoding is a rapidly developing method for efficiently identifying samples to species level by means of short standard DNA sequences. However, reliable species assignment requires the availability of a comprehensive DNA barcode reference library, and hence numerous initiatives aim at generating such barcode databases for particular taxa or geographic regions. Historical museum collections represent a potentially invaluable source for the DNA-barcoding of many taxa. This is particularly true for birds and mammals, for which collecting fresh (voucher) material is often very difficult to (nearly) impossible due to the special animal welfare and conservation regulations that apply to vertebrates in general, and birds and mammals in particular. Moreover, even great efforts might not guarantee sufficiently complete sampling of fresh material in a short period of time. DNA extracted from historical samples is usually degraded, such that only short fragments can be amplified, rendering the recovery of the barcoding region as a single fragment impossible. Here, we present a new set of primers that allows the efficient amplification and sequencing of the entire barcoding region in most higher taxa of Central European birds and mammals in six overlapping fragments, thus greatly increasing the value of historical museum collections for generating DNA barcode reference libraries. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens. PMID:28358863

Opening the treasure chest: A DNA-barcoding primer set for most higher taxa of Central European birds and mammals from museum collections.

PubMed

Schäffer, Sylvia; Zachos, Frank E; Koblmüller, Stephan

2017-01-01

DNA-barcoding is a rapidly developing method for efficiently identifying samples to species level by means of short standard DNA sequences. However, reliable species assignment requires the availability of a comprehensive DNA barcode reference library, and hence numerous initiatives aim at generating such barcode databases for particular taxa or geographic regions. Historical museum collections represent a potentially invaluable source for the DNA-barcoding of many taxa. This is particularly true for birds and mammals, for which collecting fresh (voucher) material is often very difficult to (nearly) impossible due to the special animal welfare and conservation regulations that apply to vertebrates in general, and birds and mammals in particular. Moreover, even great efforts might not guarantee sufficiently complete sampling of fresh material in a short period of time. DNA extracted from historical samples is usually degraded, such that only short fragments can be amplified, rendering the recovery of the barcoding region as a single fragment impossible. Here, we present a new set of primers that allows the efficient amplification and sequencing of the entire barcoding region in most higher taxa of Central European birds and mammals in six overlapping fragments, thus greatly increasing the value of historical museum collections for generating DNA barcode reference libraries. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens.

DNA Barcoding of the Endangered Aquilaria (Thymelaeaceae) and Its Application in Species Authentication of Agarwood Products Traded in the Market

PubMed Central

Lee, Shiou Yih; Ng, Wei Lun; Mahat, Mohd Noor; Nazre, Mohd; Mohamed, Rozi

2016-01-01

The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication. PMID:27128309

Highlights of DNA Barcoding in identification of salient microorganisms like fungi.

PubMed

Dulla, E L; Kathera, C; Gurijala, H K; Mallakuntla, T R; Srinivasan, P; Prasad, V; Mopati, R D; Jasti, P K

2016-12-01

Fungi, the second largest kingdom of eukaryotic life, are diverse and widespread. Fungi play a distinctive role in the production of different products on industrial scale, like fungal enzymes, antibiotics, fermented foods, etc., to give storage stability and improved health to meet major global challenges. To utilize algae perfectly for human needs, and to pave the way for getting a healthy relationship with fungi, it is important to identify them in a quick and robust manner with molecular-based identification system. So, there is a technique that aims to provide a well-organized method for species level identifications and to contribute powerfully to taxonomic and biodiversity research is DNA Barcoding. DNA Barcoding is generally achieved by the retrieval of a short DNA sequence - the 'barcode' - from a standard part of the genome and that barcode is then compared with a library of reference barcode sequences derived from individuals of known identity for identification. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

DNA barcoding the floras of biodiversity hotspots.

PubMed

Lahaye, Renaud; van der Bank, Michelle; Bogarin, Diego; Warner, Jorge; Pupulin, Franco; Gigot, Guillaume; Maurin, Olivier; Duthoit, Sylvie; Barraclough, Timothy G; Savolainen, Vincent

2008-02-26

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a "DNA barcoding gap" is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes.

DNA barcoding the floras of biodiversity hotspots

PubMed Central

Lahaye, Renaud; van der Bank, Michelle; Bogarin, Diego; Warner, Jorge; Pupulin, Franco; Gigot, Guillaume; Maurin, Olivier; Duthoit, Sylvie; Barraclough, Timothy G.; Savolainen, Vincent

2008-01-01

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a “DNA barcoding gap” is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes. PMID:18258745

Beyond the Colours: Discovering Hidden Diversity in the Nymphalidae of the Yucatan Peninsula in Mexico through DNA Barcoding

PubMed Central

Prado, Blanca R.; Pozo, Carmen; Valdez-Moreno, Martha; Hebert, Paul D. N.

2011-01-01

Background Recent studies have demonstrated the utility of DNA barcoding in the discovery of overlooked species and in the connection of immature and adult stages. In this study, we use DNA barcoding to examine diversity patterns in 121 species of Nymphalidae from the Yucatan Peninsula in Mexico. Our results suggest the presence of cryptic species in 8 of these 121 taxa. As well, the reference database derived from the analysis of adult specimens allowed the identification of nymphalid caterpillars providing new details on host plant use. Methodology/Principal Findings We gathered DNA barcode sequences from 857 adult Nymphalidae representing 121 different species. This total includes four species (Adelpha iphiclus, Adelpha malea, Hamadryas iphtime and Taygetis laches) that were initially overlooked because of their close morphological similarity to other species. The barcode results showed that each of the 121 species possessed a diagnostic array of barcode sequences. In addition, there was evidence of cryptic taxa; seven species included two barcode clusters showing more than 2% sequence divergence while one species included three clusters. All 71 nymphalid caterpillars were identified to a species level by their sequence congruence to adult sequences. These caterpillars represented 16 species, and included Hamadryas julitta, an endemic species from the Yucatan Peninsula whose larval stages and host plant (Dalechampia schottii, also endemic to the Yucatan Peninsula) were previously unknown. Conclusions/Significance This investigation has revealed overlooked species in a well-studied museum collection of nymphalid butterflies and suggests that there is a substantial incidence of cryptic species that await full characterization. The utility of barcoding in the rapid identification of caterpillars also promises to accelerate the assembly of information on life histories, a particularly important advance for hyperdiverse tropical insect assemblages. PMID:22132140

Reproducibility of Illumina platform deep sequencing errors allows accurate determination of DNA barcodes in cells.

PubMed

Beltman, Joost B; Urbanus, Jos; Velds, Arno; van Rooij, Nienke; Rohr, Jan C; Naik, Shalin H; Schumacher, Ton N

2016-04-02

Next generation sequencing (NGS) of amplified DNA is a powerful tool to describe genetic heterogeneity within cell populations that can both be used to investigate the clonal structure of cell populations and to perform genetic lineage tracing. For applications in which both abundant and rare sequences are biologically relevant, the relatively high error rate of NGS techniques complicates data analysis, as it is difficult to distinguish rare true sequences from spurious sequences that are generated by PCR or sequencing errors. This issue, for instance, applies to cellular barcoding strategies that aim to follow the amount and type of offspring of single cells, by supplying these with unique heritable DNA tags. Here, we use genetic barcoding data from the Illumina HiSeq platform to show that straightforward read threshold-based filtering of data is typically insufficient to filter out spurious barcodes. Importantly, we demonstrate that specific sequencing errors occur at an approximately constant rate across different samples that are sequenced in parallel. We exploit this observation by developing a novel approach to filter out spurious sequences. Application of our new method demonstrates its value in the identification of true sequences amongst spurious sequences in biological data sets.

A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region

PubMed Central

Kress, W. John; Erickson, David L.

2007-01-01

Background A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Methodology/Principal Findings Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. Conclusions/Significance A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination. PMID:17551588

DNA barcoding as an aid for species identification in austral black flies (Insecta: Diptera: Simuliidae).

PubMed

Hernández-Triana, Luis M; Montes De Oca, Fernanda; Prosser, Sean W J; Hebert, Paul D N; Gregory, T Ryan; McMurtrie, Shelley

2017-04-01

In this paper, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies in the austral region was assessed. Twenty-eight morphospecies were analyzed: eight of the genus Austrosimulium (four species in the subgenus Austrosimulium s. str., three species in the subgenus Novaustrosimulium, and one species unassigned to subgenus), two of the genus Cnesia, eight of Gigantodax, three of Paracnephia, one of Paraustrosimulium, and six of Simulium (subgenera Morops, Nevermannia, and Pternaspatha). The neighbour-joining tree derived from the DNA barcode sequences grouped most specimens according to species or species groups recognized by morphotaxonomic studies. Intraspecific sequence divergences within morphologically distinct species ranged from 0% to 1.8%, while higher divergences (2%-4.2%) in certain species suggested the presence of cryptic diversity. The existence of well-defined groups within S. simile revealed the likely inclusion of cryptic diversity. DNA barcodes also showed that specimens identified as C. dissimilis, C. nr. pussilla, and C. ornata might be conspecific, suggesting possible synonymy. DNA barcoding combined with a sound morphotaxonomic framework would provide an effective approach for the identification of black flies in the region.

Ten years of barcoding at the African Centre for DNA Barcoding.

PubMed

Bezeng, B S; Davies, T J; Daru, B H; Kabongo, R M; Maurin, O; Yessoufou, K; van der Bank, H; van der Bank, M

2017-07-01

The African Centre for DNA Barcoding (ACDB) was established in 2005 as part of a global initiative to accurately and rapidly survey biodiversity using short DNA sequences. The mitochondrial cytochrome c oxidase 1 gene (CO1) was rapidly adopted as the de facto barcode for animals. Following the evaluation of several candidate loci for plants, the Plant Working Group of the Consortium for the Barcoding of Life in 2009 recommended that two plastid genes, rbcLa and matK, be adopted as core DNA barcodes for terrestrial plants. To date, numerous studies continue to test the discriminatory power of these markers across various plant lineages. Over the past decade, we at the ACDB have used these core DNA barcodes to generate a barcode library for southern Africa. To date, the ACDB has contributed more than 21 000 plant barcodes and over 3000 CO1 barcodes for animals to the Barcode of Life Database (BOLD). Building upon this effort, we at the ACDB have addressed questions related to community assembly, biogeography, phylogenetic diversification, and invasion biology. Collectively, our work demonstrates the diverse applications of DNA barcoding in ecology, systematics, evolutionary biology, and conservation.

The HTS barcode checker pipeline, a tool for automated detection of illegally traded species from high-throughput sequencing data.

PubMed

Lammers, Youri; Peelen, Tamara; Vos, Rutger A; Gravendeel, Barbara

2014-02-06

Mixtures of internationally traded organic substances can contain parts of species protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). These mixtures often raise the suspicion of border control and customs offices, which can lead to confiscation, for example in the case of Traditional Chinese medicines (TCMs). High-throughput sequencing of DNA barcoding markers obtained from such samples provides insight into species constituents of mixtures, but manual cross-referencing of results against the CITES appendices is labor intensive. Matching DNA barcodes against NCBI GenBank using BLAST may yield misleading results both as false positives, due to incorrectly annotated sequences, and false negatives, due to spurious taxonomic re-assignment. Incongruence between the taxonomies of CITES and NCBI GenBank can result in erroneous estimates of illegal trade. The HTS barcode checker pipeline is an application for automated processing of sets of 'next generation' barcode sequences to determine whether these contain DNA barcodes obtained from species listed on the CITES appendices. This analytical pipeline builds upon and extends existing open-source applications for BLAST matching against the NCBI GenBank reference database and for taxonomic name reconciliation. In a single operation, reads are converted into taxonomic identifications matched with names on the CITES appendices. By inclusion of a blacklist and additional names databases, the HTS barcode checker pipeline prevents false positives and resolves taxonomic heterogeneity. The HTS barcode checker pipeline can detect and correctly identify DNA barcodes of CITES-protected species from reads obtained from TCM samples in just a few minutes. The pipeline facilitates and improves molecular monitoring of trade in endangered species, and can aid in safeguarding these species from extinction in the wild. The HTS barcode checker pipeline is available at https://github.com/naturalis/HTS-barcode-checker.

The HTS barcode checker pipeline, a tool for automated detection of illegally traded species from high-throughput sequencing data

PubMed Central

2014-01-01

Background Mixtures of internationally traded organic substances can contain parts of species protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). These mixtures often raise the suspicion of border control and customs offices, which can lead to confiscation, for example in the case of Traditional Chinese medicines (TCMs). High-throughput sequencing of DNA barcoding markers obtained from such samples provides insight into species constituents of mixtures, but manual cross-referencing of results against the CITES appendices is labor intensive. Matching DNA barcodes against NCBI GenBank using BLAST may yield misleading results both as false positives, due to incorrectly annotated sequences, and false negatives, due to spurious taxonomic re-assignment. Incongruence between the taxonomies of CITES and NCBI GenBank can result in erroneous estimates of illegal trade. Results The HTS barcode checker pipeline is an application for automated processing of sets of 'next generation’ barcode sequences to determine whether these contain DNA barcodes obtained from species listed on the CITES appendices. This analytical pipeline builds upon and extends existing open-source applications for BLAST matching against the NCBI GenBank reference database and for taxonomic name reconciliation. In a single operation, reads are converted into taxonomic identifications matched with names on the CITES appendices. By inclusion of a blacklist and additional names databases, the HTS barcode checker pipeline prevents false positives and resolves taxonomic heterogeneity. Conclusions The HTS barcode checker pipeline can detect and correctly identify DNA barcodes of CITES-protected species from reads obtained from TCM samples in just a few minutes. The pipeline facilitates and improves molecular monitoring of trade in endangered species, and can aid in safeguarding these species from extinction in the wild. The HTS barcode checker pipeline is available at https://github.com/naturalis/HTS-barcode-checker. PMID:24502833

DNA barcoding commercially important fish species of Turkey.

PubMed

Keskın, Emre; Atar, Hasan H

2013-09-01

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654-bp-long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2-parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour-joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries. © 2013 John Wiley & Sons Ltd.

Four years of DNA barcoding: current advances and prospects.

PubMed

Frézal, Lise; Leblois, Raphael

2008-09-01

Research using cytochrome c oxidase barcoding techniques on zoological specimens was initiated by Hebert et al. [Hebert, P.D.N., Ratnasingham, S., deWaard, J.R., 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270, S96-S99]. By March 2004, the Consortium for the Barcode of Life started to promote the use of a standardized DNA barcoding approach, consisting of identifying a specimen as belonging to a certain animal species based on a single universal marker: the DNA barcode sequence. Over the last 4 years, this approach has become increasingly popular and advances as well as limitations have clearly emerged as increasing amounts of organisms have been studied. Our purpose is to briefly expose DNA Barcode of Life principles, pros and cons, relevance and universality. The initially proposed Barcode of life framework has greatly evolved, giving rise to a flexible description of DNA barcoding and a larger range of applications.

Exploring the utility of DNA barcoding in species delimitation of Polypedilum (Tripodura) non-biting midges (Diptera: Chironomidae).

PubMed

Song, Chao; Wang, Qian; Zhang, Ruilei; Sun, Bingjiao; Wang, Xinhua

2016-02-16

In this study, we tested the utility of the mitochondrial gene cytochrome c oxidase subunit 1 (CO1) as the barcode region to deal with taxonomical problems of Polypedilum (Tripodura) non-biting midges (Diptera: Chironomidae). The 114 DNA barcodes representing 27 morphospecies are divided into 33 well separated clusters based on both Neighbor Joining and Maximum Likelihood methods. DNA barcodes revealed an 82% success rate in matching with morphospecies. The selected DNA barcode data support 37-64 operational taxonomic units (OTUs) based on the methods of Automatic Barcode Gap Discovery (ABGD) and Poisson Tree Process (PTP). Furthermore, a priori species based on consistent phenotypic variations were attested by molecular analysis, and a taxonomical misidentification of barcode sequences from GenBank was found. We could not observe a distinct barcode gap but an overlap ranged from 9-12%. Our results supported DNA barcoding as an ideal method to detect cryptic species, delimit sibling species, and associate different life stages in non-biting midges.

DNA Barcoding of Marine Metazoa

NASA Astrophysics Data System (ADS)

Bucklin, Ann; Steinke, Dirk; Blanco-Bercial, Leocadio

2011-01-01

More than 230,000 known species representing 31 metazoan phyla populate the world's oceans. Perhaps another 1,000,000 or more species remain to be discovered. There is reason for concern that species extinctions may outpace discovery, especially in diverse and endangered marine habitats such as coral reefs. DNA barcodes (i.e., short DNA sequences for species recognition and discrimination) are useful tools to accelerate species-level analysis of marine biodiversity and to facilitate conservation efforts. This review focuses on the usual barcode region for metazoans: a ˜648 base-pair region of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Barcodes have also been used for population genetic and phylogeographic analysis, identification of prey in gut contents, detection of invasive species, forensics, and seafood safety. More controversially, barcodes have been used to delimit species boundaries, reveal cryptic species, and discover new species. Emerging frontiers are the use of barcodes for rapid and increasingly automated biodiversity assessment by high-throughput sequencing, including environmental barcoding and the use of barcodes to detect species for which formal identification or scientific naming may never be possible.

Molecular Approach to the Identification of Fish in the South China Sea

PubMed Central

Zhang, Junbin; Hanner, Robert

2012-01-01

Background DNA barcoding is one means of establishing a rapid, accurate, and cost-effective system for the identification of species. It involves the use of short, standard gene targets to create sequence profiles of known species against sequences of unknowns that can be matched and subsequently identified. The Fish Barcode of Life (FISH-BOL) campaign has the primary goal of gathering DNA barcode records for all the world's fish species. As a contribution to FISH-BOL, we examined the degree to which DNA barcoding can discriminate marine fishes from the South China Sea. Methodology/Principal Findings DNA barcodes of cytochrome oxidase subunit I (COI) were characterized using 1336 specimens that belong to 242 species fishes from the South China Sea. All specimen provenance data (including digital specimen images and geospatial coordinates of collection localities) and collateral sequence information were assembled using Barcode of Life Data System (BOLD; www.barcodinglife.org). Small intraspecific and large interspecific differences create distinct genetic boundaries among most species. In addition, the efficiency of two mitochondrial genes, 16S rRNA (16S) and cytochrome b (cytb), and one nuclear ribosomal gene, 18S rRNA (18S), was also evaluated for a few select groups of species. Conclusions/Significance The present study provides evidence for the effectiveness of DNA barcoding as a tool for monitoring marine biodiversity. Open access data of fishes from the South China Sea can benefit relative applications in ecology and taxonomy. PMID:22363454

Molecular Authentication of the Traditional Medicinal Plant "Lakshman Booti" (Smithia conferta Sm.) and Its Adulterants through DNA Barcoding.

PubMed

Umdale, Suraj D; Kshirsagar, Parthraj R; Lekhak, Manoj M; Gaikwad, Nikhil B

2017-07-01

Smithia conferta Sm. is an annual herb widely used in Indian traditional medical practice and commonly known as "Lakshman booti" in Sanskrit. Morphological resemblance among the species of genus Smithia Aiton . leads to inaccurate identification and adulteration. This causes inconsistent therapeutic effects and also affects the quality of herbal medicine. This study aimed to generate potential barcode for authentication of S. conferta and its adulterants through DNA barcoding technique. Genomic DNA extracted from S. conferta and its adulterants was used as templates for polymerase chain reaction amplification of the barcoding regions. The amplicons were directed for sequencing, and species identification was conducted using BLASTn and unweighted pair-group method with arithmetic mean trees. In addition, the secondary structures of internal transcribed spacer (ITS) 2 region were predicted. The nucleotide sequence of ITS provides species-specific single nucleotide polymorphisms and sequence divergence (22%) than psb A- trn H (10.9%) and rbc L (3.1%) sequences. The ITS barcode indicates that S. conferta and Smithia sensitiva are closely related compared to other species. ITS is the most applicable barcode for molecular authentication of S. conferta , and further chloroplast barcodes should be tested for phylogenetic analysis of genus Smithia. The present investigation is the first effort of utilization of DNA barcode for molecular authentication of S. conferta and its adulterants. Also, this study expanded the application of the ITS2 sequence data in the authentication. The ITS has been proved as a potential and reliable candidate barcode for the authentication of S. conferta . Abbreviations used: BLASTn: Basic Local Alignment Search Tool for Nucleotide; MEGA: Molecular Evolutionary Genetic Analysis; EMBL: European Molecular Biology Laboratory; psb A- trn H: Photosystem II protein D1- stuctural RNA: His tRNA gene; rbcL: Ribulose 1,5 bi-phosphate carboxylase/oxygenase large subunit gene.

BOKP: A DNA Barcode Reference Library for Monitoring Herbal Drugs in the Korean Pharmacopeia

PubMed Central

Liu, Jinxin; Shi, Linchun; Song, Jingyuan; Sun, Wei; Han, Jianping; Liu, Xia; Hou, Dianyun; Yao, Hui; Li, Mingyue; Chen, Shilin

2017-01-01

Herbal drug authentication is an important task in traditional medicine; however, it is challenged by the limitations of traditional authentication methods and the lack of trained experts. DNA barcoding is conspicuous in almost all areas of the biological sciences and has already been added to the British pharmacopeia and Chinese pharmacopeia for routine herbal drug authentication. However, DNA barcoding for the Korean pharmacopeia still requires significant improvements. Here, we present a DNA barcode reference library for herbal drugs in the Korean pharmacopeia and developed a species identification engine named KP-IDE to facilitate the adoption of this DNA reference library for the herbal drug authentication. Using taxonomy records, specimen records, sequence records, and reference records, KP-IDE can identify an unknown specimen. Currently, there are 6,777 taxonomy records, 1,054 specimen records, 30,744 sequence records (ITS2 and psbA-trnH) and 285 reference records. Moreover, 27 herbal drug materials were collected from the Seoul Yangnyeongsi herbal medicine market to give an example for real herbal drugs authentications. Our study demonstrates the prospects of the DNA barcode reference library for the Korean pharmacopeia and provides future directions for the use of DNA barcoding for authenticating herbal drugs listed in other modern pharmacopeias. PMID:29326593

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

PubMed

Yang, Jun-Bo; Wang, Yi-Ping; Möller, Michael; Gao, Lian-Ming; Wu, Ding

2012-03-01

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. © 2011 Blackwell Publishing Ltd.

Advances in DNA metabarcoding for food and wildlife forensic species identification.

PubMed

Staats, Martijn; Arulandhu, Alfred J; Gravendeel, Barbara; Holst-Jensen, Arne; Scholtens, Ingrid; Peelen, Tamara; Prins, Theo W; Kok, Esther

2016-07-01

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders' needs.

DNA barcoding insect–host plant associations

PubMed Central

Jurado-Rivera, José A.; Vogler, Alfried P.; Reid, Chris A.M.; Petitpierre, Eduard; Gómez-Zurita, Jesús

2008-01-01

Short-sequence fragments (‘DNA barcodes’) used widely for plant identification and inventorying remain to be applied to complex biological problems. Host–herbivore interactions are fundamental to coevolutionary relationships of a large proportion of species on the Earth, but their study is frequently hampered by limited or unreliable host records. Here we demonstrate that DNA barcodes can greatly improve this situation as they (i) provide a secure identification of host plant species and (ii) establish the authenticity of the trophic association. Host plants of leaf beetles (subfamily Chrysomelinae) from Australia were identified using the chloroplast trnL(UAA) intron as barcode amplified from beetle DNA extracts. Sequence similarity and phylogenetic analyses provided precise identifications of each host species at tribal, generic and specific levels, depending on the available database coverage in various plant lineages. The 76 species of Chrysomelinae included—more than 10 per cent of the known Australian fauna—feed on 13 plant families, with preference for Australian radiations of Myrtaceae (eucalypts) and Fabaceae (acacias). Phylogenetic analysis of beetles shows general conservation of host association but with rare host shifts between distant plant lineages, including a few cases where barcodes supported two phylogenetically distant host plants. The study demonstrates that plant barcoding is already feasible with the current publicly available data. By sequencing plant barcodes directly from DNA extractions made from herbivorous beetles, strong physical evidence for the host association is provided. Thus, molecular identification using short DNA fragments brings together the detection of species and the analysis of their interactions. PMID:19004756

DNA Barcoding in Fragaria L. (Strawberry) Species

USDA-ARS?s Scientific Manuscript database

DNA barcoding for species identification using a short DNA sequence has been successful in animals due to rapid mutation rates of the mitochondrial genome where the animal DNA barocode, cytochrome c oxidase 1 gene is located. The chloroplast PsbA-trnH spacer and the nuclear ribosomal internal transc...

Scaling up discovery of hidden diversity in fungi: impacts of barcoding approaches.

PubMed

Yahr, Rebecca; Schoch, Conrad L; Dentinger, Bryn T M

2016-09-05

The fungal kingdom is a hyperdiverse group of multicellular eukaryotes with profound impacts on human society and ecosystem function. The challenge of documenting and describing fungal diversity is exacerbated by their typically cryptic nature, their ability to produce seemingly unrelated morphologies from a single individual and their similarity in appearance to distantly related taxa. This multiplicity of hurdles resulted in the early adoption of DNA-based comparisons to study fungal diversity, including linking curated DNA sequence data to expertly identified voucher specimens. DNA-barcoding approaches in fungi were first applied in specimen-based studies for identification and discovery of taxonomic diversity, but are now widely deployed for community characterization based on sequencing of environmental samples. Collectively, fungal barcoding approaches have yielded important advances across biological scales and research applications, from taxonomic, ecological, industrial and health perspectives. A major outstanding issue is the growing problem of 'sequences without names' that are somewhat uncoupled from the traditional framework of fungal classification based on morphology and preserved specimens. This review summarizes some of the most significant impacts of fungal barcoding, its limitations, and progress towards the challenge of effective utilization of the exponentially growing volume of data gathered from high-throughput sequencing technologies.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

FBIS: A regional DNA barcode archival & analysis system for Indian fishes.

PubMed

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. The database is available for free at http://mail.nbfgr.res.in/fbis/

Towards a comprehensive barcode library for arctic life - Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada

PubMed Central

2009-01-01

Background This study reports progress in assembling a DNA barcode reference library for Ephemeroptera, Plecoptera, and Trichoptera ("EPTs") from a Canadian subarctic site, which is the focus of a comprehensive biodiversity inventory using DNA barcoding. These three groups of aquatic insects exhibit a moderate level of species diversity, making them ideal for testing the feasibility of DNA barcoding for routine biotic surveys. We explore the correlation between the morphological species delineations, DNA barcode-based haplotype clusters delimited by a sequence threshold (2%), and a threshold-free approach to biodiversity quantification--phylogenetic diversity. Results A DNA barcode reference library is built for 112 EPT species for the focal region, consisting of 2277 COI sequences. Close correspondence was found between EPT morphospecies and haplotype clusters as designated using a standard threshold value. Similarly, the shapes of taxon accumulation curves based upon haplotype clusters were very similar to those generated using phylogenetic diversity accumulation curves, but were much more computationally efficient. Conclusion The results of this study will facilitate other lines of research on northern EPTs and also bode well for rapidly conducting initial biodiversity assessments in unknown EPT faunas. PMID:20003245

Does a global DNA barcoding gap exist in Annelida?

PubMed

Kvist, Sebastian

2016-05-01

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications - false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for "Polychaeta", "Oligochaeta", and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 ("Polychaeta"), 3639 ("Oligochaeta"), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

Noise reduction in single time frame optical DNA maps

PubMed Central

Müller, Vilhelm; Westerlund, Fredrik

2017-01-01

In optical DNA mapping technologies sequence-specific intensity variations (DNA barcodes) along stretched and stained DNA molecules are produced. These “fingerprints” of the underlying DNA sequence have a resolution of the order one kilobasepairs and the stretching of the DNA molecules are performed by surface adsorption or nano-channel setups. A post-processing challenge for nano-channel based methods, due to local and global random movement of the DNA molecule during imaging, is how to align different time frames in order to produce reproducible time-averaged DNA barcodes. The current solutions to this challenge are computationally rather slow. With high-throughput applications in mind, we here introduce a parameter-free method for filtering a single time frame noisy barcode (snap-shot optical map), measured in a fraction of a second. By using only a single time frame barcode we circumvent the need for post-processing alignment. We demonstrate that our method is successful at providing filtered barcodes which are less noisy and more similar to time averaged barcodes. The method is based on the application of a low-pass filter on a single noisy barcode using the width of the Point Spread Function of the system as a unique, and known, filtering parameter. We find that after applying our method, the Pearson correlation coefficient (a real number in the range from -1 to 1) between the single time-frame barcode and the time average of the aligned kymograph increases significantly, roughly by 0.2 on average. By comparing to a database of more than 3000 theoretical plasmid barcodes we show that the capabilities to identify plasmids is improved by filtering single time-frame barcodes compared to the unfiltered analogues. Since snap-shot experiments and computational time using our method both are less than a second, this study opens up for high throughput optical DNA mapping with improved reproducibility. PMID:28640821

DNA barcoding the native flowering plants and conifers of Wales.

PubMed

de Vere, Natasha; Rich, Tim C G; Ford, Col R; Trinder, Sarah A; Long, Charlotte; Moore, Chris W; Satterthwaite, Danielle; Davies, Helena; Allainguillaume, Joel; Ronca, Sandra; Tatarinova, Tatiana; Garbett, Hannah; Walker, Kevin; Wilkinson, Mike J

2012-01-01

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification.

Evaluating ethanol-based sample preservation to facilitate use of DNA barcoding in routine freshwater biomonitoring programs using benthic macroinvertebrates.

PubMed

Stein, Eric D; White, Bryan P; Mazor, Raphael D; Miller, Peter E; Pilgrim, Erik M

2013-01-01

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93-99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.

Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

PubMed Central

Stein, Eric D.; White, Bryan P.; Mazor, Raphael D.; Miller, Peter E.; Pilgrim, Erik M.

2013-01-01

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93–99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity. PMID:23308097

DNA Barcoding the Native Flowering Plants and Conifers of Wales

PubMed Central

de Vere, Natasha; Rich, Tim C. G.; Ford, Col R.; Trinder, Sarah A.; Long, Charlotte; Moore, Chris W.; Satterthwaite, Danielle; Davies, Helena; Allainguillaume, Joel; Ronca, Sandra; Tatarinova, Tatiana; Garbett, Hannah; Walker, Kevin; Wilkinson, Mike J.

2012-01-01

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification. PMID:22701588

Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques.

PubMed

Xu, Chao; Dong, Wenpan; Shi, Shuo; Cheng, Tao; Li, Changhao; Liu, Yanlei; Wu, Ping; Wu, Hongkun; Gao, Peng; Zhou, Shiliang

2015-11-01

A well-covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self-primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four-sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400-500 bp without bringing or inducing any sequence errors. About one-third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well-covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA-labelled next-generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality. © 2015 John Wiley & Sons Ltd.

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

PubMed

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

Multiplex single-molecule interaction profiling of DNA barcoded proteins

PubMed Central

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

Forensic identification of CITES protected slimming cactus (Hoodia) using DNA barcoding.

PubMed

Gathier, Gerard; van der Niet, Timotheus; Peelen, Tamara; van Vugt, Rogier R; Eurlings, Marcel C M; Gravendeel, Barbara

2013-11-01

Slimming cactus (Hoodia), found only in southwestern Africa, is a well-known herbal product for losing weight. Consequently, Hoodia extracts are sought-after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA-trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI-MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade. © 2013 American Academy of Forensic Sciences.

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms.

PubMed

Dentinger, Bryn T M; Margaritescu, Simona; Moncalvo, Jean-Marc

2010-07-01

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field. © 2009 Blackwell Publishing Ltd.

A DNA Barcode Library for Korean Chironomidae (Insecta: Diptera) and Indexes for Defining Barcode Gap

PubMed Central

Kim, Sungmin; Song, Kyo-Hong; Ree, Han-Il; Kim, Won

2012-01-01

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis’ sequences to the library. PMID:22138764

Defining operational taxonomic units using DNA barcode data.

PubMed

Blaxter, Mark; Mann, Jenna; Chapman, Tom; Thomas, Fran; Whitton, Claire; Floyd, Robin; Abebe, Eyualem

2005-10-29

The scale of diversity of life on this planet is a significant challenge for any scientific programme hoping to produce a complete catalogue, whatever means is used. For DNA barcoding studies, this difficulty is compounded by the realization that any chosen barcode sequence is not the gene 'for' speciation and that taxa have evolutionary histories. How are we to disentangle the confounding effects of reticulate population genetic processes? Using the DNA barcode data from meiofaunal surveys, here we discuss the benefits of treating the taxa defined by barcodes without reference to their correspondence to 'species', and suggest that using this non-idealist approach facilitates access to taxon groups that are not accessible to other methods of enumeration and classification. Major issues remain, in particular the methodologies for taxon discrimination in DNA barcode data.

Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes.

PubMed

Chen, He; Yao, Jiacheng; Fu, Yusi; Pang, Yuhong; Wang, Jianbin; Huang, Yanyi

2018-04-11

The next generation sequencing (NGS) technologies have been rapidly evolved and applied to various research fields, but they often suffer from losing long-range information due to short library size and read length. Here, we develop a simple, cost-efficient, and versatile NGS library preparation method, called tagmentation on microbeads (TOM). This method is capable of recovering long-range information through tagmentation mediated by microbead-immobilized transposomes. Using transposomes with DNA barcodes to identically label adjacent sequences during tagmentation, we can restore inter-read connection of each fragment from original DNA molecule by fragment-barcode linkage after sequencing. In our proof-of-principle experiment, more than 4.5% of the reads are linked with their adjacent reads, and the longest linkage is over 1112 bp. We demonstrate TOM with eight barcodes, but the number of barcodes can be scaled up by an ultrahigh complexity construction. We also show this method has low amplification bias and effectively fits the applications to identify copy number variations.

Creation of reference DNA barcode library and authentication of medicinal plant raw drugs used in Ayurvedic medicine.

PubMed

Vassou, Sophie Lorraine; Nithaniyal, Stalin; Raju, Balaji; Parani, Madasamy

2016-07-18

Ayurveda is a system of traditional medicine that originated in ancient India, and it is still in practice. Medicinal plants are the backbone of Ayurveda, which heavily relies on the plant-derived therapeutics. While Ayurveda is becoming more popular in several countries throughout the World, lack of authenticated medicinal plant raw drugs is a growing concern. Our aim was to DNA barcode the medicinal plants that are listed in the Ayurvedic Pharmacopoeia of India (API) to create a reference DNA barcode library, and to use the same to authenticate the raw drugs that are sold in markets. We have DNA barcoded 347 medicinal plants using rbcL marker, and curated rbcL DNA barcodes for 27 medicinal plants from public databases. These sequences were used to create Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL). This library was used to authenticate 100 medicinal plant raw drugs, which were in the form of powders (82) and seeds (18). Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL) was created with high quality and authentic rbcL barcodes for 374 out of the 395 medicinal plants that are included in the API. The rbcL DNA barcode differentiated 319 species (85 %) with the pairwise divergence ranging between 0.2 and 29.9 %. PCR amplification and DNA sequencing success rate of rbcL marker was 100 % even for the poorly preserved medicinal plant raw drugs that were collected from local markets. DNA barcoding revealed that only 79 % raw drugs were authentic, and the remaining 21 % samples were adulterated. Further, adulteration was found to be much higher with powders (ca. 25 %) when compared to seeds (ca. 5 %). The present study demonstrated the utility of DNA barcoding in authenticating medicinal plant raw drugs, and found that approximately one fifth of the market samples were adulterated. Powdered raw drugs, which are very difficult to be identified by taxonomists as well as common people, seem to be the easy target for adulteration. Developing a quality control protocol for medicinal plant raw drugs by incorporating DNA barcoding as a component is essential to ensure safety to the consumers.

Machine Learned Replacement of N-Labels for Basecalled Sequences in DNA Barcoding.

PubMed

Ma, Eddie Y T; Ratnasingham, Sujeevan; Kremer, Stefan C

2018-01-01

This study presents a machine learning method that increases the number of identified bases in Sanger Sequencing. The system post-processes a KB basecalled chromatogram. It selects a recoverable subset of N-labels in the KB-called chromatogram to replace with basecalls (A,C,G,T). An N-label correction is defined given an additional read of the same sequence, and a human finished sequence. Corrections are added to the dataset when an alignment determines the additional read and human agree on the identity of the N-label. KB must also rate the replacement with quality value of in the additional read. Corrections are only available during system training. Developing the system, nearly 850,000 N-labels are obtained from Barcode of Life Datasystems, the premier database of genetic markers called DNA Barcodes. Increasing the number of correct bases improves reference sequence reliability, increases sequence identification accuracy, and assures analysis correctness. Keeping with barcoding standards, our system maintains an error rate of percent. Our system only applies corrections when it estimates low rate of error. Tested on this data, our automation selects and recovers: 79 percent of N-labels from COI (animal barcode); 80 percent from matK and rbcL (plant barcodes); and 58 percent from non-protein-coding sequences (across eukaryotes).

Building a DNA barcode library of Alaska's non-marine arthropods.

PubMed

Sikes, Derek S; Bowser, Matthew; Morton, John M; Bickford, Casey; Meierotto, Sarah; Hildebrandt, Kyndall

2017-03-01

Climate change may result in ecological futures with novel species assemblages, trophic mismatch, and mass extinction. Alaska has a limited taxonomic workforce to address these changes. We are building a DNA barcode library to facilitate a metabarcoding approach to monitoring non-marine arthropods. Working with the Canadian Centre for DNA Barcoding, we obtained DNA barcodes from recently collected and authoritatively identified specimens in the University of Alaska Museum (UAM) Insect Collection and the Kenai National Wildlife Refuge collection. We submitted tissues from 4776 specimens, of which 81% yielded DNA barcodes representing 1662 species and 1788 Barcode Index Numbers (BINs), of primarily terrestrial, large-bodied arthropods. This represents 84% of the species available for DNA barcoding in the UAM Insect Collection. There are now 4020 Alaskan arthropod species represented by DNA barcodes, after including all records in Barcode of Life Data Systems (BOLD) of species that occur in Alaska - i.e., 48.5% of the 8277 Alaskan, non-marine-arthropod, named species have associated DNA barcodes. An assessment of the identification power of the library in its current state yielded fewer species-level identifications than expected, but the results were not discouraging. We believe we are the first to deliberately begin development of a DNA barcode library of the entire arthropod fauna for a North American state or province. Although far from complete, this library will become increasingly valuable as more species are added and costs to obtain DNA sequences fall.

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes.

PubMed

Iftikhar, Romana; Ashfaq, Muhammad; Rasool, Akhtar; Hebert, Paul D N

2016-01-01

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5' (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.

Wolbachia and DNA barcoding insects: patterns, potential, and problems.

PubMed

Smith, M Alex; Bertrand, Claudia; Crosby, Kate; Eveleigh, Eldon S; Fernandez-Triana, Jose; Fisher, Brian L; Gibbs, Jason; Hajibabaei, Mehrdad; Hallwachs, Winnie; Hind, Katharine; Hrcek, Jan; Huang, Da-Wei; Janda, Milan; Janzen, Daniel H; Li, Yanwei; Miller, Scott E; Packer, Laurence; Quicke, Donald; Ratnasingham, Sujeevan; Rodriguez, Josephine; Rougerie, Rodolphe; Shaw, Mark R; Sheffield, Cory; Stahlhut, Julie K; Steinke, Dirk; Whitfield, James; Wood, Monty; Zhou, Xin

2012-01-01

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein--wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor--which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.

Design of character-based DNA barcode motif for species identification: A computational approach and its validation in fishes.

PubMed

Chakraborty, Mohua; Dhar, Bishal; Ghosh, Sankar Kumar

2017-11-01

The DNA barcodes are generally interpreted using distance-based and character-based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance-based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character-based approach more accurately defines this using a unique set of nucleotide characters. The character-based analysis of full-length barcode has some inherent limitations, like sequencing of the full-length barcode, use of a sparse-data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154-bp fragment, from the transversion-rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species-specific barcode motifs for 109 species by the character-based method, which successfully identifies the correct species using a pattern-matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species-specific mini-barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini-barcode approach will greatly benefit the field-based system of rapid species identification. © 2017 John Wiley & Sons Ltd.

DNA barcode-based molecular identification system for fish species.

PubMed

Kim, Sungmin; Eo, Hae-Seok; Koo, Hyeyoung; Choi, Jun-Kil; Kim, Won

2010-12-01

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 http://bioinfosys.snu.ac.kr:8080/MISF/misf.jsp .

Profiling Nematode Communities in Unmanaged Flowerbed and Agricultural Field Soils in Japan by DNA Barcode Sequencing

PubMed Central

Morise, Hisashi; Miyazaki, Erika; Yoshimitsu, Shoko; Eki, Toshihiko

2012-01-01

Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis. PMID:23284767

rbcL and matK earn two thumbs up as the core DNA barcode for ferns.

PubMed

Li, Fay-Wei; Kuo, Li-Yaung; Rothfels, Carl J; Ebihara, Atsushi; Chiou, Wen-Liang; Windham, Michael D; Pryer, Kathleen M

2011-01-01

DNA barcoding will revolutionize our understanding of fern ecology, most especially because the accurate identification of the independent but cryptic gametophyte phase of the fern's life history--an endeavor previously impossible--will finally be feasible. In this study, we assess the discriminatory power of the core plant DNA barcode (rbcL and matK), as well as alternatively proposed fern barcodes (trnH-psbA and trnL-F), across all major fern lineages. We also present plastid barcode data for two genera in the hyperdiverse polypod clade--Deparia (Woodsiaceae) and the Cheilanthes marginata group (currently being segregated as a new genus of Pteridaceae)--to further evaluate the resolving power of these loci. Our results clearly demonstrate the value of matK data, previously unavailable in ferns because of difficulties in amplification due to a major rearrangement of the plastid genome. With its high sequence variation, matK complements rbcL to provide a two-locus barcode with strong resolving power. With sequence variation comparable to matK, trnL-F appears to be a suitable alternative barcode region in ferns, and perhaps should be added to the core barcode region if universal primer development for matK fails. In contrast, trnH-psbA shows dramatically reduced sequence variation for the majority of ferns. This is likely due to the translocation of this segment of the plastid genome into the inverted repeat regions, which are known to have a highly constrained substitution rate. Our study provides the first endorsement of the two-locus barcode (rbcL+matK) in ferns, and favors trnL-F over trnH-psbA as a potential back-up locus. Future work should focus on gathering more fern matK sequence data to facilitate universal primer development.

The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates.

PubMed

Stoeck, T; Przybos, E; Dunthorn, M

2014-05-01

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL barcode for ciliated protists. © 2013 John Wiley & Sons Ltd.

Applications of three DNA barcodes in assorting intertidal red macroalgal flora in Qingdao, China

NASA Astrophysics Data System (ADS)

Zhao, Xiaobo; Pang, Shaojun; Shan, Tifeng; Liu, Feng

2013-03-01

This study is part of the endeavor to construct a comprehensive DNA barcoding database for common seaweeds in China. Identifications of red seaweeds, which have simple morphology and anatomy, are sometimes difficult solely depending on morphological characteristics. In recent years, DNA barcode technique has become a more and more effective tool to help solve some of the taxonomic difficulties. Some DNA markers such as COI (cytochrome oxidase subunit I) are proposed as standardized DNA barcodes for all seaweed species. In this study, COI, UPA (universal plastid amplicon, domain V of 23S rRNA), and ITS (nuclear internal transcribed spacer) were employed to analyze common species of intertidal red seaweeds in Qingdao (119.3°-121°E, 35.35°-37.09°N). The applicability of using one or a few combined barcodes to identify red seaweed species was tested. The results indicated that COI is a sensitive marker at species level. However, not all the tested species gave PCR amplification products due to lack of the universal primers. The second barcode UPA had effective universal primers but needed to be tested for the effectiveness of resolving closely related species. More than one ITS sequence types were found in some species in this investigation, which might lead to confusion in further analysis. Therefore ITS sequence is not recommended as a universal barcode for seaweeds identification.

Diversity in a Cold Hot-Spot: DNA-Barcoding Reveals Patterns of Evolution among Antarctic Demosponges (Class Demospongiae, Phylum Porifera).

PubMed

Vargas, Sergio; Kelly, Michelle; Schnabel, Kareen; Mills, Sadie; Bowden, David; Wörheide, Gert

2015-01-01

The approximately 350 demosponge species that have been described from Antarctica represent a faunistic component distinct from that of neighboring regions. Sponges provide structure to the Antarctic benthos and refuge to other invertebrates, and can be dominant in some communities. Despite the importance of sponges in the Antarctic subtidal environment, sponge DNA barcodes are scarce but can provide insight into the evolutionary relationships of this unique biogeographic province. We sequenced the standard barcoding COI region for a comprehensive selection of sponges collected during expeditions to the Ross Sea region in 2004 and 2008, and produced DNA-barcodes for 53 demosponge species covering about 60% of the species collected. The Antarctic sponge communities are phylogenetically diverse, matching the diversity of well-sampled sponge communities in the Lusitanic and Mediterranean marine provinces in the Temperate Northern Atlantic for which molecular data are readily available. Additionally, DNA-barcoding revealed levels of in situ molecular evolution comparable to those present among Caribbean sponges. DNA-barcoding using the Segregating Sites Algorithm correctly assigned approximately 54% of the barcoded species to the morphologically determined species. A barcode library for Antarctic sponges was assembled and used to advance the systematic and evolutionary research of Antarctic sponges. We provide insights on the evolutionary forces shaping Antarctica's diverse sponge communities, and a barcode library against which future sequence data from other regions or depth strata of Antarctica can be compared. The opportunity for rapid taxonomic identification of sponge collections for ecological research is now at the horizon.

DNA Barcode Sequence Identification Incorporating Taxonomic Hierarchy and within Taxon Variability

PubMed Central

Little, Damon P.

2011-01-01

For DNA barcoding to succeed as a scientific endeavor an accurate and expeditious query sequence identification method is needed. Although a global multiple–sequence alignment can be generated for some barcoding markers (e.g. COI, rbcL), not all barcoding markers are as structurally conserved (e.g. matK). Thus, algorithms that depend on global multiple–sequence alignments are not universally applicable. Some sequence identification methods that use local pairwise alignments (e.g. BLAST) are unable to accurately differentiate between highly similar sequences and are not designed to cope with hierarchic phylogenetic relationships or within taxon variability. Here, I present a novel alignment–free sequence identification algorithm–BRONX–that accounts for observed within taxon variability and hierarchic relationships among taxa. BRONX identifies short variable segments and corresponding invariant flanking regions in reference sequences. These flanking regions are used to score variable regions in the query sequence without the production of a global multiple–sequence alignment. By incorporating observed within taxon variability into the scoring procedure, misidentifications arising from shared alleles/haplotypes are minimized. An explicit treatment of more inclusive terminals allows for separate identifications to be made for each taxonomic level and/or for user–defined terminals. BRONX performs better than all other methods when there is imperfect overlap between query and reference sequences (e.g. mini–barcode queries against a full–length barcode database). BRONX consistently produced better identifications at the genus–level for all query types. PMID:21857897

Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae)

PubMed Central

Wen, Jun; Ebihara, Atsushi; Li, De-Zhu

2016-01-01

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure. PMID:27603700

Defining operational taxonomic units using DNA barcode data

PubMed Central

Blaxter, Mark; Mann, Jenna; Chapman, Tom; Thomas, Fran; Whitton, Claire; Floyd, Robin; Abebe, Eyualem

2005-01-01

Abstract The scale of diversity of life on this planet is a significant challenge for any scientific programme hoping to produce a complete catalogue, whatever means is used. For DNA barcoding studies, this difficulty is compounded by the realization that any chosen barcode sequence is not the gene ‘for’ speciation and that taxa have evolutionary histories. How are we to disentangle the confounding effects of reticulate population genetic processes? Using the DNA barcode data from meiofaunal surveys, here we discuss the benefits of treating the taxa defined by barcodes without reference to their correspondence to ‘species’, and suggest that using this non-idealist approach facilitates access to taxon groups that are not accessible to other methods of enumeration and classification. Major issues remain, in particular the methodologies for taxon discrimination in DNA barcode data. PMID:16214751

Use of DNA barcodes to identify flowering plants.

PubMed

Kress, W John; Wurdack, Kenneth J; Zimmer, Elizabeth A; Weigt, Lee A; Janzen, Daniel H

2005-06-07

Methods for identifying species by using short orthologous DNA sequences, known as "DNA barcodes," have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short ( approximately 450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.

Nuclear genomes distinguish cryptic species suggested by their DNA barcodes and ecology

PubMed Central

Janzen, Daniel H.; Burns, John M.; Cong, Qian; Hallwachs, Winnie; Dapkey, Tanya; Manjunath, Ramya; Hajibabaei, Mehrdad; Hebert, Paul D. N.; Grishin, Nick V.

2017-01-01

DNA sequencing brings another dimension to exploration of biodiversity, and large-scale mitochondrial DNA cytochrome oxidase I barcoding has exposed many potential new cryptic species. Here, we add complete nuclear genome sequencing to DNA barcoding, ecological distribution, natural history, and subtleties of adult color pattern and size to show that a widespread neotropical skipper butterfly known as Udranomia kikkawai (Weeks) comprises three different species in Costa Rica. Full-length barcodes obtained from all three century-old Venezuelan syntypes of U. kikkawai show that it is a rainforest species occurring from Costa Rica to Brazil. The two new species are Udranomia sallydaleyae Burns, a dry forest denizen occurring from Costa Rica to Mexico, and Udranomia tomdaleyi Burns, which occupies the junction between the rainforest and dry forest and currently is known only from Costa Rica. Whereas the three species are cryptic, differing but slightly in appearance, their complete nuclear genomes totaling 15 million aligned positions reveal significant differences consistent with their 0.00065-Mbp (million base pair) mitochondrial barcodes and their ecological diversification. DNA barcoding of tropical insects reared by a massive inventory suggests that the presence of cryptic species is a widespread phenomenon and that further studies will substantially increase current estimates of insect species richness. PMID:28716927

FBIS: A regional DNA barcode archival & analysis system for Indian fishes

PubMed Central

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. Availability The database is available for free at http://mail.nbfgr.res.in/fbis/ PMID:22715304

A DNA Barcode Library for North American Pyraustinae (Lepidoptera: Pyraloidea: Crambidae).

PubMed

Yang, Zhaofu; Landry, Jean-François; Hebert, Paul D N

2016-01-01

Although members of the crambid subfamily Pyraustinae are frequently important crop pests, their identification is often difficult because many species lack conspicuous diagnostic morphological characters. DNA barcoding employs sequence diversity in a short standardized gene region to facilitate specimen identifications and species discovery. This study provides a DNA barcode reference library for North American pyraustines based upon the analysis of 1589 sequences recovered from 137 nominal species, 87% of the fauna. Data from 125 species were barcode compliant (>500bp, <1% n), and 99 of these taxa formed a distinct cluster that was assigned to a single BIN. The other 26 species were assigned to 56 BINs, reflecting frequent cases of deep intraspecific sequence divergence and a few instances of barcode sharing, creating a total of 155 BINs. Two systems for OTU designation, ABGD and BIN, were examined to check the correspondence between current taxonomy and sequence clusters. The BIN system performed better than ABGD in delimiting closely related species, while OTU counts with ABGD were influenced by the value employed for relative gap width. Different species with low or no interspecific divergence may represent cases of unrecognized synonymy, whereas those with high intraspecific divergence require further taxonomic scrutiny as they may involve cryptic diversity. The barcode library developed in this study will also help to advance understanding of relationships among species of Pyraustinae.

Quantitative phenotyping via deep barcode sequencing.

PubMed

Smith, Andrew M; Heisler, Lawrence E; Mellor, Joseph; Kaper, Fiona; Thompson, Michael J; Chee, Mark; Roth, Frederick P; Giaever, Guri; Nislow, Corey

2009-10-01

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

[Identification of antler powder components based on DNA barcoding technology].

PubMed

Jia, Jing; Shi, Lin-chun; Xu, Zhi-chao; Xin, Tian-yi; Song, Jing-yuan; Chen Shi, Lin

2015-10-01

In order to authenticate the components of antler powder in the market, DNA barcoding technology coupled with cloning method were used. Cytochrome c oxidase subunit I (COI) sequences were obtained according to the DNA barcoding standard operation procedure (SOP). For antler powder with possible mixed components, the cloning method was used to get each COI sequence. 65 COI sequences were successfully obtained from commercial antler powders via sequencing PCR products. The results indicates that only 38% of these samples were derived from Cervus nippon Temminck or Cervus elaphus Linnaeus which is recorded in the 2010 edition of "Chinese Pharmacopoeia", while 62% of them were derived from other species. Rangifer tarandus Linnaeus was the most frequent species among the adulterants. Further analysis showed that some samples collected from different regions, companies and prices, contained adulterants. Analysis of 36 COI sequences obtained by the cloning method showed that C. elaphus and C. nippon were main components. In addition, some samples were marked clearly as antler powder on the label, however, C. elaphus or R. tarandus were their main components. In summary, DNA barcoding can accurately and efficiently distinguish the exact content in the commercial antler powder, which provides a new technique to ensure clinical safety and improve quality control of Chinese traditional medicine

A comprehensive DNA barcode database for Central European beetles with a focus on Germany: adding more than 3500 identified species to BOLD.

PubMed

Hendrich, Lars; Morinière, Jérôme; Haszprunar, Gerhard; Hebert, Paul D N; Hausmann, Axel; Köhler, Frank; Balke, Michael

2015-07-01

Beetles are the most diverse group of animals and are crucial for ecosystem functioning. In many countries, they are well established for environmental impact assessment, but even in the well-studied Central European fauna, species identification can be very difficult. A comprehensive and taxonomically well-curated DNA barcode library could remedy this deficit and could also link hundreds of years of traditional knowledge with next generation sequencing technology. However, such a beetle library is missing to date. This study provides the globally largest DNA barcode reference library for Coleoptera for 15 948 individuals belonging to 3514 well-identified species (53% of the German fauna) with representatives from 97 of 103 families (94%). This study is the first comprehensive regional test of the efficiency of DNA barcoding for beetles with a focus on Germany. Sequences ≥500 bp were recovered from 63% of the specimens analysed (15 948 of 25 294) with short sequences from another 997 specimens. Whereas most specimens (92.2%) could be unambiguously assigned to a single known species by sequence diversity at CO1, 1089 specimens (6.8%) were assigned to more than one Barcode Index Number (BIN), creating 395 BINs which need further study to ascertain if they represent cryptic species, mitochondrial introgression, or simply regional variation in widespread species. We found 409 specimens (2.6%) that shared a BIN assignment with another species, most involving a pair of closely allied species as 43 BINs were involved. Most of these taxa were separated by barcodes although sequence divergences were low. Only 155 specimens (0.97%) show identical or overlapping clusters. © 2014 John Wiley & Sons Ltd.

DNA barcodes of the native ray-finned fishes in Taiwan.

PubMed

Chang, Chia-Hao; Shao, Kwang-Tsao; Lin, Han-Yang; Chiu, Yung-Chieh; Lee, Mao-Ying; Liu, Shih-Hui; Lin, Pai-Lei

2017-07-01

Species identification based on the DNA sequence of a fragment of the cytochrome c oxidase subunit I gene in the mitochondrial genome, DNA barcoding, is widely applied to assist in sustainable exploitation of fish resources and the protection of fish biodiversity. The aim of this study was to establish a reliable barcoding reference database of the native ray-finned fishes in Taiwan. A total of 2993 individuals, belonging to 1245 species within 637 genera, 184 families and 29 orders of ray-finned fishes and representing approximately 40% of the recorded ray-finned fishes in Taiwan, were PCR amplified at the barcode region and bidirectionally sequenced. The mean length of the 2993 barcodes is 549 bp. Mean congeneric K2P distance (15.24%) is approximately 10-fold higher than the mean conspecific one (1.51%), but approximately 1.4-fold less than the mean genetic distance between families (20.80%). The Barcode Index Number (BIN) discordance report shows that 2993 specimens represent 1275 BINs and, among them, 86 BINs are singletons, 570 BINs are taxonomically concordant, and the other 619 BINs are taxonomically discordant. Barcode gap analysis also revealed that more than 90% of the collected fishes in this study can be discriminated by DNA barcoding. Overall, the barcoding reference database established by this study reveals the need for taxonomic revisions and voucher specimen rechecks, in addition to assisting in the management of Taiwan's fish resources and diversity. © 2016 John Wiley & Sons Ltd.

Barcoding of fresh water fishes from Pakistan.

PubMed

Karim, Asma; Iqbal, Asad; Akhtar, Rehan; Rizwan, Muhammad; Amar, Ali; Qamar, Usman; Jahan, Shah

2016-07-01

DNA bar-coding is a taxonomic method that uses small genetic markers in organisms' mitochondrial DNA (mt DNA) for identification of particular species. It uses sequence diversity in a 658-base pair fragment near the 5' end of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene as a tool for species identification. DNA barcoding is more accurate and reliable method as compared with the morphological identification. It is equally useful in juveniles as well as adult stages of fishes. The present study was conducted to identify three farm fish species of Pakistan (Cyprinus carpio, Cirrhinus mrigala, and Ctenopharyngodon idella) genetically. All of them belonged to family cyprinidae. CO1 gene was amplified. PCR products were sequenced and analyzed by bioinformatic software. Conspecific, congenric, and confamilial k2P nucleotide divergence was estimated. From these findings, it was concluded that the gene sequence, CO1, may serve as milestone for the identification of related species at molecular level.

Ecological niche modelling and nDNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding.

PubMed

Hawlitschek, Oliver; Porch, Nick; Hendrich, Lars; Balke, Michael

2011-02-09

DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species.

Ecological Niche Modelling and nDNA Sequencing Support a New, Morphologically Cryptic Beetle Species Unveiled by DNA Barcoding

PubMed Central

Hawlitschek, Oliver; Porch, Nick; Hendrich, Lars; Balke, Michael

2011-01-01

Background DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. Methodology/Principal Findings The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. Conclusion/Significance In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species. PMID:21347370

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

DNA barcoding for identification of consumer-relevant mushrooms: A partial solution for product certification?

PubMed

Raja, Huzefa A; Baker, Timothy R; Little, Jason G; Oberlies, Nicholas H

2017-01-01

One challenge in the dietary supplement industry is confirmation of species identity for processed raw materials, i.e. those modified by milling, drying, or extraction, which move through a multilevel supply chain before reaching the finished product. This is particularly difficult for samples containing fungal mycelia, where processing removes morphological characteristics, such that they do not present sufficient variation to differentiate species by traditional techniques. To address this issue, we have demonstrated the utility of DNA barcoding to verify the taxonomic identity of fungi found commonly in the food and dietary supplement industry; such data are critical for protecting consumer health, by assuring both safety and quality. By using DNA barcoding of nuclear ribosomal internal transcribed spacer (ITS) of the rRNA gene with fungal specific ITS primers, ITS barcodes were generated for 33 representative fungal samples, all of which could be used by consumers for food and/or dietary supplement purposes. In the majority of cases, we were able to sequence the ITS region from powdered mycelium samples, grocery store mushrooms, and capsules from commercial dietary supplements. After generating ITS barcodes utilizing standard procedures accepted by the Consortium for the Barcode of Life, we tested their utility by performing a BLAST search against authenticate published ITS sequences in GenBank. In some cases, we also downloaded published, homologous sequences of the ITS region of fungi inspected in this study and examined the phylogenetic relationships of barcoded fungal species in light of modern taxonomic and phylogenetic studies. We anticipate that these data will motivate discussions on DNA barcoding based species identification as applied to the verification/certification of mushroom-containing dietary supplements. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A DNA Barcode Library for North American Ephemeroptera: Progress and Prospects

PubMed Central

Webb, Jeffrey M.; Jacobus, Luke M.; Funk, David H.; Zhou, Xin; Kondratieff, Boris; Geraci, Christy J.; DeWalt, R. Edward; Baird, Donald J.; Richard, Barton; Phillips, Iain; Hebert, Paul D. N.

2012-01-01

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3–24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species. PMID:22666447

Moorea BIOCODE barcode library as a tool for understanding predator-prey interactions: insights into the diet of common predatory coral reef fishes

NASA Astrophysics Data System (ADS)

Leray, M.; Boehm, J. T.; Mills, S. C.; Meyer, C. P.

2012-06-01

Identifying species involved in consumer-resource interactions is one of the main limitations in the construction of food webs. DNA barcoding of prey items in predator guts provides a valuable tool for characterizing trophic interactions, but the method relies on the availability of reference sequences to which prey sequences can be matched. In this study, we demonstrate that the COI sequence library of the Moorea BIOCODE project, an ecosystem-level barcode initiative, enables the identification of a large proportion of semi-digested fish, crustacean and mollusks found in the guts of three Hawkfish and two Squirrelfish species. While most prey remains lacked diagnostic morphological characters, 94% of the prey found in 67 fishes had >98% sequence similarity with BIOCODE reference sequences. Using this species-level prey identification, we demonstrate how DNA barcoding can provide insights into resource partitioning, predator feeding behaviors and the consequences of predation on ecosystem function.

Identification of species based on DNA barcode using k-mer feature vector and Random forest classifier.

PubMed

Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Rao, A R

2016-11-05

DNA barcoding is a molecular diagnostic method that allows automated and accurate identification of species based on a short and standardized fragment of DNA. To this end, an attempt has been made in this study to develop a computational approach for identifying the species by comparing its barcode with the barcode sequence of known species present in the reference library. Each barcode sequence was first mapped onto a numeric feature vector based on k-mer frequencies and then Random forest methodology was employed on the transformed dataset for species identification. The proposed approach outperformed similarity-based, tree-based, diagnostic-based approaches and found comparable with existing supervised learning based approaches in terms of species identification success rate, while compared using real and simulated datasets. Based on the proposed approach, an online web interface SPIDBAR has also been developed and made freely available at http://cabgrid.res.in:8080/spidbar/ for species identification by the taxonomists. Copyright © 2016 Elsevier B.V. All rights reserved.

The changing epitome of species identification – DNA barcoding

PubMed Central

Ajmal Ali, M.; Gyulai, Gábor; Hidvégi, Norbert; Kerti, Balázs; Al Hemaid, Fahad M.A.; Pandey, Arun K.; Lee, Joongku

2014-01-01

The discipline taxonomy (the science of naming and classifying organisms, the original bioinformatics and a basis for all biology) is fundamentally important in ensuring the quality of life of future human generation on the earth; yet over the past few decades, the teaching and research funding in taxonomy have declined because of its classical way of practice which lead the discipline many a times to a subject of opinion, and this ultimately gave birth to several problems and challenges, and therefore the taxonomist became an endangered race in the era of genomics. Now taxonomy suddenly became fashionable again due to revolutionary approaches in taxonomy called DNA barcoding (a novel technology to provide rapid, accurate, and automated species identifications using short orthologous DNA sequences). In DNA barcoding, complete data set can be obtained from a single specimen irrespective to morphological or life stage characters. The core idea of DNA barcoding is based on the fact that the highly conserved stretches of DNA, either coding or non coding regions, vary at very minor degree during the evolution within the species. Sequences suggested to be useful in DNA barcoding include cytoplasmic mitochondrial DNA (e.g. cox1) and chloroplast DNA (e.g. rbcL, trnL-F, matK, ndhF, and atpB rbcL), and nuclear DNA (ITS, and house keeping genes e.g. gapdh). The plant DNA barcoding is now transitioning the epitome of species identification; and thus, ultimately helping in the molecularization of taxonomy, a need of the hour. The ‘DNA barcodes’ show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, forensic analysis, and many more. PMID:24955007

Mini-DNA barcode in identification of the ornamental fish: A case study from Northeast India.

PubMed

Dhar, Bishal; Ghosh, Sankar Kumar

2017-09-05

The ornamental fishes were exported under the trade names or generic names, thus creating problems in species identification. In this regard, DNA barcoding could effectively elucidate the actual species status. However, the problem arises if the specimen is having taxonomic disputes, falsified by trade/generic names, etc., On the other hand, barcoding the archival museum specimens would be of greater benefit to address such issues as it would create firm, error-free reference database for rapid identification of any species. This can be achieved only by generating short sequences as DNA from chemically preserved are mostly degraded. Here we aimed to identify a short stretch of informative sites within the full-length barcode segment, capable of delineating diverse group of ornamental fish species, commonly traded from NE India. We analyzed 287 full-length barcode sequences from the major fish orders and compared the interspecific K2P distance with nucleotide substitutions patterns and found a strong correlation of interspecies distance with transversions (0.95, p<0.001). We, therefore, proposed a short stretch of 171bp (transversion rich) segment as mini-barcode. The proposed segment was compared with the full-length barcodes and found to delineate the species effectively. Successful PCR amplification and sequencing of the 171bp segment using designed primers for different orders validated it as mini-barcodes for ornamental fishes. Thus, our findings would be helpful in strengthening the global database with the sequence of archived fish species as well as an effective identification tool of the traded ornamental fish species, as a less time consuming, cost effective field-based application. Copyright © 2017 Elsevier B.V. All rights reserved.

A report on identification of sequence polymorphism in barcode region of six commercially important Cymbopogon species.

PubMed

Bishoyi, Ashok Kumar; Kavane, Aarti; Sharma, Anjali; Geetha, K A

2017-02-01

CYMBOPOGON: is an important member of grass family Poaceae, cultivated for essential oils which have greater medicinal and industrial value. Taxonomic identification of Cymbopogon species is determined mainly by morphological markers, odour of essential oils and concentration of bioactive compounds present in the oil matrices which are highly influenced by environment. Authenticated molecular marker based taxonomical identification is also lacking in the genus; hence effort was made to evaluate potential DNA barcode loci in six commercially important Cymbopogon species for their individual discrimination and authentication at the species level. Four widely used DNA barcoding regions viz., ITS 1 & ITS 2 spacers, matK, psbA-trnH and rbcL were taken for the study. Gene sequences of the same or related genera of the concerned loci were mined from NCBI domain and primers were designed and validated for barcode loci amplification. Out of the four loci studied, sequences from matK and ITS spacer loci revealed 0.46% and 5.64% nucleotide sequence diversity, respectively whereas the other two loci i.e., psbA-trnH and rbcL showed 100% sequence homology. The newly developed primers can be used for barcode loci amplification in the genus Cymbopogon. The identified Single Nucleotide Polymorphisms from the studied sequences may be used as barcodes for the six Cymbopogon species. The information generated can also be utilized for barcode development of the genus by including more number of Cymbopgon species in future.

The chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data.

PubMed

Links, Matthew G; Dumonceaux, Tim J; Hemmingsen, Sean M; Hill, Janet E

2012-01-01

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life. Using the framework of the International Barcode of Life we evaluated DNA barcodes for Bacteria. Candidates from the 16S rRNA gene and the protein coding cpn60 gene were evaluated. Within complete bacterial genomes in the public domain representing 983 species from 21 phyla, the largest difference between median pairwise inter- and intra-specific distances ("barcode gap") was found from cpn60. Distribution of sequence diversity along the ∼555 bp cpn60 target region was remarkably uniform. The barcode gap of the cpn60 universal target facilitated the faithful de novo assembly of full-length operational taxonomic units from pyrosequencing data from a synthetic microbial community. Analysis supported the recognition of both 16S rRNA and cpn60 as DNA barcodes for Bacteria. The cpn60 universal target was found to have a much larger barcode gap than 16S rRNA suggesting cpn60 as a preferred barcode for Bacteria. A large barcode gap for cpn60 provided a robust target for species-level characterization of data. The assembly of consensus sequences for barcodes was shown to be a reliable method for the identification and tracking of novel microbes in metagenomic studies.

Highly-sensitive microRNA detection based on bio-bar-code assay and catalytic hairpin assembly two-stage amplification.

PubMed

Tang, Songsong; Gu, Yuan; Lu, Huiting; Dong, Haifeng; Zhang, Kai; Dai, Wenhao; Meng, Xiangdan; Yang, Fan; Zhang, Xueji

2018-04-03

Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a "sandwich" structure. After "sandwich" structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Deciphering amphibian diversity through DNA barcoding: chances and challenges.

PubMed

Vences, Miguel; Thomas, Meike; Bonett, Ronald M; Vieites, David R

2005-10-29

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.

DNA Barcoding Identifies Argentine Fishes from Marine and Brackish Waters

PubMed Central

Mabragaña, Ezequiel; Díaz de Astarloa, Juan Martín; Hanner, Robert; Zhang, Junbin; González Castro, Mariano

2011-01-01

Background DNA barcoding has been advanced as a promising tool to aid species identification and discovery through the use of short, standardized gene targets. Despite extensive taxonomic studies, for a variety of reasons the identification of fishes can be problematic, even for experts. DNA barcoding is proving to be a useful tool in this context. However, its broad application is impeded by the need to construct a comprehensive reference sequence library for all fish species. Here, we make a regional contribution to this grand challenge by calibrating the species discrimination efficiency of barcoding among 125 Argentine fish species, representing nearly one third of the known fauna, and examine the utility of these data to address several key taxonomic uncertainties pertaining to species in this region. Methodology/Principal Findings Specimens were collected and morphologically identified during crusies conducted between 2005 and 2008. The standard BARCODE fragment of COI was amplified and bi-directionally sequenced from 577 specimens (mean of 5 specimens/species), and all specimens and sequence data were archived and interrogated using analytical tools available on the Barcode of Life Data System (BOLD; www.barcodinglife.org). Nearly all species exhibited discrete clusters of closely related haplogroups which permitted the discrimination of 95% of the species (i.e. 119/125) examined while cases of shared haplotypes were detected among just three species-pairs. Notably, barcoding aided the identification of a new species of skate, Dipturus argentinensis, permitted the recognition of Genypterus brasiliensis as a valid species and questions the generic assignment of Paralichthys isosceles. Conclusions/Significance This study constitutes a significant contribution to the global barcode reference sequence library for fishes and demonstrates the utility of barcoding for regional species identification. As an independent assessment of alpha taxonomy, barcodes provide robust support for most morphologically based taxon concepts and also highlight key areas of taxonomic uncertainty worthy of reappraisal. PMID:22174860

"Crown of thorns" of Daphnia: an exceptional inducible defense discovered by DNA barcoding.

PubMed

Laforsch, Christian; Haas, Andreas; Jung, Nina; Schwenk, Klaus; Tollrian, Ralph; Petrusek, Adam

2009-09-01

DNA barcoding has emerged as valuable tool to document global biodiversity. Mitochondrial cytochrome oxidase I (COI) sequences serve as genetic markers to catalogue species richness in the animal kingdom and to identify cryptic and polymorphic animal species. Furthermore, DNA barcoding data serve as a fuel for ecological studies, as they provide the opportunity to unravel species interactions among hosts and parasites, predators and prey, and among competitors in unprecedented detail. In a recent paper we described how DNA barcoding in combination with morphological and ecological data unravelled a striking predator-prey interaction of organisms from temporary aquatic habitats, the predatory notostracan Triops and its prey, cladocerans of the Daphnia atkinsoni complex.

Patterns of DNA barcode variation in Canadian marine molluscs.

PubMed

Layton, Kara K S; Martel, André L; Hebert, Paul D N

2014-01-01

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area. This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0-26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%-46.5%), and showed a significant positive correlation with nearest neighbour distances. DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.

Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building.

PubMed

Pomerantz, Aaron; Peñafiel, Nicolás; Arteaga, Alejandro; Bustamante, Lucas; Pichardo, Frank; Coloma, Luis A; Barrio-Amorós, César L; Salazar-Valenzuela, David; Prost, Stefan

2018-04-01

Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field. We demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding. Overall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

Telling plant species apart with DNA: from barcodes to genomes

PubMed Central

Li, De-Zhu; van der Bank, Michelle

2016-01-01

Land plants underpin a multitude of ecosystem functions, support human livelihoods and represent a critically important component of terrestrial biodiversity—yet many tens of thousands of species await discovery, and plant identification remains a substantial challenge, especially where material is juvenile, fragmented or processed. In this opinion article, we tackle two main topics. Firstly, we provide a short summary of the strengths and limitations of plant DNA barcoding for addressing these issues. Secondly, we discuss options for enhancing current plant barcodes, focusing on increasing discriminatory power via either gene capture of nuclear markers or genome skimming. The former has the advantage of establishing a defined set of target loci maximizing efficiency of sequencing effort, data storage and analysis. The challenge is developing a probe set for large numbers of nuclear markers that works over sufficient phylogenetic breadth. Genome skimming has the advantage of using existing protocols and being backward compatible with existing barcodes; and the depth of sequence coverage can be increased as sequencing costs fall. Its non-targeted nature does, however, present a major informatics challenge for upscaling to large sample sets. This article is part of the themed issue ‘From DNA barcodes to biomes’. PMID:27481790

INTERNAL TRANSCRIBED SPACER (ITS), AN IDEAL DNA BARCODE FOR SPECIES DISCRIMINATION IN CRAWFURDIA WALL. (GENTIANACEAE).

PubMed

Zhang, Dequan; Jiang, Bei; Duan, Lizhen; Zhou, Nong

2016-01-01

DNA barcoding is a technique used to identify species based on species-specific differences in short regions of their DNA. It is widely used in species discrimination of medicinal plants and traditional medicines. In the present study, four potential DNA barcodes, namely rbcL , matK , trnH-psbA and ITS (nuclear ribosomal internal transcribed spacer) were adopted for species discrimination in Crawfurdia Wall (Genetiaceae). Identification ability of these DNA barcodes and combinations were evaluated using three classic methods (Distance, Blast and Tree-Building). As a result, ITS, trnH-psbA and rbcL regions showed great universality for a success rate of 100%; whereas matK was disappointing for which only 65% samples gained useful DNA sequences. ITS region, which could clearly and effectively identify the five species in Crawfurdia , performed very well in this study. On the contrary, trnH-psbA and rbcL performed poorly in discrimination among these species. ITS marker was an ideal DNA barcode in Crawfurdia and it should be incorporated into one of the core barcodes for seed plants.

Increasing global participation in genetics research through DNA barcoding.

PubMed

Adamowicz, Sarah J; Steinke, Dirk

2015-12-01

DNA barcoding--the sequencing of short, standardized DNA regions for specimen identification and species discovery--has promised to facilitate rapid access to biodiversity knowledge by diverse users. Here, we advance our opinion that increased global participation in genetics research is beneficial, both to scientists and for science, and explore the premise that DNA barcoding can help to democratize participation in genetics research. We examine publication patterns (2003-2014) in the DNA barcoding literature and compare trends with those in the broader, related domain of genomics. While genomics is the older and much larger field, the number of nations contributing to the published literature is similar between disciplines. Meanwhile, DNA barcoding exhibits a higher pace of growth in the number of publications as well as greater evenness among nations in their proportional contribution to total authorships. This exploration revealed DNA barcoding to be a highly international discipline, with growing participation by researchers in especially biodiverse nations. We briefly consider several of the challenges that may hinder further participation in genetics research, including access to training and molecular facilities as well as policy relating to the movement of genetic resources.

DNA barcoding of vouchered xylarium wood specimens of nine endangered Dalbergia species.

PubMed

Yu, Min; Jiao, Lichao; Guo, Juan; Wiedenhoeft, Alex C; He, Tuo; Jiang, Xiaomei; Yin, Yafang

2017-12-01

ITS2+ trnH - psbA was the best combination of DNA barcode to resolve the Dalbergia wood species studied. We demonstrate the feasibility of building a DNA barcode reference database using xylarium wood specimens. The increase in illegal logging and timber trade of CITES-listed tropical species necessitates the development of unambiguous identification methods at the species level. For these methods to be fully functional and deployable for law enforcement, they must work using wood or wood products. DNA barcoding of wood has been promoted as a promising tool for species identification; however, the main barrier to extensive application of DNA barcoding to wood is the lack of a comprehensive and reliable DNA reference library of barcodes from wood. In this study, xylarium wood specimens of nine Dalbergia species were selected from the Wood Collection of the Chinese Academy of Forestry and DNA was then extracted from them for further PCR amplification of eight potential DNA barcode sequences (ITS2, matK, trnL, trnH-psbA, trnV-trnM1, trnV-trnM2, trnC-petN, and trnS-trnG). The barcodes were tested singly and in combination for species-level discrimination ability by tree-based [neighbor-joining (NJ)] and distance-based (TaxonDNA) methods. We found that the discrimination ability of DNA barcodes in combination was higher than any single DNA marker among the Dalbergia species studied, with the best two-marker combination of ITS2+trnH-psbA analyzed with NJ trees performing the best (100% accuracy). These barcodes are relatively short regions (<350 bp) and amplification reactions were performed with high success (≥90%) using wood as the source material, a necessary factor to apply DNA barcoding to timber trade. The present results demonstrate the feasibility of using vouchered xylarium specimens to build DNA barcoding reference databases.

Evaluation of the DNA barcodes in Dendrobium (Orchidaceae) from mainland Asia.

PubMed

Xu, Songzhi; Li, Dezhu; Li, Jianwu; Xiang, Xiaoguo; Jin, Weitao; Huang, Weichang; Jin, Xiaohua; Huang, Luqi

2015-01-01

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera.

Evaluation of the DNA Barcodes in Dendrobium (Orchidaceae) from Mainland Asia

PubMed Central

Xu, Songzhi; Li, Dezhu; Li, Jianwu; Xiang, Xiaoguo; Jin, Weitao; Huang, Weichang; Jin, Xiaohua; Huang, Luqi

2015-01-01

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera. PMID:25602282

DNA barcoding discriminates freshwater fishes from southeastern Nigeria and provides river system-level phylogeographic resolution within some species.

PubMed

Nwani, Christopher D; Becker, Sven; Braid, Heather E; Ude, Emmanuel F; Okogwu, Okechukwu I; Hanner, Robert

2011-10-01

Fishes are the main animal protein source for human beings and play a vital role in aquatic ecosystems and food webs. Fish identification can be challenging, especially in the tropics (due to high diversity), and this is particularly true for larval forms or fragmentary remains. DNA barcoding, which uses the 5' region of the mitochondrial cytochrome c oxidase subunit I (COI) as a target gene, is an efficient method for standardized species-level identification for biodiversity assessment and conservation, pending the establishment of reference sequence libraries. In this study, fishes were collected from three rivers in southeastern Nigeria, identified morphologically, and imaged digitally. DNA was extracted, PCR-amplified, and the standard barcode region was bidirectionally sequenced for 363 individuals belonging to 70 species in 38 genera. All specimen provenance data and associated sequence information were recorded in the barcode of life data systems (BOLD; www.barcodinglife.org ). Analytical tools on BOLD were used to assess the performance of barcoding to identify species. Using neighbor-joining distance comparison, the average genetic distance was 60-fold higher between species than within species, as pairwise genetic distance estimates averaged 10.29% among congeners and only 0.17% among conspecifics. Despite low levels of divergence within species, we observed river system-specific haplotype partitioning within eight species (11.4% of all species). Our preliminary results suggest that DNA barcoding is very effective for species identification of Nigerian freshwater fishes.

Classification of Sharks in the Egyptian Mediterranean Waters Using Morphological and DNA Barcoding Approaches

PubMed Central

Moftah, Marie; Abdel Aziz, Sayeda H.; Elramah, Sara; Favereaux, Alexandre

2011-01-01

The identification of species constitutes the first basic step in phylogenetic studies, biodiversity monitoring and conservation. DNA barcoding, i.e. the sequencing of a short standardized region of DNA, has been proposed as a new tool for animal species identification. The present study provides an update on the composition of shark in the Egyptian Mediterranean waters off Alexandria, since the latest study to date was performed 30 years ago, DNA barcoding was used in addition to classical taxonomical methodologies. Thus, 51 specimen were DNA barcoded for a 667 bp region of the mitochondrial COI gene. Although DNA barcoding aims at developing species identification systems, some phylogenetic signals were apparent in the data. In the neighbor-joining tree, 8 major clusters were apparent, each of them containing individuals belonging to the same species, and most with 100% bootstrap value. This study is the first to our knowledge to use DNA barcoding of the mitochondrial COI gene in order to confirm the presence of species Squalus acanthias, Oxynotus centrina, Squatina squatina, Scyliorhinus canicula, Scyliorhinus stellaris, Mustelus mustelus, Mustelus punctulatus and Carcharhinus altimus in the Egyptian Mediterranean waters. Finally, our study is the starting point of a new barcoding database concerning shark composition in the Egyptian Mediterranean waters (Barcoding of Egyptian Mediterranean Sharks [BEMS], http://www.boldsystems.org/views/projectlist.php?&#Barcoding%20Fish%20%28FishBOL%29). PMID:22087242

Authentication of Botanical Origin in Herbal Teas by Plastid Noncoding DNA Length Polymorphisms.

PubMed

Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami

2015-07-01

The aim of this study was to develop a DNA barcode assay to authenticate the botanical origin of herbal teas. To reach this aim, we tested the efficiency of a PCR-capillary electrophoresis (PCR-CE) approach on commercial herbal tea samples using two noncoding plastid barcodes, the trnL intron and the intergenic spacer between trnL and trnF. Barcode DNA length polymorphisms proved successful in authenticating the species origin of herbal teas. We verified the validity of our approach by sequencing species-specific barcode amplicons from herbal tea samples. Moreover, we displayed the utility of PCR-CE assays coupled with sequencing to identify the origin of undeclared plant material in herbal tea samples. The PCR-CE assays proposed in this work can be applied as routine tests for the verification of botanical origin in herbal teas and can be extended to authenticate all types of herbal foodstuffs.

Use of DNA barcodes to identify flowering plants

PubMed Central

Kress, W. John; Wurdack, Kenneth J.; Zimmer, Elizabeth A.; Weigt, Lee A.; Janzen, Daniel H.

2005-01-01

Methods for identifying species by using short orthologous DNA sequences, known as “DNA barcodes,” have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short (≈450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes. PMID:15928076

DNA barcoding as a useful tool in the systematic study of wild bees of the tribe Augochlorini (Hymenoptera: Halictidae).

PubMed

González-Vaquero, Rocío Ana; Roig-Alsina, Arturo; Packer, Laurence

2016-10-01

Special care is needed in the delimitation and identification of halictid bee species, which are renowned for being morphologically monotonous. Corynura Spinola and Halictillus Moure (Halictidae: Augochlorini) contain species that are key elements in southern South American ecosystems. These bees are very difficult to identify due to close morphological similarity among species and high sexual dimorphism. We analyzed 170 barcode-compliant COI sequences from 19 species. DNA barcodes were useful to confirm gender associations and to detect two new cryptic species. Interspecific distances were significantly higher than those reported for other bees. Maximum intraspecific divergence was less than 1% in 14 species. Barcode index numbers (BINs) were useful to identify putative species that need further study. More than one BIN was assigned to five species. The name Corynura patagonica (Cockerell) probably refers to two cryptic species. The results suggest that Corynura and Halictillus species can be identified using DNA barcodes. The sequences of the species included in this study can be used as a reference to assess the identification of unknown specimens. This study provides additional support for the use of DNA barcodes in bee taxonomy and the identification of specimens, which is particularly relevant in insects of ecological importance such as pollinators.

A single mini-barcode test to screen for Australian mammalian predators from environmental samples

PubMed Central

MacDonald, Anna J; Sarre, Stephen D

2017-01-01

Abstract Identification of species from trace samples is now possible through the comparison of diagnostic DNA fragments against reference DNA sequence databases. DNA detection of animals from non-invasive samples, such as predator faeces (scats) that contain traces of DNA from their species of origin, has proved to be a valuable tool for the management of elusive wildlife. However, application of this approach can be limited by the availability of appropriate genetic markers. Scat DNA is often degraded, meaning that longer DNA sequences, including standard DNA barcoding markers, are difficult to recover. Instead, targeted short diagnostic markers are required to serve as diagnostic mini-barcodes. The mitochondrial genome is a useful source of such trace DNA markers because it provides good resolution at the species level and occurs in high copy numbers per cell. We developed a mini-barcode based on a short (178 bp) fragment of the conserved 12S ribosomal ribonucleic acid mitochondrial gene sequence, with the goal of discriminating amongst the scats of large mammalian predators of Australia. We tested the sensitivity and specificity of our primers and can accurately detect and discriminate amongst quolls, cats, dogs, foxes, and devils from trace DNA samples. Our approach provides a cost-effective, time-efficient, and non-invasive tool that enables identification of all 8 medium-large mammal predators in Australia, including native and introduced species, using a single test. With modification, this approach is likely to be of broad applicability elsewhere. PMID:28810700

Quantitative phenotyping via deep barcode sequencing

PubMed Central

Smith, Andrew M.; Heisler, Lawrence E.; Mellor, Joseph; Kaper, Fiona; Thompson, Michael J.; Chee, Mark; Roth, Frederick P.; Giaever, Guri; Nislow, Corey

2009-01-01

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or “Bar-seq,” outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that ∼20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene–environment interactions on a genome-wide scale. PMID:19622793

[Identification and analysis of Corydalis boweri, Meconopsis horridula and their close related species of the same genus by using ITS2 DNA barcode].

PubMed

Dou, Rong-kun; Bi, Zhen-fei; Bai, Rui-xue; Ren, Yao-yao; Tan, Rui; Song, Liang-ke; Li, Di-qiang; Mao, Can-quan

2015-04-01

The study is aimed to ensure the quality and safety of medicinal plants by using ITS2 DNA barcode technology to identify Corydalis boweri, Meconopsis horridula and their close related species. The DNA of 13 herb samples including C. boweri and M. horridula from Lhasa of Tibet was extracted, ITS PCR were amplified and sequenced. Both assembled and web downloaded 71 ITS2 sequences were removed of 5. 8S and 28S. Multiple sequence alignment was completed and the intraspecific and interspecific genetic distances were calculated by MEGA 5.0, while the neighbor-joining phylogenetic trees were constructed. We also predicted the ITS2 secondary structure of C. boweri, M. horridula and their close related species. The results showed that ITS2 as DNA barcode was able to identify C. boweri, M. horridula as well as well as their close related species effectively. The established based on ITS2 barcode method provides the regular and safe detection technology for identification of C. boweri, M. horridula and their close related species, adulterants and counterfeits, in order to ensure their quality control, safe medication, reasonable development and utilization.

Use of mitochondrial COI gene for the identification of family Salticidae and Lycosidae of spiders.

PubMed

Naseem, Sajida; Tahir, Hafiz Muhammad

2018-01-01

In recent years, DNA barcoding has become quite popular for molecular identification of species because it is simple, quick and an affordable method. Present study was conducted to identify spiders of most abundant families, i.e. Salticidae and Lycosidae from citrus orchards in Sargodha district using DNA barcoding. A total of 160 specimens were subjected to DNA barcoding but, sequences up to 600 bp were recovered for 156 specimens. This molecular approach proved helpful to assign the exact taxon to those specimens which were misidentified through morphological characters in the study. We were succeeded to discriminate six species of Lycosidae and nine species of Salticidae through DNA barcoding. Results revealed the presence of clear barcode gap (discontinuity in intra- and inter-specific divergences) for members of both families. Furthermore, the maximum intra-specific divergence was less than NN (nearest neighbour) distance for all species. This suggested the reliability of DNA barcoding for spider's identification up to species level. We got 98% success in our study. It is concluded from present study that DNA barcoding is more reliable tool especially for immature spiders, when morphological characters are ambiguous.

DNA barcode identification of Podocarpaceae--the second largest conifer family.

PubMed

Little, Damon P; Knopf, Patrick; Schulz, Christian

2013-01-01

We have generated matK, rbcL, and nrITS2 DNA barcodes for 320 specimens representing all 18 extant genera of the conifer family Podocarpaceae. The sample includes 145 of the 198 recognized species. Comparative analyses of sequence quality and species discrimination were conducted on the 159 individuals from which all three markers were recovered (representing 15 genera and 97 species). The vast majority of sequences were of high quality (B 30 = 0.596-0.989). Even the lowest quality sequences exceeded the minimum requirements of the BARCODE data standard. In the few instances that low quality sequences were generated, the responsible mechanism could not be discerned. There were no statistically significant differences in the discriminatory power of markers or marker combinations (p = 0.05). The discriminatory power of the barcode markers individually and in combination is low (56.7% of species at maximum). In some instances, species discrimination failed in spite of ostensibly useful variation being present (genotypes were shared among species), but in many cases there was simply an absence of sequence variation. Barcode gaps (maximum intraspecific p-distance > minimum interspecific p-distance) were observed in 50.5% of species when all three markers were considered simultaneously. The presence of a barcode gap was not predictive of discrimination success (p = 0.02) and there was no statistically significant difference in the frequency of barcode gaps among markers (p = 0.05). In addition, there was no correlation between number of individuals sampled per species and the presence of a barcode gap (p = 0.27).

Competitive Genomic Screens of Barcoded Yeast Libraries

PubMed Central

Urbanus, Malene; Proctor, Michael; Heisler, Lawrence E.; Giaever, Guri; Nislow, Corey

2011-01-01

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment. PMID:21860376

DNA Barcode Identification of Podocarpaceae—The Second Largest Conifer Family

PubMed Central

Little, Damon P.; Knopf, Patrick; Schulz, Christian

2013-01-01

We have generated matK, rbcL, and nrITS2 DNA barcodes for 320 specimens representing all 18 extant genera of the conifer family Podocarpaceae. The sample includes 145 of the 198 recognized species. Comparative analyses of sequence quality and species discrimination were conducted on the 159 individuals from which all three markers were recovered (representing 15 genera and 97 species). The vast majority of sequences were of high quality (B 30 = 0.596–0.989). Even the lowest quality sequences exceeded the minimum requirements of the BARCODE data standard. In the few instances that low quality sequences were generated, the responsible mechanism could not be discerned. There were no statistically significant differences in the discriminatory power of markers or marker combinations (p = 0.05). The discriminatory power of the barcode markers individually and in combination is low (56.7% of species at maximum). In some instances, species discrimination failed in spite of ostensibly useful variation being present (genotypes were shared among species), but in many cases there was simply an absence of sequence variation. Barcode gaps (maximum intraspecific p–distance > minimum interspecific p–distance) were observed in 50.5% of species when all three markers were considered simultaneously. The presence of a barcode gap was not predictive of discrimination success (p = 0.02) and there was no statistically significant difference in the frequency of barcode gaps among markers (p = 0.05). In addition, there was no correlation between number of individuals sampled per species and the presence of a barcode gap (p = 0.27). PMID:24312258

Services of DNA barcoding in different fields.

PubMed

Muhammad Tahir, Hafiz; Akhtar, Samreen

2016-11-01

DNA barcoding is a new master key for species identification and has greatly accelerated the pace of species discovery. In this novel and cost-effective technique, a short DNA sequence from a standard region of mitochondrial "CO1" gene called "barcode" is used. At present, researchers all over the world are utilizing this powerful tool for investigating biodiversity, differentiating cryptic species, testing food authenticity, identifying parasites, vectors, insect pests, and predators, monitoring of illegal trade of animals and their products, and identifying forensically important insects. In addition, this technique can potentially be used to monitor quality of drinking water, quickly identify the indicator species of lakes, rivers, and streams, identify species with harmful attributes or medicinal properties, monitor smuggling of endangered plants and animals and their products, and disease investigations. Despite non-favorable criticism from a few researchers, DNA barcoding has achieved immense popularity in the scientific community, especially among biologists. The present review provides an overview of DNA barcoding and its practical applications. The limitation, future prospective and main informative platforms for DNA barcoding have also been discussed.

A comparative study of COI and 16 S rRNA genes for DNA barcoding of cultivable carps in India.

PubMed

Mohanty, Mausumee; Jayasankar, Pallipuram; Sahoo, Lakshman; Das, Paramananda

2015-02-01

The 5' region of the mitochondrial DNA gene cytochrome c oxidase subunit I (COI) is the standard marker for DNA barcoding. However, 16 S rRNA has also been advocated for DNA barcoding in many animal species. Herein, we directly compare the usefulness of COI and 16 S rRNA in discriminating six cultivable carp species: Labeo rohita, Catla catla, Cirrhinus mrigala, Labeo fimbriatus, Labeo bata and Cirrhinus reba from India. Analysis of partial sequences of these two gene fragments from 171 individuals indicated close genetic relationship between Catla catla and Labeo rohita. The results of the present study indicated COI to be more useful than 16 S rRNA for DNA barcoding of Indian carps.

Application of DNA Barcodes in Asian Tropical Trees--A Case Study from Xishuangbanna Nature Reserve, Southwest China.

PubMed

Huang, Xiao-cui; Ci, Xiu-qin; Conran, John G; Li, Jie

2015-01-01

Within a regional floristic context, DNA barcoding is more useful to manage plant diversity inventories on a large scale and develop valuable conservation strategies. However, there are no DNA barcode studies from tropical areas of China, which represents one of the biodiversity hotspots around the world. A DNA barcoding database of an Asian tropical trees with high diversity was established at Xishuangbanna Nature Reserve, Yunnan, southwest China using rbcL and matK as standard barcodes, as well as trnH-psbA and ITS as supplementary barcodes. The performance of tree species identification success was assessed using 2,052 accessions from four plots belonging to two vegetation types in the region by three methods: Neighbor-Joining, Maximum-Likelihood and BLAST. We corrected morphological field identification errors (9.6%) for the three plots using rbcL and matK based on Neighbor-Joining tree. The best barcode region for PCR and sequencing was rbcL (97.6%, 90.8%), followed by trnH-psbA (93.6%, 85.6%), while matK and ITS obtained relative low PCR and sequencing success rates. However, ITS performed best for both species (44.6-58.1%) and genus (72.8-76.2%) identification. With trnH-psbA slightly less effective for species identification. The two standard barcode rbcL and matK gave poor results for species identification (24.7-28.5% and 31.6-35.3%). Compared with other studies from comparable tropical forests (e.g. Cameroon, the Amazon and India), the overall performance of the four barcodes for species identification was lower for the Xishuangbanna Nature Reserve, possibly because of species/genus ratios and species composition between these tropical areas. Although the core barcodes rbcL and matK were not suitable for species identification of tropical trees from Xishuangbanna Nature Reserve, they could still help with identification at the family and genus level. Considering the relative sequence recovery and the species identification performance, we recommend the use of trnH-psbA and ITS in combination as the preferred barcodes for tropical tree species identification in China.

Rapidly evolving homing CRISPR barcodes

PubMed Central

Kalhor, Reza; Mali, Prashant; Church, George M.

2017-01-01

We present here an approach for engineering evolving DNA barcodes in living cells. The methodology entails using a homing guide RNA (hgRNA) scaffold that directs the Cas9-hgRNA complex to target the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show the barcode RNAs can be assayed as single molecules in situ . This integrated approach will have wide ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping. PMID:27918539

DNA barcoding and morphological analysis for rapid identification of most economically important crop-infesting Sunn pests belonging to Eurygaster Laporte, 1833 (Hemiptera, Scutelleridae).

PubMed

Syromyatnikov, Mikhail Y; Golub, Victor B; Kokina, Anastasia V; Victoria A Soboleva; Popov, Vasily N

2017-01-01

The genus Eurygaster Laporte, 1833 includes ten species five of which inhabit the European part of Russia. The harmful species of the genus is E. integriceps . Eurygaster species identification based on the morphological traits is very difficult, while that of the species at the egg or larval stages is extremely difficult or impossible. Eurygaster integriceps , E. maura , and E. testudinaria differ only slightly between each other morphologically, E. maura and E. testudinaria being almost indiscernible. DNA barcoding based on COI sequences have shown that E. integriceps differs significantly from these closely related species, which enables its rapid and accurate identification. Based on COI nucleotide sequences, three species of Sunn pests, E. maura , E. testudinarius , E. dilaticollis , could not be differentiated from each other through DNA barcoding. The difference in the DNA sequences between the COI gene of E. integriceps and COI genes of E. maura and E. testudinarius was more than 4%. In the present study DNA barcoding of two Eurygaster species was performed for the first time on E. integriceps , the most dangerous pest in the genus, and E. dilaticollis that only inhabits natural ecosystems. The PCR-RFLP method was developed in this work for the rapid identification of E. integriceps .

DNA barcoding and morphological analysis for rapid identification of most economically important crop-infesting Sunn pests belonging to Eurygaster Laporte, 1833 (Hemiptera, Scutelleridae)

PubMed Central

Syromyatnikov, Mikhail Y.; Golub, Victor B.; Kokina, Anastasia V.; Victoria A. Soboleva; Popov, Vasily N.

2017-01-01

Abstract The genus Eurygaster Laporte, 1833 includes ten species five of which inhabit the European part of Russia. The harmful species of the genus is E. integriceps. Eurygaster species identification based on the morphological traits is very difficult, while that of the species at the egg or larval stages is extremely difficult or impossible. Eurygaster integriceps, E. maura, and E. testudinaria differ only slightly between each other morphologically, E. maura and E. testudinaria being almost indiscernible. DNA barcoding based on COI sequences have shown that E. integriceps differs significantly from these closely related species, which enables its rapid and accurate identification. Based on COI nucleotide sequences, three species of Sunn pests, E. maura, E. testudinarius, E. dilaticollis, could not be differentiated from each other through DNA barcoding. The difference in the DNA sequences between the COI gene of E. integriceps and COI genes of E. maura and E. testudinarius was more than 4%. In the present study DNA barcoding of two Eurygaster species was performed for the first time on E. integriceps, the most dangerous pest in the genus, and E. dilaticollis that only inhabits natural ecosystems. The PCR-RFLP method was developed in this work for the rapid identification of E. integriceps. PMID:29118620

Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north-eastern part of the Congo basin.

PubMed

Decru, Eva; Moelants, Tuur; De Gelas, Koen; Vreven, Emmanuel; Verheyen, Erik; Snoeks, Jos

2016-01-01

This study evaluates the utility of DNA barcoding to traditional morphology-based species identifications for the fish fauna of the north-eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio 'nearest-neighbour distance/maximum intraspecific divergence' was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative. © 2015 John Wiley & Sons Ltd.

rbcL and matK Earn Two Thumbs Up as the Core DNA Barcode for Ferns

PubMed Central

Li, Fay-Wei; Kuo, Li-Yaung; Rothfels, Carl J.; Ebihara, Atsushi; Chiou, Wen-Liang; Windham, Michael D.; Pryer, Kathleen M.

2011-01-01

Background DNA barcoding will revolutionize our understanding of fern ecology, most especially because the accurate identification of the independent but cryptic gametophyte phase of the fern's life history—an endeavor previously impossible—will finally be feasible. In this study, we assess the discriminatory power of the core plant DNA barcode (rbcL and matK), as well as alternatively proposed fern barcodes (trnH-psbA and trnL-F), across all major fern lineages. We also present plastid barcode data for two genera in the hyperdiverse polypod clade—Deparia (Woodsiaceae) and the Cheilanthes marginata group (currently being segregated as a new genus of Pteridaceae)—to further evaluate the resolving power of these loci. Principal Findings Our results clearly demonstrate the value of matK data, previously unavailable in ferns because of difficulties in amplification due to a major rearrangement of the plastid genome. With its high sequence variation, matK complements rbcL to provide a two-locus barcode with strong resolving power. With sequence variation comparable to matK, trnL-F appears to be a suitable alternative barcode region in ferns, and perhaps should be added to the core barcode region if universal primer development for matK fails. In contrast, trnH-psbA shows dramatically reduced sequence variation for the majority of ferns. This is likely due to the translocation of this segment of the plastid genome into the inverted repeat regions, which are known to have a highly constrained substitution rate. Conclusions Our study provides the first endorsement of the two-locus barcode (rbcL+matK) in ferns, and favors trnL-F over trnH-psbA as a potential back-up locus. Future work should focus on gathering more fern matK sequence data to facilitate universal primer development. PMID:22028918

DNA barcode analysis of butterfly species from Pakistan points towards regional endemism

PubMed Central

Ashfaq, Muhammad; Akhtar, Saleem; Khan, Arif M; Adamowicz, Sarah J; Hebert, Paul D N

2013-01-01

DNA barcodes were obtained for 81 butterfly species belonging to 52 genera from sites in north-central Pakistan to test the utility of barcoding for their identification and to gain a better understanding of regional barcode variation. These species represent 25% of the butterfly fauna of Pakistan and belong to five families, although the Nymphalidae were dominant, comprising 38% of the total specimens. Barcode analysis showed that maximum conspecific divergence was 1.6%, while there was 1.7–14.3% divergence from the nearest neighbour species. Barcode records for 55 species showed <2% sequence divergence to records in the Barcode of Life Data Systems (BOLD), but only 26 of these cases involved specimens from neighbouring India and Central Asia. Analysis revealed that most species showed little incremental sequence variation when specimens from other regions were considered, but a threefold increase was noted in a few cases. There was a clear gap between maximum intraspecific and minimum nearest neighbour distance for all 81 species. Neighbour-joining cluster analysis showed that members of each species formed a monophyletic cluster with strong bootstrap support. The barcode results revealed two provisional species that could not be clearly linked to known taxa, while 24 other species gained their first coverage. Future work should extend the barcode reference library to include all butterfly species from Pakistan as well as neighbouring countries to gain a better understanding of regional variation in barcode sequences in this topographically and climatically complex region. PMID:23789612

Interbreeding among deeply divergent mitochondrial lineages in the American cockroach (Periplaneta americana)

NASA Astrophysics Data System (ADS)

von Beeren, Christoph; Stoeckle, Mark Y.; Xia, Joyce; Burke, Griffin; Kronauer, Daniel J. C.

2015-02-01

DNA barcoding promises to be a useful tool to identify pest species assuming adequate representation of genetic variants in a reference library. Here we examined mitochondrial DNA barcodes in a global urban pest, the American cockroach (Periplaneta americana). Our sampling effort generated 284 cockroach specimens, most from New York City, plus 15 additional U.S. states and six other countries, enabling the first large-scale survey of P. americana barcode variation. Periplaneta americana barcode sequences (n = 247, including 24 GenBank records) formed a monophyletic lineage separate from other Periplaneta species. We found three distinct P. americana haplogroups with relatively small differences within (<=0.6%) and larger differences among groups (2.4%-4.7%). This could be interpreted as indicative of multiple cryptic species. However, nuclear DNA sequences (n = 77 specimens) revealed extensive gene flow among mitochondrial haplogroups, confirming a single species. This unusual genetic pattern likely reflects multiple introductions from genetically divergent source populations, followed by interbreeding in the invasive range. Our findings highlight the need for comprehensive reference databases in DNA barcoding studies, especially when dealing with invasive populations that might be derived from multiple genetically distinct source populations.

DNA barcodes for 1/1000 of the animal kingdom.

PubMed

Hebert, Paul D N; Dewaard, Jeremy R; Landry, Jean-François

2010-06-23

This study reports DNA barcodes for more than 1300 Lepidoptera species from the eastern half of North America, establishing that 99.3 per cent of these species possess diagnostic barcode sequences. Intraspecific divergences averaged just 0.43 per cent among this assemblage, but most values were lower. The mean was elevated by deep barcode divergences (greater than 2%) in 5.1 per cent of the species, often involving the sympatric occurrence of two barcode clusters. A few of these cases have been analysed in detail, revealing species overlooked by the current taxonomic system. This study also provided a large-scale test of the extent of regional divergence in barcode sequences, indicating that geographical differentiation in the Lepidoptera of eastern North America is small, even when comparisons involve populations as much as 2800 km apart. The present results affirm that a highly effective system for the identification of Lepidoptera in this region can be built with few records per species because of the limited intra-specific variation. As most terrestrial and marine taxa are likely to possess a similar pattern of population structure, an effective DNA-based identification system can be developed with modest effort.

20 years since the introduction of DNA barcoding: from theory to application.

PubMed

Fišer Pečnikar, Živa; Buzan, Elena V

2014-02-01

Traditionally, taxonomic identification has relied upon morphological characters. In the last two decades, molecular tools based on DNA sequences of short standardised gene fragments, termed DNA barcodes, have been developed for species discrimination. The most common DNA barcode used in animals is a fragment of the cytochrome c oxidase (COI) mitochondrial gene, while for plants, two chloroplast gene fragments from the RuBisCo large subunit (rbcL) and maturase K (matK) genes are widely used. Information gathered from DNA barcodes can be used beyond taxonomic studies and will have far-reaching implications across many fields of biology, including ecology (rapid biodiversity assessment and food chain analysis), conservation biology (monitoring of protected species), biosecurity (early identification of invasive pest species), medicine (identification of medically important pathogens and their vectors) and pharmacology (identification of active compounds). However, it is important that the limitations of DNA barcoding are understood and techniques continually adapted and improved as this young science matures.

Calibrating snakehead diversity with DNA barcodes: expanding taxonomic coverage to enable identification of potential and established invasive species.

PubMed

Serrao, Natasha R; Steinke, Dirk; Hanner, Robert H

2014-01-01

Detecting and documenting the occurrence of invasive species outside their native range requires tools to support their identification. This can be challenging for taxa with diverse life stages and/or problematic or unresolved morphological taxonomies. DNA barcoding provides a potent method for identifying invasive species, as it allows for species identification at all life stages, including fragmentary remains. It also provides an efficient interim taxonomic framework for quantifying cryptic genetic diversity by parsing barcode sequences into discontinuous haplogroup clusters (typical of reproductively isolated species) and labelling them with unique alphanumeric identifiers. Snakehead fishes are a diverse group of opportunistic predators endemic to Asia and Africa that may potentially pose significant threats as aquatic invasive species. At least three snakehead species (Channa argus, C. maculata, and C. marulius) are thought to have entered North America through the aquarium and live-food fish markets, and have established populations, yet their origins remain unclear. The objectives of this study were to assemble a library of DNA barcode sequences derived from expert identified reference specimens in order to determine the identity and aid invasion pathway analysis of the non-indigenous species found in North America using DNA barcodes. Sequences were obtained from 121 tissue samples representing 25 species and combined with public records from GenBank for a total of 36 putative species, which then partitioned into 49 discrete haplogroups. Multiple divergent clusters were observed within C. gachua, C. marulius, C. punctata and C. striata suggesting the potential presence of cryptic species diversity within these lineages. Our findings demonstrate that DNA barcoding is a valuable tool for species identification in challenging and under-studied taxonomic groups such as snakeheads, and provides a useful framework for inferring invasion pathway analysis.

Reliable DNA Barcoding Performance Proved for Species and Island Populations of Comoran Squamate Reptiles

PubMed Central

Hawlitschek, Oliver; Nagy, Zoltán T.; Berger, Johannes; Glaw, Frank

2013-01-01

In the past decade, DNA barcoding became increasingly common as a method for species identification in biodiversity inventories and related studies. However, mainly due to technical obstacles, squamate reptiles have been the target of few barcoding studies. In this article, we present the results of a DNA barcoding study of squamates of the Comoros archipelago, a poorly studied group of oceanic islands close to and mostly colonized from Madagascar. The barcoding dataset presented here includes 27 of the 29 currently recognized squamate species of the Comoros, including 17 of the 18 endemic species. Some species considered endemic to the Comoros according to current taxonomy were found to cluster with non-Comoran lineages, probably due to poorly resolved taxonomy. All other species for which more than one barcode was obtained corresponded to distinct clusters useful for species identification by barcoding. In most species, even island populations could be distinguished using barcoding. Two cryptic species were identified using the DNA barcoding approach. The obtained barcoding topology, a Bayesian tree based on COI sequences of 5 genera, was compared with available multigene topologies, and in 3 cases, major incongruences between the two topologies became evident. Three of the multigene studies were initiated after initial screening of a preliminary version of the barcoding dataset presented here. We conclude that in the case of the squamates of the Comoros Islands, DNA barcoding has proven a very useful and efficient way of detecting isolated populations and promising starting points for subsequent research. PMID:24069192

DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

PubMed Central

Makarova, Olga; Contaldo, Nicoletta; Paltrinieri, Samanta; Kawube, Geofrey; Bertaccini, Assunta; Nicolaisen, Mogens

2012-01-01

Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification. PMID:23272216

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding.

PubMed

Versteirt, V; Nagy, Z T; Roelants, P; Denis, L; Breman, F C; Damiens, D; Dekoninck, W; Backeljau, T; Coosemans, M; Van Bortel, W

2015-03-01

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well-supported clusters. Intraspecific Kimura 2-parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra- and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species. © 2014 John Wiley & Sons Ltd.

Evaluating the feasibility of using candidate DNA barcodes in discriminating species of the large Asteraceae family.

PubMed

Gao, Ting; Yao, Hui; Song, Jingyuan; Zhu, Yingjie; Liu, Chang; Chen, Shilin

2010-10-26

Five DNA regions, namely, rbcL, matK, ITS, ITS2, and psbA-trnH, have been recommended as primary DNA barcodes for plants. Studies evaluating these regions for species identification in the large plant taxon, which includes a large number of closely related species, have rarely been reported. The feasibility of using the five proposed DNA regions was tested for discriminating plant species within Asteraceae, the largest family of flowering plants. Among these markers, ITS2 was the most useful in terms of universality, sequence variation, and identification capability in the Asteraceae family. The species discriminating power of ITS2 was also explored in a large pool of 3,490 Asteraceae sequences that represent 2,315 species belonging to 494 different genera. The result shows that ITS2 correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively. In addition, ITS2 displayed a variable ability to discriminate related species within different genera. ITS2 is the best DNA barcode for the Asteraceae family. This approach significantly broadens the application of DNA barcoding to resolve classification problems in the family Asteraceae at the genera and species levels.

Mitochondrial heteroplasmy and DNA barcoding in Hawaiian Hylaeus (Nesoprosopis) bees (Hymenoptera: Colletidae).

PubMed

Magnacca, Karl N; Brown, Mark J F

2010-06-11

The past several years have seen a flurry of papers seeking to clarify the utility and limits of DNA barcoding, particularly in areas such as species discovery and paralogy due to nuclear pseudogenes. Heteroplasmy, the coexistence of multiple mitochondrial haplotypes in a single organism, has been cited as a potentially serious problem for DNA barcoding but its effect on identification accuracy has not been tested. In addition, few studies of barcoding have tested a large group of closely-related species with a well-established morphological taxonomy. In this study we examine both of these issues, by densely sampling the Hawaiian Hylaeus bee radiation. Individuals from 21 of the 49 a priori morphologically-defined species exhibited coding sequence heteroplasmy at levels of 1-6% or more. All homoplasmic species were successfully identified by COI using standard methods of analysis, but only 71% of heteroplasmic species. The success rate in identifying heteroplasmic species was increased to 86% by treating polymorphisms as character states rather than ambiguities. Nuclear pseudogenes (numts) were also present in four species, and were distinguishable from heteroplasmic sequences by patterns of nucleotide and amino acid change. Heteroplasmy significantly decreased the reliability of species identification. In addition, the practical issue of dealing with large numbers of polymorphisms- and resulting increased time and labor required - makes the development of DNA barcode databases considerably more complex than has previously been suggested. The impact of heteroplasmy on the utility of DNA barcoding as a bulk specimen identification tool will depend upon its frequency across populations, which remains unknown. However, DNA barcoding is still likely to remain an important identification tool for those species that are difficult or impossible to identify through morphology, as is the case for the ecologically important solitary bee fauna.

Mitochondrial heteroplasmy and DNA barcoding in Hawaiian Hylaeus (Nesoprosopis) bees (Hymenoptera: Colletidae)

PubMed Central

2010-01-01

Background The past several years have seen a flurry of papers seeking to clarify the utility and limits of DNA barcoding, particularly in areas such as species discovery and paralogy due to nuclear pseudogenes. Heteroplasmy, the coexistence of multiple mitochondrial haplotypes in a single organism, has been cited as a potentially serious problem for DNA barcoding but its effect on identification accuracy has not been tested. In addition, few studies of barcoding have tested a large group of closely-related species with a well-established morphological taxonomy. In this study we examine both of these issues, by densely sampling the Hawaiian Hylaeus bee radiation. Results Individuals from 21 of the 49 a priori morphologically-defined species exhibited coding sequence heteroplasmy at levels of 1-6% or more. All homoplasmic species were successfully identified by COI using standard methods of analysis, but only 71% of heteroplasmic species. The success rate in identifying heteroplasmic species was increased to 86% by treating polymorphisms as character states rather than ambiguities. Nuclear pseudogenes (numts) were also present in four species, and were distinguishable from heteroplasmic sequences by patterns of nucleotide and amino acid change. Conclusions Heteroplasmy significantly decreased the reliability of species identification. In addition, the practical issue of dealing with large numbers of polymorphisms- and resulting increased time and labor required - makes the development of DNA barcode databases considerably more complex than has previously been suggested. The impact of heteroplasmy on the utility of DNA barcoding as a bulk specimen identification tool will depend upon its frequency across populations, which remains unknown. However, DNA barcoding is still likely to remain an important identification tool for those species that are difficult or impossible to identify through morphology, as is the case for the ecologically important solitary bee fauna. PMID:20540728

DNA barcodes for bio-surveillance: regulated and economically important arthropod plant pests.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N

2016-11-01

Many of the arthropod species that are important pests of agriculture and forestry are impossible to discriminate morphologically throughout all of their life stages. Some cannot be differentiated at any life stage. Over the past decade, DNA barcoding has gained increasing adoption as a tool to both identify known species and to reveal cryptic taxa. Although there has not been a focused effort to develop a barcode library for them, reference sequences are now available for 77% of the 409 species of arthropods documented on major pest databases. Aside from developing the reference library needed to guide specimen identifications, past barcode studies have revealed that a significant fraction of arthropod pests are a complex of allied taxa. Because of their importance as pests and disease vectors impacting global agriculture and forestry, DNA barcode results on these arthropods have significant implications for quarantine detection, regulation, and management. The current review discusses these implications in light of the presence of cryptic species in plant pests exposed by DNA barcoding.

DNA barcoding for the identification of sand fly species (Diptera, Psychodidae, Phlebotominae) in Colombia.

PubMed

Contreras Gutiérrez, María Angélica; Vivero, Rafael J; Vélez, Iván D; Porter, Charles H; Uribe, Sandra

2014-01-01

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia.

DNA Barcoding for the Identification of Sand Fly Species (Diptera, Psychodidae, Phlebotominae) in Colombia

PubMed Central

Contreras Gutiérrez, María Angélica; Vivero, Rafael J.; Vélez, Iván D.; Porter, Charles H.; Uribe, Sandra

2014-01-01

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia. PMID:24454877

Barcode extension for analysis and reconstruction of structures

NASA Astrophysics Data System (ADS)

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L.; Gootenberg, Jonathan S.; Yin, Peng

2017-03-01

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.

Barcode extension for analysis and reconstruction of structures.

PubMed

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L; Gootenberg, Jonathan S; Yin, Peng

2017-03-13

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.

Barcode extension for analysis and reconstruction of structures

PubMed Central

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L; Gootenberg, Jonathan S; Yin, Peng

2017-01-01

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures. PMID:28287117

DNA barcoding of Rhodiola (crassulaceae): a case study on a group of recently diversified medicinal plants from the Qinghai-Tibetan Plateau.

PubMed

Zhang, Jian-Qiang; Meng, Shi-Yong; Wen, Jun; Rao, Guang-Yuan

2015-01-01

DNA barcoding, the identification of species using one or a few short standardized DNA sequences, is an important complement to traditional taxonomy. However, there are particular challenges for barcoding plants, especially for species with complex evolutionary histories. We herein evaluated the utility of five candidate sequences - rbcL, matK, trnH-psbA, trnL-F and the internal transcribed spacer (ITS) - for barcoding Rhodiola species, a group of high-altitude plants frequently used as adaptogens, hemostatics and tonics in traditional Tibetan medicine. Rhodiola was suggested to have diversified rapidly recently. The genus is thus a good model for testing DNA barcoding strategies for recently diversified medicinal plants. This study analyzed 189 accessions, representing 47 of the 55 recognized Rhodiola species in the Flora of China treatment. Based on intraspecific and interspecific divergence and degree of monophyly statistics, ITS was the best single-locus barcode, resolving 66% of the Rhodiola species. The core combination rbcL+matK resolved only 40.4% of them. Unsurprisingly, the combined use of all five loci provided the highest discrimination power, resolving 80.9% of the species. However, this is weaker than the discrimination power generally reported in barcoding studies of other plant taxa. The observed complications may be due to the recent diversification, incomplete lineage sorting and reticulate evolution of the genus. These processes are common features of numerous plant groups in the high-altitude regions of the Qinghai-Tibetan Plateau.

New taxonomy and old collections: integrating DNA barcoding into the collection curation process.

PubMed

Puillandre, N; Bouchet, P; Boisselier-Dubayle, M-C; Brisset, J; Buge, B; Castelin, M; Chagnoux, S; Christophe, T; Corbari, L; Lambourdière, J; Lozouet, P; Marani, G; Rivasseau, A; Silva, N; Terryn, Y; Tillier, S; Utge, J; Samadi, S

2012-05-01

Because they house large biodiversity collections and are also research centres with sequencing facilities, natural history museums are well placed to develop DNA barcoding best practices. The main difficulty is generally the vouchering system: it must ensure that all data produced remain attached to the corresponding specimen, from the field to publication in articles and online databases. The Museum National d'Histoire Naturelle in Paris is one of the leading laboratories in the Marine Barcode of Life (MarBOL) project, which was used as a pilot programme to include barcode collections for marine molluscs and crustaceans. The system is based on two relational databases. The first one classically records the data (locality and identification) attached to the specimens. In the second one, tissue-clippings, DNA extractions (both preserved in 2D barcode tubes) and PCR data (including primers) are linked to the corresponding specimen. All the steps of the process [sampling event, specimen identification, molecular processing, data submission to Barcode Of Life Database (BOLD) and GenBank] are thus linked together. Furthermore, we have developed several web-based tools to automatically upload data into the system, control the quality of the sequences produced and facilitate the submission to online databases. This work is the result of a joint effort from several teams in the Museum National d'Histoire Naturelle (MNHN), but also from a collaborative network of taxonomists and molecular systematists outside the museum, resulting in the vouchering so far of ∼41,000 sequences and the production of ∼11,000 COI sequences. © 2012 Blackwell Publishing Ltd.

Barcode Identifiers as a Practical Tool for Reliable Species Assignment of Medically Important Black Yeast Species

PubMed Central

Heinrichs, Guido; de Hoog, G. Sybren

2012-01-01

Herpotrichiellaceous black yeasts and relatives comprise severe pathogens flanked by nonpathogenic environmental siblings. Reliable identification by conventional methods is notoriously difficult. Molecular identification is hampered by the sequence variability in the internal transcribed spacer (ITS) domain caused by difficult-to-sequence homopolymeric regions and by poor taxonomic attribution of sequences deposited in GenBank. Here, we present a potential solution using short barcode identifiers (27 to 50 bp) based on ITS2 ribosomal DNA (rDNA), which allows unambiguous definition of species-specific fragments. Starting from proven sequences of ex-type and authentic strains, we were able to describe 103 identifiers. Multiple BLAST searches of these proposed barcode identifiers in GenBank revealed uniqueness for 100 taxonomic entities, whereas the three remaining identifiers each matched with two entities, but the species of these identifiers could easily be discriminated by differences in the remaining ITS regions. Using the proposed barcode identifiers, a 4.1-fold increase of 100% matches in GenBank was achieved in comparison to the classical approach using the complete ITS sequences. The proposed barcode identifiers will be made accessible for the diagnostic laboratory in a permanently updated online database, thereby providing a highly practical, reliable, and cost-effective tool for identification of clinically important black yeasts and relatives. PMID:22785187

Morphological description and DNA barcoding of Hydrobaenus majus sp. nov. (Diptera: Chironomidae: Orthocladiinae) from the Russian Far East.

PubMed

Makarchenko, Eugenyi A; Makarchenko, Marina A; Semenchenko, Alexander A

2015-08-14

Illustrated descriptions of adult male, pupa and fourth instar larva, as well as DNA barcoding, of Hydrobaenus majus sp. nov. in comparison with the close related species H. sikhotealinensis Makarchenko et Makarchenko from the Russian Far East are provided. The species-specificity of H. majus sp. nov. COI sequences is analyzed and the sequences are presented as diagnostic characters--molecular markers of H. majus and H. sikhotealinensis.

Assessing universality of DNA barcoding in geographically isolated selected desert medicinal species of Fabaceae and Poaceae

PubMed Central

Hussain, Fatma; Ahmed, Nisar; Ghorbani, Abdolbaset

2018-01-01

In pursuit of developing fast and accurate species-level molecular identification methods, we tested six DNA barcodes, namely ITS2, matK, rbcLa, ITS2+matK, ITS2+rbcLa, matK+rbcLa and ITS2+matK+rbcLa, for their capacity to identify frequently consumed but geographically isolated medicinal species of Fabaceae and Poaceae indigenous to the desert of Cholistan. Data were analysed by BLASTn sequence similarity, pairwise sequence divergence in TAXONDNA, and phylogenetic (neighbour-joining and maximum-likelihood trees) methods. Comparison of six barcode regions showed that ITS2 has the highest number of variable sites (209/360) for tested Fabaceae and (106/365) Poaceae species, the highest species-level identification (40%) in BLASTn procedure, distinct DNA barcoding gap, 100% correct species identification in BM and BCM functions of TAXONDNA, and clear cladding pattern with high nodal support in phylogenetic trees in both families. ITS2+matK+rbcLa followed ITS2 in its species-level identification capacity. The study was concluded with advocating the DNA barcoding as an effective tool for species identification and ITS2 as the best barcode region in identifying medicinal species of Fabaceae and Poaceae. Current research has practical implementation potential in the fields of pharmaco-vigilance, trade of medicinal plants and biodiversity conservation. PMID:29576968

Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan.

PubMed

Ebihara, Atsushi; Nitta, Joel H; Ito, Motomi

2010-12-08

DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking. The Japanese pteridophyte flora (733 taxa including subspecies and varieties) was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA) with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0%) taxa for rbcL and 617 (84.2%) taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances) did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52%) was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only. This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially anticipated to be helpful for identification of non-hybridizing gametophytes.

A DNA barcode library for ground beetles of Germany: the genus Amara Bonelli, 1810 (Insecta, Coleoptera, Carabidae)

PubMed Central

Raupach, Michael J.; Hannig, Karsten; Moriniére, Jérôme; Hendrich, Lars

2018-01-01

Abstract The genus Amara Bonelli, 1810 is a very speciose and taxonomically difficult genus of the Carabidae. The identification of many of the species is accomplished with considerable difficulty, in particular for females and immature stages. In this study the effectiveness of DNA barcoding, the most popular method for molecular species identification, was examined to discriminate various species of this genus from Central Europe. DNA barcodes from 690 individuals and 47 species were analysed, including sequences from previous studies and more than 350 newly generated DNA barcodes. Our analysis revealed unique BINs for 38 species (81%). Interspecific K2P distances below 2.2% were found for three species pairs and one species trio, including haplotype sharing between Amara alpina/Amara torrida and Amara communis/Amara convexior/Amara makolskii. This study represents another step in generating an extensive reference library of DNA barcodes for carabids, highly valuable bioindicators for characterizing disturbances in various habitats. PMID:29853775

Fungi in Thailand: a case study of the efficacy of an ITS barcode for automatically identifying species within the Annulohypoxylon and Hypoxylon genera.

PubMed

Suwannasai, Nuttika; Martín, María P; Phosri, Cherdchai; Sihanonth, Prakitsin; Whalley, Anthony J S; Spouge, John L

2013-01-01

Thailand, a part of the Indo-Burma biodiversity hotspot, has many endemic animals and plants. Some of its fungal species are difficult to recognize and separate, complicating assessments of biodiversity. We assessed species diversity within the fungal genera Annulohypoxylon and Hypoxylon, which produce biologically active and potentially therapeutic compounds, by applying classical taxonomic methods to 552 teleomorphs collected from across Thailand. Using probability of correct identification (PCI), we also assessed the efficacy of automated species identification with a fungal barcode marker, ITS, in the model system of Annulohypoxylon and Hypoxylon. The 552 teleomorphs yielded 137 ITS sequences; in addition, we examined 128 GenBank ITS sequences, to assess biases in evaluating a DNA barcode with GenBank data. The use of multiple sequence alignment in a barcode database like BOLD raises some concerns about non-protein barcode markers like ITS, so we also compared species identification using different alignment methods. Our results suggest the following. (1) Multiple sequence alignment of ITS sequences is competitive with pairwise alignment when identifying species, so BOLD should be able to preserve its present bioinformatics workflow for species identification for ITS, and possibly therefore with at least some other non-protein barcode markers. (2) Automated species identification is insensitive to a specific choice of evolutionary distance, contributing to resolution of a current debate in DNA barcoding. (3) Statistical methods are available to address, at least partially, the possibility of expert misidentification of species. Phylogenetic trees discovered a cryptic species and strongly supported monophyletic clades for many Annulohypoxylon and Hypoxylon species, suggesting that ITS can contribute usefully to a barcode for these fungi. The PCIs here, derived solely from ITS, suggest that a fungal barcode will require secondary markers in Annulohypoxylon and Hypoxylon, however. The URL http://tinyurl.com/spouge-barcode contains computer programs and other supplementary material relevant to this article.

Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy.

PubMed

Levy-Sakin, Michal; Ebenstein, Yuval

2013-08-01

Next generation sequencing (NGS) is revolutionizing all fields of biological research but it fails to extract the full range of information associated with genetic material. Optical mapping of DNA grants access to genetic and epigenetic information on individual DNA molecules up to ∼1 Mbp in length. Fluorescent labeling of specific sequence motifs, epigenetic marks and other genomic information on individual DNA molecules generates a high content optical barcode along the DNA. By stretching the DNA to a linear configuration this barcode may be directly visualized by fluorescence microscopy. We discuss the advances of these methods in light of recent developments in nano-fabrication and super-resolution optical imaging (nanoscopy) and review the latest achievements of optical mapping in the context of genomic analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

DNA barcode analysis of butterfly species from Pakistan points towards regional endemism.

PubMed

Ashfaq, Muhammad; Akhtar, Saleem; Khan, Arif M; Adamowicz, Sarah J; Hebert, Paul D N

2013-09-01

DNA barcodes were obtained for 81 butterfly species belonging to 52 genera from sites in north-central Pakistan to test the utility of barcoding for their identification and to gain a better understanding of regional barcode variation. These species represent 25% of the butterfly fauna of Pakistan and belong to five families, although the Nymphalidae were dominant, comprising 38% of the total specimens. Barcode analysis showed that maximum conspecific divergence was 1.6%, while there was 1.7-14.3% divergence from the nearest neighbour species. Barcode records for 55 species showed <2% sequence divergence to records in the Barcode of Life Data Systems (BOLD), but only 26 of these cases involved specimens from neighbouring India and Central Asia. Analysis revealed that most species showed little incremental sequence variation when specimens from other regions were considered, but a threefold increase was noted in a few cases. There was a clear gap between maximum intraspecific and minimum nearest neighbour distance for all 81 species. Neighbour-joining cluster analysis showed that members of each species formed a monophyletic cluster with strong bootstrap support. The barcode results revealed two provisional species that could not be clearly linked to known taxa, while 24 other species gained their first coverage. Future work should extend the barcode reference library to include all butterfly species from Pakistan as well as neighbouring countries to gain a better understanding of regional variation in barcode sequences in this topographically and climatically complex region. © 2013 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.

The practical evaluation of DNA barcode efficacy.

PubMed

Spouge, John L; Mariño-Ramírez, Leonardo

2012-01-01

This chapter describes a workflow for measuring the efficacy of a barcode in identifying species. First, assemble individual sequence databases corresponding to each barcode marker. A controlled collection of taxonomic data is preferable to GenBank data, because GenBank data can be problematic, particularly when comparing barcodes based on more than one marker. To ensure proper controls when evaluating species identification, specimens not having a sequence in every marker database should be discarded. Second, select a computer algorithm for assigning species to barcode sequences. No algorithm has yet improved notably on assigning a specimen to the species of its nearest neighbor within a barcode database. Because global sequence alignments (e.g., with the Needleman-Wunsch algorithm, or some related algorithm) examine entire barcode sequences, they generally produce better species assignments than local sequence alignments (e.g., with BLAST). No neighboring method (e.g., global sequence similarity, global sequence distance, or evolutionary distance based on a global alignment) has yet shown a notable superiority in identifying species. Finally, "the probability of correct identification" (PCI) provides an appropriate measurement of barcode efficacy. The overall PCI for a data set is the average of the species PCIs, taken over all species in the data set. This chapter states explicitly how to calculate PCI, how to estimate its statistical sampling error, and how to use data on PCR failure to set limits on how much improvements in PCR technology can improve species identification.

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

PubMed Central

Hadi, Sámed I. I. A.; Santana, Hugo; Brunale, Patrícia P. M.; Gomes, Taísa G.; Oliveira, Márcia D.; Matthiensen, Alexandre; Oliveira, Marcos E. C.; Silva, Flávia C. P.; Brasil, Bruno S. A. F.

2016-01-01

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker. PMID:26900844

Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building

PubMed Central

Peñafiel, Nicolás; Arteaga, Alejandro; Bustamante, Lucas; Pichardo, Frank; Coloma, Luis A; Barrio-Amorós, César L; Salazar-Valenzuela, David; Prost, Stefan

2018-01-01

Abstract Background Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field. Findings We demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding. Conclusions Overall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism. PMID:29617771

First Molecular Identification and Phylogeny of Moroccan Anopheles sergentii (Diptera: Culicidae) Based on Second Internal Transcribed Spencer (ITS2) and Cytochrome c Oxidase I (COI) Sequences.

PubMed

Benabdelkrim Filali, Oumama; Kabine, Mostafa; El Hamouchi, Adil; Lemrani, Meryem; Debboun, Mustapha; Sarih, M'hammed

2018-06-05

Anopheles sergentii known as the "oasis vector" or the "desert malaria vector" is considered the main vector of malaria in the southern parts of Morocco. Its presence in Morocco is confirmed for the first time through sequencing of mitochondrial DNA (mDNA) cytochrome c oxidase subunit I (COI) barcodes and nuclear ribosomal DNA (rDNA) second internal transcribed spacer (ITS2) sequences and direct comparison with specimens of A. sergentii of other countries. The DNA barcodes (n = 39) obtained from A. sergentii collected in 2015 and 2016 showed more diversity with 10 haplotypes, compared with 3 haplotypes obtained from ITS2 sequences (n = 59). Moreover, the comparison using the ITS2 sequences showed closer evolutionary relationship between the Moroccan and Egyptian strains than the Iranian strain. Nevertheless, genetic differences due to geographical segregation were also observed. This study provides the first report on the sequence of rDNA-ITS2 and mtDNA COI, which could be used to better understand the biodiversity of A. sergentii.

Contrasting morphological and DNA barcode-suggested species boundaries among shallow-water amphipod fauna from the southern European Atlantic coast.

PubMed

Lobo, Jorge; Ferreira, Maria S; Antunes, Ilisa C; Teixeira, Marcos A L; Borges, Luisa M S; Sousa, Ronaldo; Gomes, Pedro A; Costa, Maria Helena; Cunha, Marina R; Costa, Filipe O

2017-02-01

In this study we compared DNA barcode-suggested species boundaries with morphology-based species identifications in the amphipod fauna of the southern European Atlantic coast. DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) were generated for 43 morphospecies (178 specimens) collected along the Portuguese coast which, together with publicly available COI-5P sequences, produced a final dataset comprising 68 morphospecies and 295 sequences. Seventy-five BINs (Barcode Index Numbers) were assigned to these morphospecies, of which 48 were concordant (i.e., 1 BIN = 1 species), 8 were taxonomically discordant, and 19 were singletons. Twelve species had matching sequences (<2% distance) with conspecifics from distant locations (e.g., North Sea). Seven morphospecies were assigned to multiple, and highly divergent, BINs, including specimens of Corophium multisetosum (18% divergence) and Dexamine spiniventris (16% divergence), which originated from sampling locations on the west coast of Portugal (only about 36 and 250 km apart, respectively). We also found deep divergence (4%-22%) among specimens of seven species from Portugal compared to those from the North Sea and Italy. The detection of evolutionarily meaningful divergence among populations of several amphipod species from southern Europe reinforces the need for a comprehensive re-assessment of the diversity of this faunal group.

DNA Barcoding analysis of seafood accuracy in Washington, D.C. restaurants

PubMed Central

Stern, David B.; Castro Nallar, Eduardo; Rathod, Jason

2017-01-01

In Washington D.C., recent legislation authorizes citizens to test if products are properly represented and, if they are not, to bring a lawsuit for the benefit of the general public. Recent studies revealing the widespread phenomenon of seafood substitution across the United States make it a fertile area for consumer protection testing. DNA barcoding provides an accurate and cost-effective way to perform these tests, especially when tissue alone is available making species identification based on morphology impossible. In this study, we sequenced the 5′ barcoding region of the Cytochrome Oxidase I gene for 12 samples of vertebrate and invertebrate food items across six restaurants in Washington, D.C. and used multiple analytical methods to make identifications. These samples included several ambiguous menu listings, sequences with little genetic variation among closely related species and one sequence with no available reference sequence. Despite these challenges, we were able to make identifications for all samples and found that 33% were potentially mislabeled. While we found a high degree of mislabeling, the errors involved closely related species and we did not identify egregious substitutions as have been found in other cities. This study highlights the efficacy of DNA barcoding and robust analyses in identifying seafood items for consumer protection. PMID:28462038

Identification of Rays through DNA Barcoding: An Application for Ecologists

PubMed Central

Cerutti-Pereyra, Florencia; Meekan, Mark G.; Wei, Nu-Wei V.; O'Shea, Owen; Bradshaw, Corey J. A.; Austin, Chris M.

2012-01-01

DNA barcoding potentially offers scientists who are not expert taxonomists a powerful tool to support the accuracy of field studies involving taxa that are diverse and difficult to identify. The taxonomy of rays has received reasonable attention in Australia, although the fauna in remote locations such as Ningaloo Reef, Western Australia is poorly studied and the identification of some species in the field is problematic. Here, we report an application of DNA-barcoding to the identification of 16 species (from 10 genera) of tropical rays as part of an ecological study. Analysis of the dataset combined across all samples grouped sequences into clearly defined operational taxonomic units, with two conspicuous exceptions: the Neotrygon kuhlii species complex and the Aetobatus species complex. In the field, the group that presented the most difficulties for identification was the spotted whiptail rays, referred to as the ‘uarnak’ complex. Two sets of problems limited the successful application of DNA barcoding: (1) the presence of cryptic species, species complexes with unresolved taxonomic status and intra-specific geographical variation, and (2) insufficient numbers of entries in online databases that have been verified taxonomically, and the presence of lodged sequences in databases with inconsistent names. Nevertheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to highlight species complexes where taxonomic uncertainty might confound ecological data. PMID:22701556

Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

PubMed

Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

2017-06-15

Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R 2 =0.9984) between the fluorescent intensity and the target DNA concentration in the samples. Copyright © 2016. Published by Elsevier B.V.

Land plants and DNA barcodes: short-term and long-term goals.

PubMed

Chase, Mark W; Salamin, Nicolas; Wilkinson, Mike; Dunwell, James M; Kesanakurthi, Rao Prasad; Haider, Nadia; Haidar, Nadia; Savolainen, Vincent

2005-10-29

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.

Land plants and DNA barcodes: short-term and long-term goals

PubMed Central

Chase, Mark W; Salamin, Nicolas; Wilkinson, Mike; Dunwell, James M; Kesanakurthi, Rao Prasad; Haidar, Nadia; Savolainen, Vincent

2005-01-01

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the ‘genetic gaps’ that are useful in assessing species limits. PMID:16214746

Application of DNA Barcodes in Asian Tropical Trees – A Case Study from Xishuangbanna Nature Reserve, Southwest China

PubMed Central

Conran, John G.; Li, Jie

2015-01-01

Background Within a regional floristic context, DNA barcoding is more useful to manage plant diversity inventories on a large scale and develop valuable conservation strategies. However, there are no DNA barcode studies from tropical areas of China, which represents one of the biodiversity hotspots around the world. Methodology and Principal Findings A DNA barcoding database of an Asian tropical trees with high diversity was established at Xishuangbanna Nature Reserve, Yunnan, southwest China using rbcL and matK as standard barcodes, as well as trnH–psbA and ITS as supplementary barcodes. The performance of tree species identification success was assessed using 2,052 accessions from four plots belonging to two vegetation types in the region by three methods: Neighbor-Joining, Maximum-Likelihood and BLAST. We corrected morphological field identification errors (9.6%) for the three plots using rbcL and matK based on Neighbor-Joining tree. The best barcode region for PCR and sequencing was rbcL (97.6%, 90.8%), followed by trnH–psbA (93.6%, 85.6%), while matK and ITS obtained relative low PCR and sequencing success rates. However, ITS performed best for both species (44.6–58.1%) and genus (72.8–76.2%) identification. With trnH–psbA slightly less effective for species identification. The two standard barcode rbcL and matK gave poor results for species identification (24.7–28.5% and 31.6–35.3%). Compared with other studies from comparable tropical forests (e.g. Cameroon, the Amazon and India), the overall performance of the four barcodes for species identification was lower for the Xishuangbanna Nature Reserve, possibly because of species/genus ratios and species composition between these tropical areas. Conclusions/Significance Although the core barcodes rbcL and matK were not suitable for species identification of tropical trees from Xishuangbanna Nature Reserve, they could still help with identification at the family and genus level. Considering the relative sequence recovery and the species identification performance, we recommend the use of trnH–psbA and ITS in combination as the preferred barcodes for tropical tree species identification in China. PMID:26121045

DNA Barcoding of Sigmodontine Rodents: Identifying Wildlife Reservoirs of Zoonoses

PubMed Central

Müller, Lívia; Gonçalves, Gislene L.; Cordeiro-Estrela, Pedro; Marinho, Jorge R.; Althoff, Sérgio L.; Testoni, André. F.; González, Enrique M.; Freitas, Thales R. O.

2013-01-01

Species identification through DNA barcoding is a tool to be added to taxonomic procedures, once it has been validated. Applying barcoding techniques in public health would aid in the identification and correct delimitation of the distribution of rodents from the subfamily Sigmodontinae. These rodents are reservoirs of etiological agents of zoonoses including arenaviruses, hantaviruses, Chagas disease and leishmaniasis. In this study we compared distance-based and probabilistic phylogenetic inference methods to evaluate the performance of cytochrome c oxidase subunit I (COI) in sigmodontine identification. A total of 130 sequences from 21 field-trapped species (13 genera), mainly from southern Brazil, were generated and analyzed, together with 58 GenBank sequences (24 species; 10 genera). Preliminary analysis revealed a 9.5% rate of misidentifications in the field, mainly of juveniles, which were reclassified after examination of external morphological characters and chromosome numbers. Distance and model-based methods of tree reconstruction retrieved similar topologies and monophyly for most species. Kernel density estimation of the distance distribution showed a clear barcoding gap with overlapping of intraspecific and interspecific densities < 1% and 21 species with mean intraspecific distance < 2%. Five species that are reservoirs of hantaviruses could be identified through DNA barcodes. Additionally, we provide information for the description of a putative new species, as well as the first COI sequence of the recently described genus Drymoreomys. The data also indicated an expansion of the distribution of Calomys tener. We emphasize that DNA barcoding should be used in combination with other taxonomic and systematic procedures in an integrative framework and based on properly identified museum collections, to improve identification procedures, especially in epidemiological surveillance and ecological assessments. PMID:24244670

Potential efficacy of mitochondrial genes for animal DNA barcoding: a case study using eutherian mammals.

PubMed

Luo, Arong; Zhang, Aibing; Ho, Simon Yw; Xu, Weijun; Zhang, Yanzhou; Shi, Weifeng; Cameron, Stephen L; Zhu, Chaodong

2011-01-28

A well-informed choice of genetic locus is central to the efficacy of DNA barcoding. Current DNA barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase 1 gene (CO1) to diagnose and delimit species. However, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal CO1 barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. Four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation. Based on 1,179 mitochondrial genomes of eutherians, we found that the universal CO1 barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate (> 90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. Our results also demonstrated that any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels. We suggest that the CO1 barcoding region, the universal DNA barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. Nevertheless, DNA barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups.

Potential efficacy of mitochondrial genes for animal DNA barcoding: a case study using eutherian mammals

PubMed Central

2011-01-01

Background A well-informed choice of genetic locus is central to the efficacy of DNA barcoding. Current DNA barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase 1 gene (CO1) to diagnose and delimit species. However, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal CO1 barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. Four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation. Results Based on 1,179 mitochondrial genomes of eutherians, we found that the universal CO1 barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate (> 90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. Our results also demonstrated that any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels. Conclusions We suggest that the CO1 barcoding region, the universal DNA barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. Nevertheless, DNA barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups. PMID:21276253

Evaluating the feasibility of using candidate DNA barcodes in discriminating species of the large Asteraceae family

PubMed Central

2010-01-01

Background Five DNA regions, namely, rbcL, matK, ITS, ITS2, and psbA-trnH, have been recommended as primary DNA barcodes for plants. Studies evaluating these regions for species identification in the large plant taxon, which includes a large number of closely related species, have rarely been reported. Results The feasibility of using the five proposed DNA regions was tested for discriminating plant species within Asteraceae, the largest family of flowering plants. Among these markers, ITS2 was the most useful in terms of universality, sequence variation, and identification capability in the Asteraceae family. The species discriminating power of ITS2 was also explored in a large pool of 3,490 Asteraceae sequences that represent 2,315 species belonging to 494 different genera. The result shows that ITS2 correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively. In addition, ITS2 displayed a variable ability to discriminate related species within different genera. Conclusions ITS2 is the best DNA barcode for the Asteraceae family. This approach significantly broadens the application of DNA barcoding to resolve classification problems in the family Asteraceae at the genera and species levels. PMID:20977734

Integrating early detection with DNA barcoding: species identification of a non-native monitor lizard (Squamata: Varanidae) carcass in Mississippi, U.S.A.

USGS Publications Warehouse

Reed, Robert N.; Hopken, Matthew W.; Steen, David A.; Falk, Bryan G.; Piaggio, Antoinette J.

2016-01-01

Early detection of invasive species is critical to increasing the probability of successful management. At the primary stage of an invasion, invasive species are easier to control as the population is likely represented by just a few individuals. Detection of these first few individuals can be challenging, particularly if they are cryptic or otherwise characterized by low detectability. The engagement of members of the public may be critical to early detection as there are far more citizen s on the landscape than trained biologists. However, it can be difficult to assess the credibility of public reporting, especially when a diagnostic digital image or a physical specimen in good condition are lacking. DNA barcoding can be used for verification when morphological identification of a specimen is not possible or uncertain (i.e., degraded or partial specimen). DNA barcoding relies on obtaining a DNA sequence from a relatively small fragment of mitochondrial DNA and comparing it to a database of sequences containing a variety of expertly identified species. He rein we report the successful identification of a degraded specimen of a non-native, potentially invasive reptile species (Varanus niloticus) via DNA barcoding, after discovery and reporting by a member of the public.

A new species of the genus Hyalessa (Hemiptera, Cicadidae) from China, with DNA barcoding data and a key to related species.

PubMed

Wang, Xu; Qiu, Yue; Wei, Cong

2016-03-02

One new species of the genus Hyalessa China, H. wangi sp. nov., from Yunnan, China is described. Partial mitochondrial COI gene (DNA barcoding) of this new species is sequenced and uploaded to GenBank. A key to all species of Hyalessa is provided.

Efficiency of ITS Sequences for DNA Barcoding in Passiflora (Passifloraceae)

PubMed Central

Giudicelli, Giovanna Câmara; Mäder, Geraldo; de Freitas, Loreta Brandão

2015-01-01

DNA barcoding is a technique for discriminating and identifying species using short, variable, and standardized DNA regions. Here, we tested for the first time the performance of plastid and nuclear regions as DNA barcodes in Passiflora. This genus is a largely variable, with more than 900 species of high ecological, commercial, and ornamental importance. We analyzed 1034 accessions of 222 species representing the four subgenera of Passiflora and evaluated the effectiveness of five plastid regions and three nuclear datasets currently employed as DNA barcodes in plants using barcoding gap, applied similarity-, and tree-based methods. The plastid regions were able to identify less than 45% of species, whereas the nuclear datasets were efficient for more than 50% using “best match” and “best close match” methods of TaxonDNA software. All subgenera presented higher interspecific pairwise distances and did not fully overlap with the intraspecific distance, and similarity-based methods showed better results than tree-based methods. The nuclear ribosomal internal transcribed spacer 1 (ITS1) region presented a higher discrimination power than the other datasets and also showed other desirable characteristics as a DNA barcode for this genus. Therefore, we suggest that this region should be used as a starting point to identify Passiflora species. PMID:25837628

DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market.

PubMed

Mishra, Priyanka; Kumar, Amit; Nagireddy, Akshitha; Mani, Daya N; Shukla, Ashutosh K; Tiwari, Rakesh; Sundaresan, Velusamy

2016-01-01

The past couple of decades have witnessed global resurgence of herbal-based health care. As a result, the trade of raw drugs has surged globally. Accurate and fast scientific identification of the plant(s) is the key to success for the herbal drug industry. The conventional approach is to engage an expert taxonomist, who uses a mix of traditional and modern techniques for precise plant identification. However, for bulk identification at industrial scale, the process is protracted and time-consuming. DNA barcoding, on the other hand, offers an alternative and feasible taxonomic tool box for rapid and robust species identification. For the success of DNA barcode, the barcode loci must have sufficient information to differentiate unambiguously between closely related plant species and discover new cryptic species. For herbal plant identification, matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA and 18S-rRNA have been used as successful DNA barcodes. Emerging advances in DNA barcoding coupled with next-generation sequencing and high-resolution melting curve analysis have paved the way for successful species-level resolution recovered from finished herbal products. Further, development of multilocus strategy and its application has provided new vistas to the DNA barcode-based plant identification for herbal drug industry. For successful and acceptable identification of herbal ingredients and a holistic quality control of the drug, DNA barcoding needs to work harmoniously with other components of the systems biology approach. We suggest that for effectively resolving authentication challenges associated with the herbal market, DNA barcoding must be used in conjunction with metabolomics along with need-based transcriptomics and proteomics. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

DNA Barcoding of Rhodiola (Crassulaceae): A Case Study on a Group of Recently Diversified Medicinal Plants from the Qinghai-Tibetan Plateau

PubMed Central

Zhang, Jian-Qiang; Meng, Shi-Yong; Wen, Jun; Rao, Guang-Yuan

2015-01-01

DNA barcoding, the identification of species using one or a few short standardized DNA sequences, is an important complement to traditional taxonomy. However, there are particular challenges for barcoding plants, especially for species with complex evolutionary histories. We herein evaluated the utility of five candidate sequences — rbcL, matK, trnH-psbA, trnL-F and the internal transcribed spacer (ITS) — for barcoding Rhodiola species, a group of high-altitude plants frequently used as adaptogens, hemostatics and tonics in traditional Tibetan medicine. Rhodiola was suggested to have diversified rapidly recently. The genus is thus a good model for testing DNA barcoding strategies for recently diversified medicinal plants. This study analyzed 189 accessions, representing 47 of the 55 recognized Rhodiola species in the Flora of China treatment. Based on intraspecific and interspecific divergence and degree of monophyly statistics, ITS was the best single-locus barcode, resolving 66% of the Rhodiola species. The core combination rbcL+matK resolved only 40.4% of them. Unsurprisingly, the combined use of all five loci provided the highest discrimination power, resolving 80.9% of the species. However, this is weaker than the discrimination power generally reported in barcoding studies of other plant taxa. The observed complications may be due to the recent diversification, incomplete lineage sorting and reticulate evolution of the genus. These processes are common features of numerous plant groups in the high-altitude regions of the Qinghai-Tibetan Plateau. PMID:25774915

DNA barcoding reveal patterns of species diversity among northwestern Pacific molluscs

PubMed Central

Sun, Shao’e; Li, Qi; Kong, Lingfeng; Yu, Hong; Zheng, Xiaodong; Yu, Ruihai; Dai, Lina; Sun, Yan; Chen, Jun; Liu, Jun; Ni, Lehai; Feng, Yanwei; Yu, Zhenzhen; Zou, Shanmei; Lin, Jiping

2016-01-01

This study represents the first comprehensive molecular assessment of northwestern Pacific molluscs. In total, 2801 DNA barcodes belonging to 569 species from China, Japan and Korea were analyzed. An overlap between intra- and interspecific genetic distances was present in 71 species. We tested the efficacy of this library by simulating a sequence-based specimen identification scenario using Best Match (BM), Best Close Match (BCM) and All Species Barcode (ASB) criteria with three threshold values. BM approach returned 89.15% true identifications (95.27% when excluding singletons). The highest success rate of congruent identifications was obtained with BCM at 0.053 threshold. The analysis of our barcode library together with public data resulted in 582 Barcode Index Numbers (BINs), 72.2% of which was found to be concordantly with morphology-based identifications. The discrepancies were divided in two groups: sequences from different species clustered in a single BIN and conspecific sequences divided in one more BINs. In Neighbour-Joining phenogram, 2,320 (83.0%) queries fromed 355 (62.4%) species-specific barcode clusters allowing their successful identification. 33 species showed paraphyletic and haplotype sharing. 62 cases are represented by deeply diverged lineages. This study suggest an increased species diversity in this region, highlighting taxonomic revision and conservation strategy for the cryptic complexes. PMID:27640675

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

PubMed Central

Miner, Brooks E.; Stöger, Reinhard J.; Burden, Alice F.; Laird, Charles D.; Hansen, R. Scott

2004-01-01

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular ‘batch-stamps’ that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes. PMID:15459281

DNA barcoding of Bemisia tabaci complex (Hemiptera: Aleyrodidae) reveals southerly expansion of the dominant whitefly species on cotton in Pakistan.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N; Mirza, M Sajjad; Khan, Arif M; Mansoor, Shahid; Shah, Ghulam S; Zafar, Yusuf

2014-01-01

Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan. Sequence diversity in the DNA barcode region (mtCOI-5') was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3' to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage "Pakistan". The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and "Pakistan" were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan. DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region.

DNA barcoding of freshwater fishes and the development of a quantitative qPCR assay for the species-specific detection and quantification of fish larvae from plankton samples.

PubMed

Loh, W K W; Bond, P; Ashton, K J; Roberts, D T; Tibbetts, I R

2014-08-01

The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0.52 ± 0.10 and 23.8 ± 2.20% respectively, indicating that barcodes can be used to discriminate most of the fish species accurately. The two terapontids, Amniataba percoides and Leiopotherapon unicolor, however, shared coI DNA sequences and could not be differentiated using this gene. A barcoding database was established and a qPCR assay was developed using coI sequences to identify and quantify proportional abundances of fish species in ichthyoplankton samples from Lake Wivenhoe. These methods provide a viable alternative to the time-consuming process of manually enumerating and identifying ichthyoplankton samples. © 2014 The Fisheries Society of the British Isles.

DNA barcoding of perennial fruit tree species of agronomic interest in the genus Annona (Annonaceae)

PubMed Central

Larranaga, Nerea; Hormaza, José I.

2015-01-01

The DNA barcode initiative aims to establish a universal protocol using short genetic sequences to discriminate among animal and plant species. Although many markers have been proposed to become the barcode of plants, the Consortium for the Barcode of Life (CBOL) Plant Working Group recommended using as a core the combination of two portions of plastid coding region, rbcL and matK. In this paper, specific markers based on matK sequences were developed for 7 closely related Annona species of agronomic interest (Annona cherimola, A. reticulata, A. squamosa, A. muricata, A. macroprophyllata, A. glabra, and A. purpurea) and the discrimination power of both rbcL and matK was tested using also sequences of the genus Annona available in the Barcode of Life Database (BOLD) data systems. The specific sequences developed allowed the discrimination among all those species tested. Moreover, the primers generated were validated in six additional species of the genus (A. liebmanniana, A. longiflora, A. montana, A. senegalensis, A. emarginata and A. neosalicifolia) and in an interspecific hybrid (A. cherimola x A. squamosa). The development of a fast, reliable and economic approach for species identification in these underutilized subtropical fruit crops in a very initial state of domestication is of great importance in order to optimize genetic resource management. PMID:26284104

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

PubMed Central

Sonet, Gontran; Jordaens, Kurt; Braet, Yves; Bourguignon, Luc; Dupont, Eréna; Backeljau, Thierry; De Meyer, Marc; Desmyter, Stijn

2013-01-01

Abstract Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available. We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes. PMID:24453564

Suitable DNA Barcoding for Identification and Supervision of Piper kadsura in Chinese Medicine Markets.

PubMed

Yu, Ning; Gu, Hong; Wei, Yulong; Zhu, Ning; Wang, Yanli; Zhang, Haiping; Zhu, Yue; Zhang, Xin; Ma, Chao; Sun, Aidong

2016-09-12

Piper kadsura is a vine-like medicinal plant which is widely used in clinical treatment. However, P. kadsura is often substituted by other materials in the markets, thereby causing health risks. In this study, 38 P. kadsura samples and eight sequences from GenBank, including a closely-related species and common adulterants were collected. This study aimed to identify an effective DNA barcode from four popular DNA loci for P. kadsura authentication. The success rates of PCR amplification, sequencing, and sequence acquisition of matK were 10.5%, 75%, and 7.9%, respectively; for rbcL they were 89.5%, 8.8%, and 7.9%, respectively; ITS2 rates were 86.8%, 3.0%, and 2.6%, respectively, while for psbA-trnH they were all 100%, which is much higher than for the other three loci. The sequences were aligned using Muscle, genetic distances were computed using MEGA 5.2.2, and barcoding gap was performed using TAXON DNA. Phylogenetic analysis showed that psbA-trnH could clearly distinguish P. kadsura from its closely related species and the common adulterant. psbA-trnH was then used to evaluate the fake proportions of P. kadsura. Results showed that 18.4% of P. kadsura samples were fake, indicating that adulterant species exist in the Chinese markets. Two-dimensional DNA barcoding imaging of P. kadsura was conducted, which was beneficial to the management of P. kadsura. We conclude that the psbA-trnH region is a powerful tool for P. kadsura identification and supervision in the current medicine markets.

[Identification of common medicinal snakes in medicated liquor of Guangdong by COI barcode sequence].

PubMed

Liao, Jing; Chao, Zhi; Zhang, Liang

2013-11-01

To identify the common snakes in medicated liquor of Guangdong using COI barcode sequence,and to test the feasibility. The COI barcode sequences of collected medicinal snakes were amplified and sequenced. The sequences combined with the data from GenBank were analyzed for divergence and building a neighbor-joining(NJ) tree with MEGA 5.0. The genetic distance and NJ tree demonstrated that there were 241 variable sites in these species, and the average (A + T) content of 56.2% was higher than the average (G + C) content of 43.7%. The maximum interspecific genetic distance was 0.2568, and the minimum was 0. 1519. In the NJ tree,each species formed a monophyletic clade with bootstrap supports of 100%. DNA barcoding identification method based on the COI sequence is accurate and can be applied to identify the common medicinal snakes.

Prospects and Problems for Identification of Poisonous Plants in China using DNA Barcodes.

PubMed

Xie, Lei; Wang, Ying Wei; Guan, Shan Yue; Xie, Li Jing; Long, Xin; Sun, Cheng Ye

2014-10-01

Poisonous plants are a deadly threat to public health in China. The traditional clinical diagnosis of the toxic plants is inefficient, fallible, and dependent upon experts. In this study, we tested the performance of DNA barcodes for identification of the most threatening poisonous plants in China. Seventy-four accessions of 27 toxic plant species in 22 genera and 17 families were sampled and three DNA barcodes (matK, rbcL, and ITS) were amplified, sequenced and tested. Three methods, Blast, pairwise global alignment (PWG) distance, and Tree-Building were tested for discrimination power. The primer universality of all the three markers was high. Except in the case of ITS for Hemerocallis minor, the three barcodes were successfully generated from all the selected species. Among the three methods applied, Blast showed the lowest discrimination rate, whereas PWG Distance and Tree-Building methods were equally effective. The ITS barcode showed highest discrimination rates using the PWG Distance and Tree-Building methods. When the barcodes were combined, discrimination rates were increased for the Blast method. DNA barcoding technique provides us a fast tool for clinical identification of poisonous plants in China. We suggest matK, rbcL, ITS used in combination as DNA barcodes for authentication of poisonous plants. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Utility of DNA barcoding for rapid and accurate assessment of bat diversity in Malaysia in the absence of formally described species.

PubMed

Wilson, J-J; Sing, K-W; Halim, M R A; Ramli, R; Hashim, R; Sofian-Azirun, M

2014-02-19

Bats are important flagship species for biodiversity research; however, diversity in Southeast Asia is considerably underestimated in the current checklists and field guides. Incorporation of DNA barcoding into surveys has revealed numerous species-level taxa overlooked by conventional methods. Inclusion of these taxa in inventories provides a more informative record of diversity, but is problematic as these species lack formal description. We investigated how frequently documented, but undescribed, bat taxa are encountered in Peninsular Malaysia. We discuss whether a barcode library provides a means of recognizing and recording these taxa across biodiversity inventories. Tissue was sampled from bats trapped at Pasir Raja, Dungun Terengganu, Peninsular Malaysia. The DNA was extracted and the COI barcode region amplified and sequenced. We identified 9 species-level taxa within our samples, based on analysis of the DNA barcodes. Six specimens matched to four previously documented taxa considered candidate species but currently lacking formal taxonomic status. This study confirms the high diversity of bats within Peninsular Malaysia (9 species in 13 samples) and demonstrates how DNA barcoding allows for inventory and documentation of known taxa lacking formal taxonomic status.

Cytochrome C oxidase subunit I barcodes provide an efficient tool for Jinqian Baihua She (Bungarus parvus) authentication

PubMed Central

Chao, Zhi; Liao, Jing; Liang, Zhenbiao; Huang, Suhua; Zhang, Liang; Li, Junde

2014-01-01

Objective: To test the feasibility of DNA barcoding for accurate identification of Jinqian Baihua She and its adulterants. Materials and Methods: Standard cytochrome C oxidase subunit I (COI) gene fragments were sequenced for DNA barcoding of 39 samples from 9 snake species, including Bungarus multicinctus, the officially recognized origin animal by Chinese Pharmacopoeia, and other 8 adulterate species. The aligned sequences, 658 base pairs in length, were analyzed for divergence using the Kimura-2-parameter (K2P) distance model with MEGA5.0. Results: The mean intraspecific K2P distance was 0.0103 and the average interspecific genetic distance was 0.2178 in B. multicinctus, far greater than the minimal interspecific genetic distance of 0.027 recommended for species identification. A neighbor-joining (NJ) tree was constructed, in which each species formed a monophyletic clade with bootstrap supports of 100%. All the data were submitted to Barcode of Life Data system version 3.0 (BOLD, http://www.barcodinglife.org) under the project title “DNA barcoding Bungarus multicinctus and its adulterants”. Ten samples of commercially available crude drugs of JBS were identified using the identification engine provided by BOLD. All the samples were clearly identified at the species level, among which five were found to be the adulterants and identified as Dinodon rufozonatum. Conclusion: DNA barcoding using the standard COI gene fragments provides an effective and accurate means for JBS identification and authentication. PMID:25422545

Application of DNA barcoding in biodiversity studies of shallow-water octocorals: molecular proxies agree with morphological estimates of species richness in Palau

NASA Astrophysics Data System (ADS)

McFadden, C. S.; Brown, A. S.; Brayton, C.; Hunt, C. B.; van Ofwegen, L. P.

2014-06-01

The application of DNA barcoding to anthozoan cnidarians has been hindered by their slow rates of mitochondrial gene evolution and the failure to identify alternative molecular markers that distinguish species reliably. Among octocorals, however, multilocus barcodes can distinguish up to 70 % of morphospecies, thereby facilitating the identification of species that are ecologically important but still very poorly known taxonomically. We tested the ability of these imperfect DNA barcodes to estimate species richness in a biodiversity survey of the shallow-water octocoral fauna of Palau using multilocus ( COI, mtMutS, 28S rDNA) sequences obtained from 305 specimens representing 38 genera of octocorals. Numbers and identities of species were estimated independently (1) by a taxonomic expert using morphological criteria and (2) by assigning sequences to molecular operational taxonomic units (MOTUs) using predefined genetic distance thresholds. Estimated numbers of MOTUs ranged from 73 to 128 depending on the barcode and distance threshold applied, bracketing the estimated number of 118 morphospecies. Concordance between morphospecies identifications and MOTUs ranged from 71 to 75 % and differed little among barcodes. For the speciose and ecologically dominant genus Sinularia, however, we were able to identify 95 % of specimens correctly simply by comparing mtMutS sequences and in situ photographs of colonies to an existing vouchered database. Because we lack a clear understanding of species boundaries in most of these taxa, numbers of morphospecies and MOTUs are both estimates of the true species diversity, and we cannot currently determine which is more accurate. Our results suggest, however, that the two methods provide comparable estimates of species richness for shallow-water Indo-Pacific octocorals. Use of molecular barcodes in biodiversity surveys will facilitate comparisons of species richness and composition among localities and over time, data that do not currently exist for any octocoral community.

A Transcontinental Challenge — A Test of DNA Barcode Performance for 1,541 Species of Canadian Noctuoidea (Lepidoptera)

PubMed Central

Zahiri, Reza; Lafontaine, J. Donald; Schmidt, B. Christian; deWaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

2014-01-01

This study provides a first, comprehensive, diagnostic use of DNA barcodes for the Canadian fauna of noctuoids or “owlet” moths (Lepidoptera: Noctuoidea) based on vouchered records for 1,541 species (99.1% species coverage), and more than 30,000 sequences. When viewed from a Canada-wide perspective, DNA barcodes unambiguously discriminate 90% of the noctuoid species recognized through prior taxonomic study, and resolution reaches 95.6% when considered at a provincial scale. Barcode sharing is concentrated in certain lineages with 54% of the cases involving 1.8% of the genera. Deep intraspecific divergence exists in 7.7% of the species, but further studies are required to clarify whether these cases reflect an overlooked species complex or phylogeographic variation in a single species. Non-native species possess higher Nearest-Neighbour (NN) distances than native taxa, whereas generalist feeders have lower NN distances than those with more specialized feeding habits. We found high concordance between taxonomic names and sequence clusters delineated by the Barcode Index Number (BIN) system with 1,082 species (70%) assigned to a unique BIN. The cases of discordance involve both BIN mergers and BIN splits with 38 species falling into both categories, most likely reflecting bidirectional introgression. One fifth of the species are involved in a BIN merger reflecting the presence of 158 species sharing their barcode sequence with at least one other taxon, and 189 species with low, but diagnostic COI divergence. A very few cases (13) involved species whose members fell into both categories. Most of the remaining 140 species show a split into two or three BINs per species, while Virbia ferruginosa was divided into 16. The overall results confirm that DNA barcodes are effective for the identification of Canadian noctuoids. This study also affirms that BINs are a strong proxy for species, providing a pathway for a rapid, accurate estimation of animal diversity. PMID:24667847

DNA barcoding and the identification of tree frogs (Amphibia: Anura: Rhacophoridae).

PubMed

Dang, Ning-Xin; Sun, Feng-Hui; Lv, Yun-Yun; Zhao, Bo-Han; Wang, Ji-Chao; Murphy, Robert W; Wang, Wen-Zhi; Li, Jia-Tang

2016-07-01

The DNA barcoding gene COI (cytochrome c oxidase subunit I) effectively identifies many species. Herein, we barcoded 172 individuals from 37 species belonging to nine genera in Rhacophoridae to test if the gene serves equally well to identify species of tree frogs. Phenetic neighbor joining and phylogenetic Bayesian inference were used to construct phylogenetic trees, which resolved all nine genera as monophyletic taxa except for Rhacophorus, two new matrilines for Liuixalus, and Polypedates leucomystax species complex. Intraspecific genetic distances ranged from 0.000 to 0.119 and interspecific genetic distances ranged from 0.015 to 0.334. Within Rhacophorus and Kurixalus, the intra- and interspecific genetic distances did not reveal an obvious barcode gap. Notwithstanding, we found that COI sequences unambiguously identified rhacophorid species and helped to discover likely new cryptic species via the synthesis of genealogical relationships and divergence patterns. Our results supported that COI is an effective DNA barcoding marker for Rhacophoridae.

An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

PubMed

Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

2013-10-15

Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. © 2013 Elsevier B.V. All rights reserved.

bold: The Barcode of Life Data System (http://www.barcodinglife.org)

PubMed Central

RATNASINGHAM, SUJEEVAN; HEBERT, PAUL D N

2007-01-01

The Barcode of Life Data System (bold) is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm. bold is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances. This paper provides a brief introduction to the key elements of bold, discusses their functional capabilities, and concludes by examining computational resources and future prospects. PMID:18784790

Developing an Apicomplexan DNA Barcoding System to Detect Blood Parasites of Small Coral Reef Fishes.

PubMed

Renoux, Lance P; Dolan, Maureen C; Cook, Courtney A; Smit, Nico J; Sikkel, Paul C

2017-08-01

Apicomplexan parasites are obligate parasites of many species of vertebrates. To date, there is very limited understanding of these parasites in the most-diverse group of vertebrates, actinopterygian fishes. While DNA barcoding targeting the eukaryotic 18S small subunit rRNA gene sequence has been useful in identifying apicomplexans in tetrapods, identification of apicomplexans infecting fishes has relied solely on morphological identification by microscopy. In this study, a DNA barcoding method was developed that targets the 18S rRNA gene primers for identifying apicomplexans parasitizing certain actinopterygian fishes. A lead primer set was selected showing no cross-reactivity to the overwhelming abundant host DNA and successfully confirmed 37 of the 41 (90.2%) microscopically verified parasitized fish blood samples analyzed in this study. Furthermore, this DNA barcoding method identified 4 additional samples that screened negative for parasitemia, suggesting this molecular method may provide improved sensitivity over morphological characterization by microscopy. In addition, this PCR screening method for fish apicomplexans, using Whatman FTA preserved DNA, was tested in efforts leading to a more simplified field collection, transport, and sample storage method as well as a streamlining sample processing important for DNA barcoding of large sample sets.

Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines

PubMed Central

2009-01-01

Background Parthenium argentatum (guayule) is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of P. argentatum was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, Lactuca sativa, Guitozia abyssinica and Helianthus annuus revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of Parthenium species and lines. Results The complete plastid genome of P. argentatum is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in L. sativa, G. abyssinica and H. annuus, we demonstrate that the P. argentatum chloroplast genome sequence is most closely related to that of H. annuus. Similar to chloroplast genomes in G. abyssinica, L. sativa and H. annuus, the plastid genome of P. argentatum has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the matK and psbA-trnH spacer chloroplast DNA barcodes, three of the four Parthenium species tested, P. tomentosum, P. hysterophorus and P. schottii, can be differentiated from P. argentatum. In addition, we identified lines within P. argentatum. Conclusion The genome sequence of the P. argentatum chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate transformation efficiency by using the precise sequence of endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially significant lines of P. argentatum that are important for producing latex. PMID:19917140

DNA barcoding of gypsy moths from China (Lepidoptera: Erebidae) reveals new haplotypes and divergence patterns within gypsy moth subspecies

Treesearch

Fang Chen; Youqing Luo; Melody A. Keena; Ying Wu; Peng Wu; Juan Shi

2015-01-01

The gypsy moth from Asia (two subspecies) is considered a greater threat to North America than European gypsy moth, because of a broader host range and females being capable of flight. Variation within and among gypsy moths from China (nine locations), one of the native countries of Asian gypsy moth, were compared using DNA barcode sequences (658 bp of mtDNA cytochrome...

Role of DNA barcoding in marine biodiversity assessment and conservation: An update

PubMed Central

Trivedi, Subrata; Aloufi, Abdulhadi A.; Ansari, Abid A.; Ghosh, Sankar K.

2015-01-01

More than two third area of our planet is covered by oceans and assessment of marine biodiversity is a challenging task. With the increasing global population, there is a tendency to exploit marine resources for food, energy and other requirements. This puts pressure on the fragile marine environment and necessitates sustainable conservation efforts. Marine species identification using traditional taxonomical methods is often burdened with taxonomic controversies. Here we discuss the comparatively new concept of DNA barcoding and its significance in marine perspective. This molecular technique can be useful in the assessment of cryptic species which is widespread in marine environment and linking the different life cycle stages to the adult which is difficult to accomplish in the marine ecosystem. Other advantages of DNA barcoding include authentication and safety assessment of seafood, wildlife forensics, conservation genetics and detection of invasive alien species (IAS). Global DNA barcoding efforts in the marine habitat include MarBOL, CeDAMar, CMarZ, SHARK-BOL, etc. An overview on DNA barcoding of different marine groups ranging from the microbes to mammals is revealed. In conjugation with newer and faster techniques like high-throughput sequencing, DNA barcoding can serve as an effective modern tool in marine biodiversity assessment and conservation. PMID:26980996

Testing the utility of matK and ITS DNA regions for discrimination of Allium species

USDA-ARS?s Scientific Manuscript database

Molecular phylogenetic analysis of the genus Allium L. has been mainly based on the nucleotide sequences of ITS region. In 2009 matK and rbcL were accepted as a two-locus DNA barcode to classify plant species by the Consortium for the Barcode of Life (CBOL) Plant Working Group. MatK region has been ...

Using DNA barcodes to identify a bird involved in a birdstrike at a Chinese airport.

PubMed

Yang, Rong; Wu, Xiaobing; Yan, Peng; Li, Xiaoqiang

2010-10-01

One day at dusk in August, 200X, an airplane was struck by a bird at a Chinese airport (M Airport). After a careful check, some blades of the plane's engine were found to be out of shape and a few feathers and some bloodstains were found in the air intake of the engine. In order to know which species of bird was involved in the birdstrike, firstly we extracted DNA from the bloodstains; secondly, the DNA barcode (portion of COI gene) of the unknown species was amplified by PCR method; thirdly, sequence divergences (K2P differences) of the DNA barcode between the unknown species and a library of 59 common bird species distributed at the airport area were analyzed. Furthermore, a neighbor-joining (NJ) tree based on COI barcodes was created to provide graphic representation of sequence divergences among the species to confirm the identification. The result showed that red-rumped swallow (Hirundo daurica) was involved in the birdstrike incident. Some suggestions to avoid birdstrikes caused by red-rumped swallows were given to the administrative department of M Airport to ensure flying safety.

International Barcode of Life: Focus on big biodiversity in South Africa.

PubMed

Adamowicz, Sarah J; Hollingsworth, Peter M; Ratnasingham, Sujeevan; van der Bank, Michelle

2017-11-01

Participants in the 7th International Barcode of Life Conference (Kruger National Park, South Africa, 20-24 November 2017) share the latest findings in DNA barcoding research and its increasingly diversified applications. Here, we review prevailing trends synthesized from among 429 invited and contributed abstracts, which are collated in this open-access special issue of Genome. Hosted for the first time on the African continent, the 7th Conference places special emphasis on the evolutionary origins, biogeography, and conservation of African flora and fauna. Within Africa and elsewhere, DNA barcoding and related techniques are being increasingly used for wildlife forensics and for the validation of commercial products, such as medicinal plants and seafood species. A striking trend of the conference is the dramatic rise of studies on environmental DNA (eDNA) and on diverse uses of high-throughput sequencing techniques. Emerging techniques in these areas are opening new avenues for environmental biomonitoring, managing species-at-risk and invasive species, and revealing species interaction networks in unprecedented detail. Contributors call for the development of validated community standards for high-throughput sequence data generation and analysis, to enable the full potential of these methods to be realized for understanding and managing biodiversity on a global scale.

DNA barcode and identification of the varieties and provenances of Taiwan's domestic and imported made teas using ribosomal internal transcribed spacer 2 sequences.

PubMed

Lee, Shih-Chieh; Wang, Chia-Hsiang; Yen, Cheng-En; Chang, Chieh

2017-04-01

The major aim of made tea identification is to identify the variety and provenance of the tea plant. The present experiment used 113 tea plants [Camellia sinensis (L.) O. Kuntze] housed at the Tea Research and Extension Substation, from which 113 internal transcribed spacer 2 (ITS2) fragments, 104 trnL intron, and 98 trnL-trnF intergenic sequence region DNA sequences were successfully sequenced. The similarity of the ITS2 nucleotide sequences between tea plants housed at the Tea Research and Extension Substation was 0.379-0.994. In this polymerase chain reaction-amplified noncoding region, no varieties possessed identical sequences. Compared with the trnL intron and trnL-trnF intergenic sequence fragments of chloroplast cpDNA, the proportion of ITS2 nucleotide sequence variation was large and is more suitable for establishing a DNA barcode database to identify tea plant varieties. After establishing the database, 30 imported teas and 35 domestic made teas were used in this model system to explore the feasibility of using ITS2 sequences to identify the varieties and provenances of made teas. A phylogenetic tree was constructed using ITS2 sequences with the unweighted pair group method with arithmetic mean, which indicated that the same variety of tea plant is likely to be successfully categorized into one cluster, but contamination from other tea plants was also detected. This result provides molecular evidence that the similarity between important tea varieties in Taiwan remains high. We suggest a direct, wide collection of made tea and original samples of tea plants to establish an ITS2 sequence molecular barcode identification database to identify the varieties and provenances of tea plants. The DNA barcode comparison method can satisfy the need for a rapid, low-cost, frontline differentiation of the large amount of made teas from Taiwan and abroad, and can provide molecular evidence of their varieties and provenances. Copyright © 2016. Published by Elsevier B.V.

Efficient distinction of invasive aquatic plant species from non-invasive related species using DNA barcoding.

PubMed

Ghahramanzadeh, R; Esselink, G; Kodde, L P; Duistermaat, H; van Valkenburg, J L C H; Marashi, S H; Smulders, M J M; van de Wiel, C C M

2013-01-01

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non-invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH-psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH-psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics. © 2012 Blackwell Publishing Ltd.

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices

NASA Astrophysics Data System (ADS)

Yan, Hao; Labean, Thomas H.; Feng, Liping; Reif, John H.

2003-07-01

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping.

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices.

PubMed

Yan, Hao; LaBean, Thomas H; Feng, Liping; Reif, John H

2003-07-08

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping.

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

PubMed Central

2018-01-01

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling. PMID:29799876

Identification of processed Chinese medicinal materials using DNA mini-barcoding.

PubMed

Song, Ming; Dong, Gang-Qiang; Zhang, Ya-Qin; Liu, Xia; Sun, Wei

2017-07-01

Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psbA-trnH, rbcL, matK, trnL (UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL (UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%-20% of the processed samples, while the amplification rates of the trnL (UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL (UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

DNA barcodes effectively identify the morphologically similar Common Opossum (Didelphis marsupialis) and Virginia Opossum (Didelphis virginiana) from areas of sympatry in Mexico.

PubMed

Cervantes, Fernando A; Arcangeli, Jésica; Hortelano-Moncada, Yolanda; Borisenko, Alex V

2010-12-01

Two morphologically similar species of opossum from the genus Didelphis-Didelphis virginiana and Didelphis marsupialis-cooccur sympatrically in Mexico. High intraspecific variation complicates their morphological discrimination, under both field and museum conditions. This study aims to evaluate the utility and reliability of using DNA barcodes (short standardized genome fragments used for DNA-based identification) to distinguish these two species. Sequences of the cytochrome c oxidase subunit I (Cox1) mitochondrial gene were obtained from 12 D. marsupialis and 29 D. virginiana individuals and were compared using the neighbor-joining (NJ) algorithm with Kimura's two-parameter (K2P) model of nucleotide substitution. Average K2P distances were 1.56% within D. virginiana and 1.65% in D. marsupialis. Interspecific distances between D. virginiana and D. marsupialis varied from 7.8 to 9.3% and their barcode sequences formed distinct non-overlapping clusters on NJ trees. All sympatric specimens of both species were effectively discriminated, confirming the utility of Cox1 barcoding as a tool for taxonomic identification of these morphologically similar taxa.

DNA barcoding based on plastid matK and RNA polymerase for assessing the genetic identity of date (Phoenix dactylifera L.) cultivars.

PubMed

Enan, M R; Ahamed, A

2014-02-14

The cultivated date palm is the most agriculturally important species of the Arecaceae family. The standard chloroplast DNA barcode for land plants recommended by the Consortium for the Barcode of Life plant working group needs to be evaluated for a wide range of plant species. Therefore, we assessed the potential of the matK and rpoC1 markers for the authentication of date cultivars. There is not one universal method to authenticate date cultivars. In this study, 11 different date cultivars were sequenced and analyzed for matK and rpoC1 genes by using bioinformatic tools to establish a cultivar-specific molecular monogram. The chloroplast matK marker was more informative than the rpoC1 chloroplast DNA markers. Phylogenetic trees were constructed on the basis of the matK and rpoC1 sequences, and the results suggested that matK alone or in combination with rpoC1 can be used for determining the levels of genetic variation and for barcoding.

Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini-barcode.

PubMed

Françoso, E; Arias, M C

2013-09-01

Bees (Apidae), of which there are more than 19 900 species, are extremely important for ecosystem services and economic purposes, so taxon identity is a major concern. The goal of this study was to optimize the DNA barcode technique based on the Cytochrome c oxidase (COI) mitochondrial gene region. This approach has previously been shown to be useful in resolving taxonomic inconsistencies and for species identification when morphological data are poor. Specifically, we designed and tested new primers and standardized PCR conditions to amplify the barcode region for bees, focusing on the corbiculate Apids. In addition, primers were designed to amplify small COI amplicons and tested with pinned specimens. Short barcode sequences were easily obtained for some Bombus century-old museum specimens and shown to be useful as mini-barcodes. The new primers and PCR conditions established in this study proved to be successful for the amplification of the barcode region for all species tested, regardless of the conditions of tissue preservation. We saw no evidence of Wolbachia or numts amplification by these primers, and so we suggest that these new primers are of broad value for corbiculate bee identification through DNA barcode. © 2013 John Wiley & Sons Ltd.

The internal transcribed spacer (ITS) region and trnH-psbA [corrected] are suitable candidate loci for DNA barcoding of tropical tree species of India.

PubMed

Tripathi, Abhinandan Mani; Tyagi, Antariksh; Kumar, Anoop; Singh, Akanksha; Singh, Shivani; Chaudhary, Lal Babu; Roy, Sribash

2013-01-01

DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. rbcL and matK are standard barcode loci while ITS, and trnH-psbA are considered as supplementary loci for plants. Plant barcode loci, namely, rbcL, matK, ITS, trnH-psbA, and the recently proposed ITS2, were tested for their efficacy as barcode loci using 300 accessions of tropical tree species. We tested these loci for PCR, sequencing success, and species discrimination ability using three methods. rbcL was the best locus as far as PCR and sequencing success rate were concerned, but not for the species discrimination ability of tropical tree species. ITS and trnH-psbA were the second best loci in PCR and sequencing success, respectively. The species discrimination ability of ITS ranged from 24.4 percent to 74.3 percent and that of trnH-psbA was 25.6 percent to 67.7 percent, depending upon the data set and the method used. matK provided the least PCR success, followed by ITS2 (59. 0%). Species resolution by ITS2 and rbcL ranged from 9.0 percent to 48.7 percent and 13.2 percent to 43.6 percent, respectively. Further, we observed that the NCBI nucleotide database is poorly represented by the sequences of barcode loci studied here for tree species. Although a conservative approach of a success rate of 60-70 percent by both ITS and trnH-psbA may not be considered as highly successful but would certainly help in large-scale biodiversity inventorization, particularly for tropical tree species, considering the standard success rate of plant DNA barcode program reported so far. The recommended matK and rbcL primers combination may not work in tropical tree species as barcode markers.

[Identification of Tibetan medicine "Dida" of Gentianaceae using DNA barcoding].

PubMed

Liu, Chuan; Zhang, Yu-Xin; Liu, Yue; Chen, Yi-Long; Fan, Gang; Xiang, Li; Xu, Jiang; Zhang, Yi

2016-02-01

The ITS2 barcode was used toidentify Tibetan medicine "Dida", and tosecure its quality and safety in medication. A total of 13 species, 151 experimental samples for the study from the Tibetan Plateau, including Gentianaceae Swertia, Halenia, Gentianopsis, Comastoma, Lomatogonium ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.7.1. The Kimura 2-Parameter (K2P) distances were calculated using MEGA 6.0. The neighbor-joining (NJ) phylogenetic trees were constructed. There are 31 haplotypes among 231 bp after alignment of all ITS2 sequence haplotypes, and the average G±C content of 61.40%. The NJ tree strongly supported that every species clustered into their own clade and high identification success rate, except that Swertia bifolia and Swertia wolfangiana could not be distinguished from each other based on the sequence divergences. DNA barcoding could be used as a fast and accurate identification method to distinguish Tibetan medicine "Dida" to ensure its safe use. Copyright© by the Chinese Pharmaceutical Association.

DNA barcoding of morphologically characterized mosquitoes belonging to the subfamily Culicinae from Sri Lanka.

PubMed

Weeraratne, Thilini Chathurika; Surendran, Sinnathamby Noble; Parakrama Karunaratne, S H P

2018-04-25

Vectors of mosquito-borne diseases in Sri Lanka, except for malaria, belong to the subfamily Culicinae, which includes nearly 84% of the mosquito fauna of the country. Hence, accurate and precise species identification of culicine mosquitoes is a crucial factor in implementing effective vector control strategies. During the present study, a combined effort using morphology and DNA barcoding was made to characterize mosquitoes of the subfamily Culicinae for the first time from nine districts of Sri Lanka. Cytochrome c oxidase subunit 1 (cox1) gene from the mitochondrial genome and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. According to morphological identification, the field collected adult mosquitoes belonged to 5 genera and 14 species, i.e. Aedes aegypti, Ae. albopictus, Ae. pallidostriatus, Aedes sp. 1, Armigeres sp. 1, Culex bitaeniorhynchus, Cx. fuscocephala, Cx. gelidus, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Cx. whitmorei, Mansonia uniformis and Mimomyia chamberlaini. Molecular analyses of 62 cox1 and 36 ITS2 sequences were exclusively comparable with the morphological identifications of all the species except for Ae. pallidostriatus and Aedes sp. 1. Although the species identification of Armigeres sp. 1 specimens using morphological features was not possible during this study, DNA barcodes of the specimens matched 100% with the publicly available Ar. subalbatus sequences, giving their species status. Analysis of all the cox1 sequences (14 clades supported by strong bootstrap value in the Neighbor-Joining tree and interspecific distances of > 3%) showed the presence of 14 different species. This is the first available DNA sequence in the GenBank records for morphologically identified Ae. pallidostriatus. Aedes sp. 1 could not be identified morphologically or by publicly available sequences. Aedes aegypti, Ae. albopictus and all Culex species reported during the current study are vectors of human diseases. All these vector species showed comparatively high diversity. The current study reflects the significance of integrated systematic approach and use of cox1 and ITS genetic markers in mosquito taxonomy. Results of DNA barcoding were comparable with morphological identifications and, more importantly, DNA barcoding could accurately identify the species in the instances where the traditional morphological identification failed due to indistinguishable characters of damaged specimens and the presence of subspecies.

The Hemiptera (Insecta) of Canada: Constructing a Reference Library of DNA Barcodes

PubMed Central

Gwiazdowski, Rodger A.; Foottit, Robert G.; Maw, H. Eric L.; Hebert, Paul D. N.

2015-01-01

DNA barcode reference libraries linked to voucher specimens create new opportunities for high-throughput identification and taxonomic re-evaluations. This study provides a DNA barcode library for about 45% of the recognized species of Canadian Hemiptera, and the publically available R workflow used for its generation. The current library is based on the analysis of 20,851 specimens including 1849 species belonging to 628 genera and 64 families. These individuals were assigned to 1867 Barcode Index Numbers (BINs), sequence clusters that often coincide with species recognized through prior taxonomy. Museum collections were a key source for identified specimens, but we also employed high-throughput collection methods that generated large numbers of unidentified specimens. Many of these specimens represented novel BINs that were subsequently identified by taxonomists, adding barcode coverage for additional species. Our analyses based on both approaches includes 94 species not listed in the most recent Canadian checklist, representing a potential 3% increase in the fauna. We discuss the development of our workflow in the context of prior DNA barcode library construction projects, emphasizing the importance of delineating a set of reference specimens to aid investigations in cases of nomenclatural and DNA barcode discordance. The identification for each specimen in the reference set can be annotated on the Barcode of Life Data System (BOLD), allowing experts to highlight questionable identifications; annotations can be added by any registered user of BOLD, and instructions for this are provided. PMID:25923328

DNA barcoding for conservation, seed banking and ecological restoration of Acacia in the Midwest of Western Australia.

PubMed

Nevill, Paul G; Wallace, Mark J; Miller, Joseph T; Krauss, Siegfried L

2013-11-01

We used DNA barcoding to address an important conservation issue in the Midwest of Western Australia, working on Australia's largest genus of flowering plant. We tested whether or not currently recommended plant DNA barcoding regions (matK and rbcL) were able to discriminate Acacia taxa of varying phylogenetic distances, and ultimately identify an ambiguously labelled seed collection from a mine-site restoration project. Although matK successfully identified the unknown seed as the rare and conservation priority listed A. karina, and was able to resolve six of the eleven study species, this region was difficult to amplify and sequence. In contrast, rbcL was straightforward to recover and align, but could not determine the origin of the seed and only resolved 3 of the 11 species. Other chloroplast regions (rpl32-trnL, psbA-trnH, trnL-F and trnK) had mixed success resolving the studied taxa. In general, species were better resolved in multilocus data sets compared to single-locus data sets. We recommend using the formal barcoding regions supplemented with data from other plastid regions, particularly rpl32-trnL, for barcoding in Acacia. Our study demonstrates the novel use of DNA barcoding for seed identification and illustrates the practical potential of DNA barcoding for the growing discipline of restoration ecology. © 2013 John Wiley & Sons Ltd.

Identification of food and beverage spoilage yeasts from DNA sequence analyses

USDA-ARS?s Scientific Manuscript database

Detection, identification, and classification of yeasts has undergone a major transformation in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of th...

How many species and under what names? Using DNA barcoding and GenBank data for west Central African amphibian conservation

PubMed Central

Mulcahy, Daniel G.; Vanthomme, Hadrien; Tobi, Elie; Wynn, Addison H.; Zimkus, Breda M.; McDiarmid, Roy W.

2017-01-01

Development projects in west Central Africa are proceeding at an unprecedented rate, often with little concern for their effects on biodiversity. In an attempt to better understand potential impacts of a road development project on the anuran amphibian community, we conducted a biodiversity assessment employing multiple methodologies (visual encounter transects, auditory surveys, leaf litter plots and pitfall traps) to inventory species prior to construction of a new road within the buffer zone of Moukalaba-Doudou National Park, Gabon. Because of difficulties in morphological identification and taxonomic uncertainty of amphibian species observed in the area, we integrated a DNA barcoding analysis into the project to improve the overall quality and accuracy of the species inventory. Based on morphology alone, 48 species were recognized in the field and voucher specimens of each were collected. We used tissue samples from specimens collected at our field site, material available from amphibians collected in other parts of Gabon and the Republic of Congo to initiate a DNA barcode library for west Central African amphibians. We then compared our sequences with material in GenBank for the genera recorded at the study site to assist in identifications. The resulting COI and 16S barcode library allowed us to update the number of species documented at the study site to 28, thereby providing a more accurate assessment of diversity and distributions. We caution that because sequence data maintained in GenBank are often poorly curated by the original submitters and cannot be amended by third-parties, these data have limited utility for identification purposes. Nevertheless, the use of DNA barcoding is likely to benefit biodiversity inventories and long-term monitoring, particularly for taxa that can be difficult to identify based on morphology alone; likewise, inventory and monitoring programs can contribute invaluable data to the DNA barcode library and the taxonomy of complex groups. Our methods provide an example of how non-taxonomists and parataxonomists working in understudied parts of the world with limited geographic sampling and comparative morphological material can use DNA barcoding and publicly available sequence data (GenBank) to rapidly identify the number of species and assign tentative names to aid in urgent conservation management actions and contribute to taxonomic resolution. PMID:29131846

Plans and progress for building a Great Lakes fauna DNA ...

EPA Pesticide Factsheets

DNA reference libraries provide researchers with an important tool for assessing regional biodiversity by allowing unknown genetic sequences to be assigned identities, while also providing a means for taxonomists to validate identifications. Expanding the representation of Great Lakes species in such reference libraries is an explicit component of research at EPA’s Mid-Continent Ecology Division. Our DNA reference library building efforts began in 2012 with the goal of providing barcodes for at least 5 specimens of each native and nonindigenous fish and aquatic invertebrate species currently present in the Great Lakes. The approach is to pull taxonomically validated specimen for sequencing from EPA led sampling efforts of adult/juvenile fish, larval fish, benthic macroinvertebrates, and zooplankton; while also soliciting aid from state and federal agencies for tissue from “shopping list” organisms. The barcodes we generate are made available through the publicly accessible BOLD (Barcode of Life) database, and help inform a planned Great Lakes biodiversity inventory. To date, our submissions to BOLD are limited to fishes; of the 88 fish species listed as being present within Lake Superior, roughly half were successfully barcoded, while only 22 species met the desired quota of 5 barcoded specimens per species. As we continue to generate genomic information from our collections and the taxonomic representations become more complete, we will continue to

The utility of mtDNA and rDNA for barcoding and phylogeny of plant-parasitic nematodes from Longidoridae (Nematoda, Enoplea).

PubMed

Palomares-Rius, J E; Cantalapiedra-Navarrete, C; Archidona-Yuste, A; Subbotin, S A; Castillo, P

2017-09-07

The traditional identification of plant-parasitic nematode species by morphology and morphometric studies is very difficult because of high morphological variability that can lead to considerable overlap of many characteristics and their ambiguous interpretation. For this reason, it is essential to implement approaches to ensure accurate species identification. DNA barcoding aids in identification and advances species discovery. This study sought to unravel the use of the mitochondrial marker cytochrome c oxidase subunit 1 (coxI) as barcode for Longidoridae species identification, and as a phylogenetic marker. The results showed that mitochondrial and ribosomal markers could be used as barcoding markers, except for some species from the Xiphinema americanum group. The ITS1 region showed a promising role in barcoding for species identification because of the clear molecular variability among species. Some species presented important molecular variability in coxI. The analysis of the newly provided sequences and the sequences deposited in GenBank showed plausible misidentifications, and the use of voucher species and topotype specimens is a priority for this group of nematodes. The use of coxI and D2 and D3 expansion segments of the 28S rRNA gene did not clarify the phylogeny at the genus level.

Mapping global biodiversity connections with DNA barcodes: Lepidoptera of Pakistan.

PubMed

Ashfaq, Muhammad; Akhtar, Saleem; Rafi, Muhammad Athar; Mansoor, Shahid; Hebert, Paul D N

2017-01-01

Sequences from the DNA barcode region of the mitochondrial COI gene are an effective tool for specimen identification and for the discovery of new species. The Barcode of Life Data Systems (BOLD) (www.boldsystems.org) currently hosts 4.5 million records from animals which have been assigned to more than 490,000 different Barcode Index Numbers (BINs), which serve as a proxy for species. Because a fourth of these BINs derive from Lepidoptera, BOLD has a strong capability to both identify specimens in this order and to support studies of faunal overlap. DNA barcode sequences were obtained from 4503 moths from 329 sites across Pakistan, specimens that represented 981 BINs from 52 families. Among 379 species with a Linnaean name assignment, all were represented by a single BIN excepting five species that showed a BIN split. Less than half (44%) of the 981 BINs had counterparts in other countries; the remaining BINs were unique to Pakistan. Another 218 BINs of Lepidoptera from Pakistan were coupled with the 981 from this study before being compared with all 116,768 BINs for this order. As expected, faunal overlap was highest with India (21%), Sri Lanka (21%), United Arab Emirates (20%) and with other Asian nations (2.1%), but it was very low with other continents including Africa (0.6%), Europe (1.3%), Australia (0.6%), Oceania (1.0%), North America (0.1%), and South America (0.1%). This study indicates the way in which DNA barcoding facilitates measures of faunal overlap even when taxa have not been assigned to a Linnean species.

DNA Barcoding for Efficient Species- and Pathovar-Level Identification of the Quarantine Plant Pathogen Xanthomonas

PubMed Central

Tian, Qian; Zhao, Wenjun; Lu, Songyu; Zhu, Shuifang; Li, Shidong

2016-01-01

Genus Xanthomonas comprises many economically important plant pathogens that affect a wide range of hosts. Indeed, fourteen Xanthomonas species/pathovars have been regarded as official quarantine bacteria for imports in China. To date, however, a rapid and accurate method capable of identifying all of the quarantine species/pathovars has yet to be developed. In this study, we therefore evaluated the capacity of DNA barcoding as a digital identification method for discriminating quarantine species/pathovars of Xanthomonas. For these analyses, 327 isolates, representing 45 Xanthomonas species/pathovars, as well as five additional species/pathovars from GenBank (50 species/pathovars total), were utilized to test the efficacy of four DNA barcode candidate genes (16S rRNA gene, cpn60, gyrB, and avrBs2). Of these candidate genes, cpn60 displayed the highest rate of PCR amplification and sequencing success. The tree-building (Neighbor-joining), ‘best close match’, and barcode gap methods were subsequently employed to assess the species- and pathovar-level resolution of each gene. Notably, all isolates of each quarantine species/pathovars formed a monophyletic group in the neighbor-joining tree constructed using the cpn60 sequences. Moreover, cpn60 also demonstrated the most satisfactory results in both barcoding gap analysis and the ‘best close match’ test. Thus, compared with the other markers tested, cpn60 proved to be a powerful DNA barcode, providing a reliable and effective means for the species- and pathovar-level identification of the quarantine plant pathogen Xanthomonas. PMID:27861494

Mapping global biodiversity connections with DNA barcodes: Lepidoptera of Pakistan

PubMed Central

Akhtar, Saleem; Rafi, Muhammad Athar; Mansoor, Shahid; Hebert, Paul D. N.

2017-01-01

Sequences from the DNA barcode region of the mitochondrial COI gene are an effective tool for specimen identification and for the discovery of new species. The Barcode of Life Data Systems (BOLD) (www.boldsystems.org) currently hosts 4.5 million records from animals which have been assigned to more than 490,000 different Barcode Index Numbers (BINs), which serve as a proxy for species. Because a fourth of these BINs derive from Lepidoptera, BOLD has a strong capability to both identify specimens in this order and to support studies of faunal overlap. DNA barcode sequences were obtained from 4503 moths from 329 sites across Pakistan, specimens that represented 981 BINs from 52 families. Among 379 species with a Linnaean name assignment, all were represented by a single BIN excepting five species that showed a BIN split. Less than half (44%) of the 981 BINs had counterparts in other countries; the remaining BINs were unique to Pakistan. Another 218 BINs of Lepidoptera from Pakistan were coupled with the 981 from this study before being compared with all 116,768 BINs for this order. As expected, faunal overlap was highest with India (21%), Sri Lanka (21%), United Arab Emirates (20%) and with other Asian nations (2.1%), but it was very low with other continents including Africa (0.6%), Europe (1.3%), Australia (0.6%), Oceania (1.0%), North America (0.1%), and South America (0.1%). This study indicates the way in which DNA barcoding facilitates measures of faunal overlap even when taxa have not been assigned to a Linnean species. PMID:28339501

Single-Molecule Denaturation Mapping of Genomic DNA in Nanofluidic Channels

NASA Astrophysics Data System (ADS)

Reisner, Walter; Larsen, Niels; Kristensen, Anders; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

2009-03-01

We have developed a new DNA barcoding technique based on the partial denaturation of extended fluorescently labeled DNA molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions and can be explained by calculations of sequence-dependent melting probability. We believe this melting mapping technology is the first optically based single molecule technique sensitive to genome wide sequence variation that does not require an additional enzymatic labeling or restriction scheme.

Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers.

PubMed

Girardot, Charles; Scholtalbers, Jelle; Sauer, Sajoscha; Su, Shu-Yi; Furlong, Eileen E M

2016-10-08

The yield obtained from next generation sequencers has increased almost exponentially in recent years, making sample multiplexing common practice. While barcodes (known sequences of fixed length) primarily encode the sample identity of sequenced DNA fragments, barcodes made of random sequences (Unique Molecular Identifier or UMIs) are often used to distinguish between PCR duplicates and transcript abundance in, for example, single-cell RNA sequencing (scRNA-seq). In paired-end sequencing, different barcodes can be inserted at each fragment end to either increase the number of multiplexed samples in the library or to use one of the barcodes as UMI. Alternatively, UMIs can be combined with the sample barcodes into composite barcodes, or with standard Illumina® indexing. Subsequent analysis must take read duplicates and sample identity into account, by identifying UMIs. Existing tools do not support these complex barcoding configurations and custom code development is frequently required. Here, we present Je, a suite of tools that accommodates complex barcoding strategies, extracts UMIs and filters read duplicates taking UMIs into account. Using Je on publicly available scRNA-seq and iCLIP data containing UMIs, the number of unique reads increased by up to 36 %, compared to when UMIs are ignored. Je is implemented in JAVA and uses the Picard API. Code, executables and documentation are freely available at http://gbcs.embl.de/Je . Je can also be easily installed in Galaxy through the Galaxy toolshed.

DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus Crataegus.

PubMed

Zarrei, Mehdi; Talent, Nadia; Kuzmina, Maria; Lee, Jeanette; Lund, Jensen; Shipley, Paul R; Stefanović, Saša; Dickinson, Timothy A

2015-04-28

DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication of hawthorn materials in natural health products. Published by Oxford University Press on behalf of the Annals of Botany Company.

DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus Crataegus

PubMed Central

Zarrei, Mehdi; Talent, Nadia; Kuzmina, Maria; Lee, Jeanette; Lund, Jensen; Shipley, Paul R.; Stefanović, Saša; Dickinson, Timothy A.

2015-01-01

DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication of hawthorn materials in natural health products. PMID:25926325

DNA Barcode Identification of Freshwater Snails in the Family Bithyniidae from Thailand

PubMed Central

Kulsantiwong, Jutharat; Prasopdee, Sattrachai; Ruangsittichai, Jiraporn; Ruangjirachuporn, Wipaporn; Boonmars, Thidarut; Viyanant, Vithoon; Pierossi, Paola; Hebert, Paul D. N.; Tesana, Smarn

2013-01-01

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5’ region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy. PMID:24223896

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices

PubMed Central

Yan, Hao; LaBean, Thomas H.; Feng, Liping; Reif, John H.

2003-01-01

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping. PMID:12821776

Assessment of three plastid DNA barcode markers for identification of Clinacanthus nutans (Acanthaceae).

PubMed

Ismail, Noor Zafirah; Arsad, Hasni; Samian, Mohammed Razip; Hamdan, Mohammad Razak; Othman, Ahmad Sofiman

2018-01-01

This study was conducted to determine the feasibility of using three plastid DNA regions ( matK , trnH - psbA , and rbcL ) as DNA barcodes to identify the medicinal plant Clinacanthus nutans . In this study, C. nutans was collected at several different locations. Total genomic DNA was extracted, amplified by polymerase chain reaction (PCR), and sequenced using matK , trnH - psbA , and rbcL , primers. DNA sequences generated from PCR were submitted to the National Center for Biotechnology Information's (NCBI) GenBank. Identification of C. nutans was carried out using NCBI's Basic Local Alignment Search Tool (BLAST). The rbcL and trnH - psbA regions successfully identified C. nutans with sequencing rates of 100% through BLAST identification. Molecular Evolutionary Genetics Analysis (MEGA) 6.0 was used to analyze interspecific and intraspecific divergence of plastid DNA sequences. rbcL and matK exhibited the lowest average interspecific distance (0.0487 and 0.0963, respectively), whereas trnH - psbA exhibited the highest average interspecific distance (0.2029). The R package Spider revealed that trnH - psbA correctly identified Barcode of Life Data System (BOLD) 96%, best close match 79%, and near neighbor 100% of the species, compared to matK (BOLD 72%; best close match 64%; near neighbor 78%) and rbcL (BOLD 77%; best close match 62%; near neighbor 88%). These results indicate that trnH - psbA is very effective at identifying C. nutans , as it performed well in discriminating species in Acanthaceae.

Barcoding Fauna Bavarica: Myriapoda – a contribution to DNA sequence-based identifications of centipedes and millipedes (Chilopoda, Diplopoda)

PubMed Central

Spelda, Jörg; Reip, Hans S.; Oliveira–Biener, Ulla; Melzer, Roland R.

2011-01-01

Abstract We give a first account of our ongoing barcoding activities on Bavarian myriapods in the framework of the Barcoding Fauna Bavarica project and IBOL, the International Barcode of Life. Having analyzed 126 taxa (including 122 species) belonging to all major German chilopod and diplopod lineages, often using four or more specimens each, at the moment our species stock includes 82% of the diplopods and 65% of the chilopods found in Bavaria, southern Germany. The partial COI sequences allow correct identification of more than 95% of the current set of Bavarian species. Moreover, most of the myriapod orders and families appear as distinct clades in neighbour-joining trees, although the phylogenetic relationships between them are not always depicted correctly. We give examples of (1) high interspecific sequence variability among closely related species; (2) low interspecific variability in some chordeumatidan genera, indicating that recent speciations cannot be resolved with certainty using COI DNA barcodes; (3) high intraspecific variation in some genera, suggesting the existence of cryptic lineages; and (4) the possible polyphyly of some taxa, i.e. the chordeumatidan genus Ochogona. This shows that, in addition to species identification, our data may be useful in various ways in the context of species delimitations, taxonomic revisions and analyses of ongoing speciation processes. PMID:22303099

DNA Barcoding the Canadian Arctic Flora: Core Plastid Barcodes (rbcL + matK) for 490 Vascular Plant Species

PubMed Central

Saarela, Jeffery M.; Sokoloff, Paul C.; Gillespie, Lynn J.; Consaul, Laurie L.; Bull, Roger D.

2013-01-01

Accurate identification of Arctic plant species is critical for understanding potential climate-induced changes in their diversity and distributions. To facilitate rapid identification we generated DNA barcodes for the core plastid barcode loci (rbcL and matK) for 490 vascular plant species, representing nearly half of the Canadian Arctic flora and 93% of the flora of the Canadian Arctic Archipelago. Sequence recovery was higher for rbcL than matK (93% and 81%), and rbcL was easier to recover than matK from herbarium specimens (92% and 77%). Distance-based and sequence-similarity analyses of combined rbcL + matK data discriminate 97% of genera, 56% of species, and 7% of infraspecific taxa. There is a significant negative correlation between the number of species sampled per genus and the percent species resolution per genus. We characterize barcode variation in detail in the ten largest genera sampled (Carex, Draba, Festuca, Pedicularis, Poa, Potentilla, Puccinellia, Ranunculus, Salix, and Saxifraga) in the context of their phylogenetic relationships and taxonomy. Discrimination with the core barcode loci in these genera ranges from 0% in Salix to 85% in Carex. Haplotype variation in multiple genera does not correspond to species boundaries, including Taraxacum, in which the distribution of plastid haplotypes among Arctic species is consistent with plastid variation documented in non-Arctic species. Introgression of Poa glauca plastid DNA into multiple individuals of P. hartzii is problematic for identification of these species with DNA barcodes. Of three supplementary barcode loci (psbA–trnH, psbK–psbI, atpF–atpH) collected for a subset of Poa and Puccinellia species, only atpF–atpH improved discrimination in Puccinellia, compared with rbcL and matK. Variation in matK in Vaccinium uliginosum and rbcL in Saxifraga oppositifolia corresponds to variation in other loci used to characterize the phylogeographic histories of these Arctic-alpine species. PMID:24348895

How effective are DNA barcodes in the identification of African rainforest trees?

PubMed

Parmentier, Ingrid; Duminil, Jérôme; Kuzmina, Maria; Philippe, Morgane; Thomas, Duncan W; Kenfack, David; Chuyong, George B; Cruaud, Corinne; Hardy, Olivier J

2013-01-01

DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species. We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95-100% success), but less for species identification (71-88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84-90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA. Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications.

How Effective Are DNA Barcodes in the Identification of African Rainforest Trees?

PubMed Central

Parmentier, Ingrid; Duminil, Jérôme; Kuzmina, Maria; Philippe, Morgane; Thomas, Duncan W.; Kenfack, David; Chuyong, George B.; Cruaud, Corinne; Hardy, Olivier J.

2013-01-01

Background DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species. Methodology/Principal Findings We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95–100% success), but less for species identification (71–88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84–90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA. Conclusions/Significance Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications. PMID:23565134

DNA barcoding the Canadian Arctic flora: core plastid barcodes (rbcL + matK) for 490 vascular plant species.

PubMed

Saarela, Jeffery M; Sokoloff, Paul C; Gillespie, Lynn J; Consaul, Laurie L; Bull, Roger D

2013-01-01

Accurate identification of Arctic plant species is critical for understanding potential climate-induced changes in their diversity and distributions. To facilitate rapid identification we generated DNA barcodes for the core plastid barcode loci (rbcL and matK) for 490 vascular plant species, representing nearly half of the Canadian Arctic flora and 93% of the flora of the Canadian Arctic Archipelago. Sequence recovery was higher for rbcL than matK (93% and 81%), and rbcL was easier to recover than matK from herbarium specimens (92% and 77%). Distance-based and sequence-similarity analyses of combined rbcL + matK data discriminate 97% of genera, 56% of species, and 7% of infraspecific taxa. There is a significant negative correlation between the number of species sampled per genus and the percent species resolution per genus. We characterize barcode variation in detail in the ten largest genera sampled (Carex, Draba, Festuca, Pedicularis, Poa, Potentilla, Puccinellia, Ranunculus, Salix, and Saxifraga) in the context of their phylogenetic relationships and taxonomy. Discrimination with the core barcode loci in these genera ranges from 0% in Salix to 85% in Carex. Haplotype variation in multiple genera does not correspond to species boundaries, including Taraxacum, in which the distribution of plastid haplotypes among Arctic species is consistent with plastid variation documented in non-Arctic species. Introgression of Poa glauca plastid DNA into multiple individuals of P. hartzii is problematic for identification of these species with DNA barcodes. Of three supplementary barcode loci (psbA-trnH, psbK-psbI, atpF-atpH) collected for a subset of Poa and Puccinellia species, only atpF-atpH improved discrimination in Puccinellia, compared with rbcL and matK. Variation in matK in Vaccinium uliginosum and rbcL in Saxifraga oppositifolia corresponds to variation in other loci used to characterize the phylogeographic histories of these Arctic-alpine species.

DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River.

PubMed

Shen, Yanjun; Guan, Lihong; Wang, Dengqiang; Gan, Xiaoni

2016-05-01

The Yangtze River is the longest river in China and is divided into upstream and mid-downstream regions by the Three Gorges (the natural barriers of the Yangtze River), resulting in a complex distribution of fish. Dramatic changes to habitat environments may ultimately threaten fish survival; thus, it is necessary to evaluate the genetic diversity and propose protective measures. Species identification is the most significant task in many fields of biological research and in conservation efforts. DNA barcoding, which constitutes the analysis of a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, has been widely used for species identification. In this study, we collected 561 COI barcode sequences from 35 fish from the midstream of the Yangtze River. The intraspecific distances of all species were below 2% (with the exception of Acheilognathus macropterus and Hemibarbus maculatus). Nevertheless, all species could be unambiguously identified from the trees, barcoding gaps and taxonomic resolution ratio values. Furthermore, the COI barcode diversity was found to be low (≤0.5%), with the exception of H. maculatus (0.87%), A. macropterus (2.02%) and Saurogobio dabryi (0.82%). No or few shared haplotypes were detected between the upstream and downstream populations for ten species with overall nucleotide diversities greater than 0.00%, which indicated the likelihood of significant population genetic structuring. Our analyses indicated that DNA barcoding is an effective tool for the identification of cyprinidae fish in the midstream of the Yangtze River. It is vital that some protective measures be taken immediately because of the low COI barcode diversity.

Review and future prospects for DNA barcoding methods in forensic palynology.

PubMed

Bell, Karen L; Burgess, Kevin S; Okamoto, Kazufusa C; Aranda, Roman; Brosi, Berry J

2016-03-01

Pollen can be a critical forensic marker in cases where determining geographic origin is important, including investigative leads, missing persons cases, and intelligence applications. However, its use has previously been limited by the need for a high level of specialization by expert palynologists, slow speeds of identification, and relatively poor taxonomic resolution (typically to the plant family or genus level). By contrast, identification of pollen through DNA barcoding has the potential to overcome all three of these limitations, and it may seem surprising that the method has not been widely implemented. Despite what might seem a straightforward application of DNA barcoding to pollen, there are technical issues that have delayed progress. However, recent developments of standard methods for DNA barcoding of pollen, along with improvements in high-throughput sequencing technology, have overcome most of these technical issues. Based on these recent methodological developments in pollen DNA barcoding, we believe that now is the time to start applying these techniques in forensic palynology. In this article, we discuss the potential for these methods, and outline directions for future research to further improve on the technology and increase its applicability to a broader range of situations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

DNA barcoding for biosecurity: case studies from the UK plant protection program.

PubMed

Hodgetts, Jennifer; Ostojá-Starzewski, Jozef C; Prior, Thomas; Lawson, Rebecca; Hall, Jayne; Boonham, Neil

2016-11-01

Since its conception, DNA barcoding has seen a rapid uptake within the research community. Nevertheless, as with many new scientific tools, progression towards the point of routine deployment within diagnostic laboratories has been slow. In this paper, we discuss the application of DNA barcoding in the Defra plant health diagnostic laboratories, where DNA barcoding is used primarily for the identification of invertebrate pests. We present a series of case studies that demonstrate the successful application of DNA barcoding but also reveal some potential limitations to expanded use. The regulated plant pest, Bursephalenchus xylophilus, and one of its vectors, Monochamus alternatus, were found in dining chairs. Some traded wood products are potentially high risk, allowing the movement of longhorn beetles; Trichoferus campestris, Leptura quadrifasciata, and Trichoferus holosericeus were found in a wooden cutlery tray, a railway sleeper, and a dining chair, respectively. An outbreak of Meloidogyne fallax was identified in Allium ampeloprasum and in three weed species. Reference sequences for UK native psyllids were generated to enable the development of rapid diagnostics to be used for monitoring following the release of Aphalara itadori as a biological control agent for Fallopia japonica.

DNA barcoding commercially important aquatic invertebrates of Turkey.

PubMed

Keskin, Emre; Atar, Hasan Hüseyin

2013-08-01

DNA barcoding was used in order to identify aquatic invertebrates sampled from fisheries bycatch and discards. A total of 440 unique cytochrome c oxidase sub unit I (COI) barcodes were generated for 22 species from three important phyla (Arthropoda, Cnidaria, and Mollusca). All the species were sequenced and submitted to GenBank and Barcode of Life Database (BOLD) databases using 654 bp-long fragment of mitochondrial COI gene. Two of them (Pontastacus leptodactylus and Rapana bezoar) were first records of the species for the BOLD database and six of them (Carcinus aestuarii, Loligo vulgaris, Melicertus kerathurus, Nephrops norvegicus, Scyllarides latus, and Scyllarus arctus) were first standard (>648 bp) COI barcode records for the GenBank database. COI barcodes were analyzed for nucleotide composition, nucleotide pair frequencies, and Kimura's two-parameter genetic distance. Mean genetic distance among species was found increasing at higher taxonomic levels. Neighbor-joining trees generated were congruent with morphometric-based taxonomic classification. Findings of this study clearly demonstrate that DNA barcodes could be used as an efficient molecular tool in identification of not only target species from fisheries but also bycatch and discard species, and so it could provide us leverage for a better understanding in monitoring and management of fisheries and biodiversity.

Publishing large DNA sequence data in reduced spaces and lasting formats, in paper or PDF.

PubMed

Aguiar, Alexandre Pires

2013-02-04

Scientific publications carry a practical moral duty: they must last. Along that line of thinking, some methods are proposed to allow economically and structurally viable publication of DNA sequence data of any size in printed matter and PDFs. The proposal is primarily aimed at contributing for preserving information for the future, while allowing authors to avoid information splitting and complement storage ex situ, that is, in server machines, outside the publication proper. The technique may also help to solve the impasse between the ICZN Code requirement that a new nomen be associated to diagnostic characters for the taxon vs. the phylogenetic definition of taxa, based on cladograms only: sequence data are characters, and can now be easily and comfortably included in taxonomic publications, with direct textual mention to their diagnostic sections. The compression level achieved allows the inclusion of all wanted DNA or RNA sequences in the same printed matter or PDF publications where the sequences are cited and discussed. Reduced font sizes, invisible fonts, and original 2D black & white and color barcodes are illustrated and briefly discussed. The level of data compression achieved can allow each full page of sequence data, or about 5000 characters, to be precisely coded into a color barcode as small as a square of 1.5 mm. A practical example is provided with Taeniogonalos woodorum Smith (Hymenoptera, Trigonalidae). Free software to generate publishable barcodes from txt or FASTA files is provided at www.systaxon.ufes.br/dna.

A DNA barcode library for Germany's mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera).

PubMed

Morinière, Jérôme; Hendrich, Lars; Balke, Michael; Beermann, Arne J; König, Tobias; Hess, Monika; Koch, Stefan; Müller, Reinhard; Leese, Florian; Hebert, Paul D N; Hausmann, Axel; Schubart, Christoph D; Haszprunar, Gerhard

2017-11-01

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well-curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost-efficient and reliable DNA-based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two-thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution. © 2017 John Wiley & Sons Ltd.

Instances of erroneous DNA barcoding of metazoan invertebrates: Are universal cox1 gene primers too "universal"?

PubMed

Mioduchowska, Monika; Czyż, Michał Jan; Gołdyn, Bartłomiej; Kur, Jarosław; Sell, Jerzy

2018-01-01

The cytochrome c oxidase subunit I (cox1) gene is the main mitochondrial molecular marker playing a pivotal role in phylogenetic research and is a crucial barcode sequence. Folmer's "universal" primers designed to amplify this gene in metazoan invertebrates allowed quick and easy barcode and phylogenetic analysis. On the other hand, the increase in the number of studies on barcoding leads to more frequent publishing of incorrect sequences, due to amplification of non-target taxa, and insufficient analysis of the obtained sequences. Consequently, some sequences deposited in genetic databases are incorrectly described as obtained from invertebrates, while being in fact bacterial sequences. In our study, in which we used Folmer's primers to amplify COI sequences of the crustacean fairy shrimp Branchipus schaefferi (Fischer 1834), we also obtained COI sequences of microbial contaminants from Aeromonas sp. However, when we searched the GenBank database for sequences closely matching these contaminations we found entries described as representatives of Gastrotricha and Mollusca. When these entries were compared with other sequences bearing the same names in the database, the genetic distance between the incorrect and correct sequences amplified from the same species was c.a. 65%. Although the responsibility for the correct molecular identification of species rests on researchers, the errors found in already published sequences data have not been re-evaluated so far. On the basis of the standard sampling technique we have estimated with 95% probability that the chances of finding incorrectly described metazoan sequences in the GenBank depend on the systematic group, and variety from less than 1% (Mollusca and Arthropoda) up to 6.9% (Gastrotricha). Consequently, the increasing popularity of DNA barcoding and metabarcoding analysis may lead to overestimation of species diversity. Finally, the study also discusses the sources of the problems with amplification of non-target sequences.

Limited performance of DNA barcoding in a diverse community of tropical butterflies

PubMed Central

Elias, Marianne; Hill, Ryan I; Willmott, Keith R; Dasmahapatra, Kanchon K; Brower, Andrew V.Z; Mallet, James; Jiggins, Chris D

2007-01-01

DNA ‘barcoding’ relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5′ half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet. PMID:17785265

DNA Barcoding of Bemisia tabaci Complex (Hemiptera: Aleyrodidae) Reveals Southerly Expansion of the Dominant Whitefly Species on Cotton in Pakistan

PubMed Central

Ashfaq, Muhammad; Hebert, Paul D. N.; Mirza, M. Sajjad; Khan, Arif M.; Mansoor, Shahid; Shah, Ghulam S.; Zafar, Yusuf

2014-01-01

Background Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan. Methods/Principal Findings Sequence diversity in the DNA barcode region (mtCOI-5′) was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3′ to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage “Pakistan”. The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and “Pakistan” were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan. Conclusions DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region. PMID:25099936

DNA barcodes for two scale insect families, mealybugs (Hemiptera: Pseudococcidae) and armored scales (Hemiptera: Diaspididae).

PubMed

Park, D-S; Suh, S-J; Hebert, P D N; Oh, H-W; Hong, K-J

2011-08-01

Although DNA barcode coverage has grown rapidly for many insect orders, there are some groups, such as scale insects, where sequence recovery has been difficult. However, using a recently developed primer set, we recovered barcode records from 373 specimens, providing coverage for 75 species from 31 genera in two families. Overall success was >90% for mealybugs and >80% for armored scale species. The G·C content was very low in most species, averaging just 16.3%. Sequence divergences (K2P) between congeneric species averaged 10.7%, while intra-specific divergences averaged 0.97%. However, the latter value was inflated by high intra-specific divergence in nine taxa, cases that may indicate species overlooked by current taxonomic treatments. Our study establishes the feasibility of developing a comprehensive barcode library for scale insects and indicates that its construction will both create an effective system for identifying scale insects and reveal taxonomic situations worthy of deeper analysis.

Ultra-barcoding in cacao (Theobroma spp.; malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA

USDA-ARS?s Scientific Manuscript database

High-throughput next-generation sequencing was used to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an indivi...

Quantitative Experimental Determination of Primer-Dimer Formation Risk by Free-Solution Conjugate Electrophoresis

PubMed Central

Desmarais, Samantha M.; Leitner, Thomas; Barron, Annelise E.

2012-01-01

DNA barcodes are short, unique ssDNA primers that “mark” individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis (FSCE) approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer-barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 basepairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive basepairs formed, yet non-consecutive basepairs did not create stable dimers even when 20 out of 30 possible basepairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation. PMID:22331820

Comparison of manual and semi-automatic DNA extraction protocols for the barcoding characterization of hematophagous louse flies (Diptera: Hippoboscidae).

PubMed

Gutiérrez-López, Rafael; Martínez-de la Puente, Josué; Gangoso, Laura; Soriguer, Ramón C; Figuerola, Jordi

2015-06-01

The barcoding of life initiative provides a universal molecular tool to distinguish animal species based on the amplification and sequencing of a fragment of the subunit 1 of the cytochrome oxidase (COI) gene. Obtaining good quality DNA for barcoding purposes is a limiting factor, especially in studies conducted on small-sized samples or those requiring the maintenance of the organism as a voucher. In this study, we compared the number of positive amplifications and the quality of the sequences obtained using DNA extraction methods that also differ in their economic costs and time requirements and we applied them for the genetic characterization of louse flies. Four DNA extraction methods were studied: chloroform/isoamyl alcohol, HotShot procedure, Qiagen DNeasy(®) Tissue and Blood Kit and DNA Kit Maxwell(®) 16LEV. All the louse flies were morphologically identified as Ornithophila gestroi and a single COI-based haplotype was identified. The number of positive amplifications did not differ significantly among DNA extraction procedures. However, the quality of the sequences was significantly lower for the case of the chloroform/isoamyl alcohol procedure with respect to the rest of methods tested here. These results may be useful for the genetic characterization of louse flies, leaving most of the remaining insect as a voucher. © 2015 The Society for Vector Ecology.

Assessment of snake DNA barcodes based on mitochondrial COI and Cytb genes revealed multiple putative cryptic species in Thailand.

PubMed

Laopichienpong, Nararat; Muangmai, Narongrit; Supikamolseni, Arrjaree; Twilprawat, Panupon; Chanhome, Lawan; Suntrarachun, Sunutcha; Peyachoknagul, Surin; Srikulnath, Kornsorn

2016-12-15

DNA barcodes of mitochondrial cytochrome c oxidase I (COI), cytochrome b (Cytb) genes, and their combined data sets were constructed from 35 snake species in Thailand. No barcoding gap was detected in either of the two genes from the observed intra- and interspecific sequence divergences. Intra- and interspecific sequence divergences of the COI gene differed 14 times, with barcode cut-off scores ranging over 2%-4% for threshold values differentiated among most of the different species; the Cytb gene differed 6 times with cut-off scores ranging over 2%-6%. Thirty-five specific nucleotide mutations were also found at interspecific level in the COI gene, identifying 18 snake species, but no specific nucleotide mutation was observed for Cytb in any single species. This suggests that COI barcoding was a better marker than Cytb. Phylogenetic clustering analysis indicated that most species were represented by monophyletic clusters, suggesting that these snake species could be clearly differentiated using COI barcodes. However, the two-marker combination of both COI and Cytb was more effective, differentiating snake species by over 2%-4%, and reducing species numbers in the overlap value between intra- and interspecific divergences. Three species delimitation algorithms (general mixed Yule-coalescent, automatic barcoding gap detection, and statistical parsimony network analysis) were extensively applied to a wide range of snakes based on both barcodes. This revealed cryptic diversity for eleven snake species in Thailand. In addition, eleven accessions from the database previously grouped under the same species were represented at different species level, suggesting either high genetic diversity, or the misidentification of these sequences in the database as a consequence of cryptic species. Copyright © 2016 Elsevier B.V. All rights reserved.

The Trichoptera barcode initiative: a strategy for generating a species-level Tree of Life.

PubMed

Zhou, Xin; Frandsen, Paul B; Holzenthal, Ralph W; Beet, Clare R; Bennett, Kristi R; Blahnik, Roger J; Bonada, Núria; Cartwright, David; Chuluunbat, Suvdtsetseg; Cocks, Graeme V; Collins, Gemma E; deWaard, Jeremy; Dean, John; Flint, Oliver S; Hausmann, Axel; Hendrich, Lars; Hess, Monika; Hogg, Ian D; Kondratieff, Boris C; Malicky, Hans; Milton, Megan A; Morinière, Jérôme; Morse, John C; Mwangi, François Ngera; Pauls, Steffen U; Gonzalez, María Razo; Rinne, Aki; Robinson, Jason L; Salokannel, Juha; Shackleton, Michael; Smith, Brian; Stamatakis, Alexandros; StClair, Ros; Thomas, Jessica A; Zamora-Muñoz, Carmen; Ziesmann, Tanja; Kjer, Karl M

2016-09-05

DNA barcoding was intended as a means to provide species-level identifications through associating DNA sequences from unknown specimens to those from curated reference specimens. Although barcodes were not designed for phylogenetics, they can be beneficial to the completion of the Tree of Life. The barcode database for Trichoptera is relatively comprehensive, with data from every family, approximately two-thirds of the genera, and one-third of the described species. Most Trichoptera, as with most of life's species, have never been subjected to any formal phylogenetic analysis. Here, we present a phylogeny with over 16 000 unique haplotypes as a working hypothesis that can be updated as our estimates improve. We suggest a strategy of implementing constrained tree searches, which allow larger datasets to dictate the backbone phylogeny, while the barcode data fill out the tips of the tree. We also discuss how this phylogeny could be used to focus taxonomic attention on ambiguous species boundaries and hidden biodiversity. We suggest that systematists continue to differentiate between 'Barcode Index Numbers' (BINs) and 'species' that have been formally described. Each has utility, but they are not synonyms. We highlight examples of integrative taxonomy, using both barcodes and morphology for species description.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

[Identification of pyrrosiae folium and its adulterants based on psbA-trnH sequence].

PubMed

Zhang, Ya-Qin; Shi, Yue; Song, Ming; Lin, Yun-Han; Ma, Xiao-Xi; Sun, Wei; Xiang, Li; Liu, Xi

2014-06-01

In this study, the psbA-trnH sequence as DNA barcode was used to evaluate the accuracy and stability for identification pteridophyte medicinal material Pyrrosiae Foliumas from adulterants. Genomic DNA from 106 samples were extracted successfully. The Kimura 2-Parameter (K2P) distances and ML tree were calculated using software MEGA 6.0. The intra-specific genetic distances of 3 original plants were lower than inter-specific genetic distances of adulterants. The ML tree indicated that Pyrrosiae Folium can be distinguished from its adulterants obviously. Therefore, the psbA-trnH sequence as a barcode of the pteridophyte, can accurately and stably distinguish Pyrrosiae Folium from its adulterants.

From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity.

PubMed

Cristescu, Melania E

2014-10-01

DNA-based species identification, known as barcoding, transformed the traditional approach to the study of biodiversity science. The field is transitioning from barcoding individuals to metabarcoding communities. This revolution involves new sequencing technologies, bioinformatics pipelines, computational infrastructure, and experimental designs. In this dynamic genomics landscape, metabarcoding studies remain insular and biodiversity estimates depend on the particular methods used. In this opinion article, I discuss the need for a coordinated advancement of DNA-based species identification that integrates taxonomic and barcoding information. Such an approach would facilitate access to almost 3 centuries of taxonomic knowledge and 1 decade of building repository barcodes. Conservation projects are time sensitive, research funding is becoming restricted, and informed decisions depend on our ability to embrace integrative approaches to biodiversity science. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identifying Canadian Freshwater Fishes through DNA Barcodes

PubMed Central

Hubert, Nicolas; Hanner, Robert; Holm, Erling; Mandrak, Nicholas E.; Taylor, Eric; Burridge, Mary; Watkinson, Douglas; Dumont, Pierre; Curry, Allen; Bentzen, Paul; Zhang, Junbin; April, Julien; Bernatchez, Louis

2008-01-01

Background DNA barcoding aims to provide an efficient method for species-level identifications using an array of species specific molecular tags derived from the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene. The efficiency of the method hinges on the degree of sequence divergence among species and species-level identifications are relatively straightforward when the average genetic distance among individuals within a species does not exceed the average genetic distance between sister species. Fishes constitute a highly diverse group of vertebrates that exhibit deep phenotypic changes during development. In this context, the identification of fish species is challenging and DNA barcoding provide new perspectives in ecology and systematics of fishes. Here we examined the degree to which DNA barcoding discriminate freshwater fish species from the well-known Canadian fauna, which currently encompasses nearly 200 species, some which are of high economic value like salmons and sturgeons. Methodology/Principal Findings We bi-directionally sequenced the standard 652 bp “barcode” region of COI for 1360 individuals belonging to 190 of the 203 Canadian freshwater fish species (95%). Most species were represented by multiple individuals (7.6 on average), the majority of which were retained as voucher specimens. The average genetic distance was 27 fold higher between species than within species, as K2P distance estimates averaged 8.3% among congeners and only 0.3% among concpecifics. However, shared polymorphism between sister-species was detected in 15 species (8% of the cases). The distribution of K2P distance between individuals and species overlapped and identifications were only possible to species group using DNA barcodes in these cases. Conversely, deep hidden genetic divergence was revealed within two species, suggesting the presence of cryptic species. Conclusions/Significance The present study evidenced that freshwater fish species can be efficiently identified through the use of DNA barcoding, especially the species complex of small-sized species, and that the present COI library can be used for subsequent applications in ecology and systematics. PMID:22423312

DNA Barcode for Identifying Folium Artemisiae Argyi from Counterfeits.

PubMed

Mei, Quanxi; Chen, Xiaolu; Xiang, Li; Liu, Yue; Su, Yanyan; Gao, Yuqiao; Dai, Weibo; Dong, Pengpeng; Chen, Shilin

2016-01-01

Folium Artemisiae Argyi is an important herb in traditional Chinese medicine. It is commonly used in moxibustion, medicine, etc. However, identifying Artemisia argyi is difficult because this herb exhibits similar morphological characteristics to closely related species and counterfeits. To verify the applicability of DNA barcoding, ITS2 and psbA-trnH were used to identify A. argyi from 15 closely related species and counterfeits. Results indicated that total DNA was easily extracted from all the samples and that both ITS2 and psbA-trnH fragments can be easily amplified. ITS2 was a more ideal barcode than psbA-trnH and ITS2+psbA-trnH to identify A. argyi from closely related species and counterfeits on the basis of sequence character, genetic distance, and tree methods. The sequence length was 225 bp for the 56 ITS2 sequences of A. argyi, and no variable site was detected. For the ITS2 sequences, A. capillaris, A. anomala, A. annua, A. igniaria, A. maximowicziana, A. princeps, Dendranthema vestitum, and D. indicum had single nucleotide polymorphisms (SNPs). The intraspecific Kimura 2-Parameter distance was zero, which is lower than the minimum interspecific distance (0.005). A. argyi, the closely related species, and counterfeits, except for Artemisia maximowicziana and Artemisia sieversiana, were separated into pairs of divergent clusters by using the neighbor joining, maximum parsimony, and maximum likelihood tree methods. Thus, the ITS2 sequence was an ideal barcode to identify A. argyi from closely related species and counterfeits to ensure the safe use of this plant.

Exploring Genetic Divergence in a Species-Rich Insect Genus Using 2790 DNA Barcodes

PubMed Central

Lin, Xiaolong; Stur, Elisabeth; Ekrem, Torbjørn

2015-01-01

DNA barcoding using a fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) has proven to be successful for species-level identification in many animal groups. However, most studies have been focused on relatively small datasets or on large datasets of taxonomically high-ranked groups. We explore the quality of DNA barcodes to delimit species in the diverse chironomid genus Tanytarsus (Diptera: Chironomidae) by using different analytical tools. The genus Tanytarsus is the most species-rich taxon of tribe Tanytarsini (Diptera: Chironomidae) with more than 400 species worldwide, some of which can be notoriously difficult to identify to species-level using morphology. Our dataset, based on sequences generated from own material and publicly available data in BOLD, consist of 2790 DNA barcodes with a fragment length of at least 500 base pairs. A neighbor joining tree of this dataset comprises 131 well separated clusters representing 121 morphological species of Tanytarsus: 77 named, 16 unnamed and 28 unidentified theoretical species. For our geographically widespread dataset, DNA barcodes unambiguously discriminate 94.6% of the Tanytarsus species recognized through prior morphological study. Deep intraspecific divergences exist in some species complexes, and need further taxonomic studies using appropriate nuclear markers as well as morphological and ecological data to be resolved. The DNA barcodes cluster into 120–242 molecular operational taxonomic units (OTUs) depending on whether Objective Clustering, Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescent model (GMYC), Poisson Tree Process (PTP), subjective evaluation of the neighbor joining tree or Barcode Index Numbers (BINs) are used. We suggest that a 4–5% threshold is appropriate to delineate species of Tanytarsus non-biting midges. PMID:26406595

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions.

PubMed

Raupach, Michael J; Barco, Andrea; Steinke, Dirk; Beermann, Jan; Laakmann, Silke; Mohrbeck, Inga; Neumann, Hermann; Kihara, Terue C; Pointner, Karin; Radulovici, Adriana; Segelken-Voigt, Alexandra; Wesse, Christina; Knebelsberger, Thomas

2015-01-01

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences.

Improving the Conservation of Mediterranean Chondrichthyans: The ELASMOMED DNA Barcode Reference Library

PubMed Central

Arculeo, Marco; Bonello, Juan J.; Bonnici, Leanne; Cannas, Rita; Carbonara, Pierluigi; Cau, Alessandro; Charilaou, Charis; El Ouamari, Najib; Fiorentino, Fabio; Follesa, Maria Cristina; Garofalo, Germana; Golani, Daniel; Guarniero, Ilaria; Hanner, Robert; Hemida, Farid; Kada, Omar; Lo Brutto, Sabrina; Mancusi, Cecilia; Morey, Gabriel; Schembri, Patrick J.; Serena, Fabrizio; Sion, Letizia; Stagioni, Marco; Tursi, Angelo; Vrgoc, Nedo; Steinke, Dirk; Tinti, Fausto

2017-01-01

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions. PMID:28107413

Establishing a community-wide DNA barcode library as a new tool for arctic research.

PubMed

Wirta, H; Várkonyi, G; Rasmussen, C; Kaartinen, R; Schmidt, N M; Hebert, P D N; Barták, M; Blagoev, G; Disney, H; Ertl, S; Gjelstrup, P; Gwiazdowicz, D J; Huldén, L; Ilmonen, J; Jakovlev, J; Jaschhof, M; Kahanpää, J; Kankaanpää, T; Krogh, P H; Labbee, R; Lettner, C; Michelsen, V; Nielsen, S A; Nielsen, T R; Paasivirta, L; Pedersen, S; Pohjoismäki, J; Salmela, J; Vilkamaa, P; Väre, H; von Tschirnhaus, M; Roslin, T

2016-05-01

DNA sequences offer powerful tools for describing the members and interactions of natural communities. In this study, we establish the to-date most comprehensive library of DNA barcodes for a terrestrial site, including all known macroscopic animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley in Northeast Greenland. To demonstrate its utility, we apply the library to identify nearly 20 000 arthropod individuals from two Malaise traps, each operated for two summers. Drawing on this material, we estimate the coverage of previous morphology-based species inventories, derive a snapshot of faunal turnover in space and time and describe the abundance and phenology of species in the rapidly changing arctic environment. Overall, 403 terrestrial animal and 160 vascular plant species were recorded by morphology-based techniques. DNA barcodes (CO1) offered high resolution in discriminating among the local animal taxa, with 92% of morphologically distinguishable taxa assigned to unique Barcode Index Numbers (BINs) and 93% to monophyletic clusters. For vascular plants, resolution was lower, with 54% of species forming monophyletic clusters based on barcode regions rbcLa and ITS2. Malaise catches revealed 122 BINs not detected by previous sampling and DNA barcoding. The insect community was dominated by a few highly abundant taxa. Even closely related taxa differed in phenology, emphasizing the need for species-level resolution when describing ongoing shifts in arctic communities and ecosystems. The DNA barcode library now established for Zackenberg offers new scope for such explorations, and for the detailed dissection of interspecific interactions throughout the community. © 2015 John Wiley & Sons Ltd.

Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

PubMed Central

Best, Katharine; Oakes, Theres; Heather, James M.; Shawe-Taylor, John; Chain, Benny

2015-01-01

The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS. PMID:26459131

Reading Mammal Diversity from Flies: The Persistence Period of Amplifiable Mammal mtDNA in Blowfly Guts (Chrysomya megacephala) and a New DNA Mini-Barcode Target.

PubMed

Lee, Ping-Shin; Sing, Kong-Wah; Wilson, John-James

2015-01-01

Most tropical mammal species are threatened or data-deficient. Data collection is impeded by the traditional monitoring approaches which can be laborious, expensive and struggle to detect cryptic diversity. Monitoring approaches using mammal DNA derived from invertebrates are emerging as cost- and time-effective alternatives. As a step towards development of blowfly-derived DNA as an effective method for mammal monitoring in the biodiversity hotspot of Peninsular Malaysia, our objectives were (i) to determine the persistence period of amplifiable mammal mtDNA in blowfly guts through a laboratory feeding experiment (ii) to design and test primers that can selectively amplify mammal COI DNA mini-barcodes in the presence of high concentrations of blowfly DNA. The persistence period of amplifiable mammal mtDNA in blowfly guts was 24 h to 96 h post-feeding indicating the need for collecting flies within 24 h of capture to detect mammal mtDNA of sufficient quantity and quality. We designed a new primer combination for a COI DNA mini-barcode that did not amplify blowfly DNA and showed 89% amplification success for a dataset of mammals from Peninsular Malaysia. The short (205 bp) DNA mini-barcode could distinguish most mammal species (including separating dark taxa) and is of suitable length for high-throughput sequencing. Our new DNA mini-barcode target and a standardized trapping protocol with retrieval of blowflies every 24 h could point the way forward in the development of blowfly-derived DNA as an effective method for mammal monitoring.

Reading Mammal Diversity from Flies: The Persistence Period of Amplifiable Mammal mtDNA in Blowfly Guts (Chrysomya megacephala) and a New DNA Mini-Barcode Target

PubMed Central

Lee, Ping-Shin; Sing, Kong-Wah; Wilson, John-James

2015-01-01

Most tropical mammal species are threatened or data-deficient. Data collection is impeded by the traditional monitoring approaches which can be laborious, expensive and struggle to detect cryptic diversity. Monitoring approaches using mammal DNA derived from invertebrates are emerging as cost- and time-effective alternatives. As a step towards development of blowfly-derived DNA as an effective method for mammal monitoring in the biodiversity hotspot of Peninsular Malaysia, our objectives were (i) to determine the persistence period of amplifiable mammal mtDNA in blowfly guts through a laboratory feeding experiment (ii) to design and test primers that can selectively amplify mammal COI DNA mini-barcodes in the presence of high concentrations of blowfly DNA. The persistence period of amplifiable mammal mtDNA in blowfly guts was 24 h to 96 h post-feeding indicating the need for collecting flies within 24 h of capture to detect mammal mtDNA of sufficient quantity and quality. We designed a new primer combination for a COI DNA mini-barcode that did not amplify blowfly DNA and showed 89% amplification success for a dataset of mammals from Peninsular Malaysia. The short (205 bp) DNA mini-barcode could distinguish most mammal species (including separating dark taxa) and is of suitable length for high-throughput sequencing. Our new DNA mini-barcode target and a standardized trapping protocol with retrieval of blowflies every 24 h could point the way forward in the development of blowfly-derived DNA as an effective method for mammal monitoring. PMID:25898278

Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library

PubMed Central

2012-01-01

Background Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. Results This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Conclusions Our results provided fast and cost-effective solution to create a comprehensive, effective DNA barcode reference library for a local flora. PMID:23190419

Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library.

PubMed

Kuzmina, Maria L; Johnson, Karen L; Barron, Hannah R; Hebert, Paul Dn

2012-11-28

Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Our results provided fast and cost-effective solution to create a comprehensive, effective DNA barcode reference library for a local flora.

Plant DNA barcodes and assessment of phylogenetic community structure of a tropical mixed dipterocarp forest in Brunei Darussalam (Borneo)

PubMed Central

Abu Salim, Kamariah; Chase, Mark W.; Dexter, Kyle G.; Pennington, R. Toby; Tan, Sylvester; Kaye, Maria Ellen; Samuel, Rosabelle

2017-01-01

DNA barcoding is a fast and reliable tool to assess and monitor biodiversity and, via community phylogenetics, to investigate ecological and evolutionary processes that may be responsible for the community structure of forests. In this study, DNA barcodes for the two widely used plastid coding regions rbcL and matK are used to contribute to identification of morphologically undetermined individuals, as well as to investigate phylogenetic structure of tree communities in 70 subplots (10 × 10m) of a 25-ha forest-dynamics plot in Brunei (Borneo, Southeast Asia). The combined matrix (rbcL + matK) comprised 555 haplotypes (from ≥154 genera, 68 families and 25 orders sensu APG, Angiosperm Phylogeny Group, 2016), making a substantial contribution to tree barcode sequences from Southeast Asia. Barcode sequences were used to reconstruct phylogenetic relationships using maximum likelihood, both with and without constraining the topology of taxonomic orders to match that proposed by the Angiosperm Phylogeny Group. A third phylogenetic tree was reconstructed using the program Phylomatic to investigate the influence of phylogenetic resolution on results. Detection of non-random patterns of community assembly was determined by net relatedness index (NRI) and nearest taxon index (NTI). In most cases, community assembly was either random or phylogenetically clustered, which likely indicates the importance to community structure of habitat filtering based on phylogenetically correlated traits in determining community structure. Different phylogenetic trees gave similar overall results, but the Phylomatic tree produced greater variation across plots for NRI and NTI values, presumably due to noise introduced by using an unresolved phylogenetic tree. Our results suggest that using a DNA barcode tree has benefits over the traditionally used Phylomatic approach by increasing precision and accuracy and allowing the incorporation of taxonomically unidentified individuals into analyses. PMID:29049301

Analyzing Mosquito (Diptera: Culicidae) Diversity in Pakistan by DNA Barcoding

PubMed Central

Ashfaq, Muhammad; Hebert, Paul D. N.; Mirza, Jawwad H.; Khan, Arif M.; Zafar, Yusuf; Mirza, M. Sajjad

2014-01-01

Background Although they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications. Methodology/Principal Findings Sequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010–2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0–2.4%, while congeneric species showed from 2.3–17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments. Conclusions As the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations. PMID:24827460

Analyzing mosquito (Diptera: culicidae) diversity in Pakistan by DNA barcoding.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N; Mirza, Jawwad H; Khan, Arif M; Zafar, Yusuf; Mirza, M Sajjad

2014-01-01

Although they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications. Sequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010-2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0-2.4%, while congeneric species showed from 2.3-17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments. As the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations.

Single molecule counting and assessment of random molecular tagging errors with transposable giga-scale error-correcting barcodes.

PubMed

Lau, Billy T; Ji, Hanlee P

2017-09-21

RNA-Seq measures gene expression by counting sequence reads belonging to unique cDNA fragments. Molecular barcodes commonly in the form of random nucleotides were recently introduced to improve gene expression measures by detecting amplification duplicates, but are susceptible to errors generated during PCR and sequencing. This results in false positive counts, leading to inaccurate transcriptome quantification especially at low input and single-cell RNA amounts where the total number of molecules present is minuscule. To address this issue, we demonstrated the systematic identification of molecular species using transposable error-correcting barcodes that are exponentially expanded to tens of billions of unique labels. We experimentally showed random-mer molecular barcodes suffer from substantial and persistent errors that are difficult to resolve. To assess our method's performance, we applied it to the analysis of known reference RNA standards. By including an inline random-mer molecular barcode, we systematically characterized the presence of sequence errors in random-mer molecular barcodes. We observed that such errors are extensive and become more dominant at low input amounts. We described the first study to use transposable molecular barcodes and its use for studying random-mer molecular barcode errors. Extensive errors found in random-mer molecular barcodes may warrant the use of error correcting barcodes for transcriptome analysis as input amounts decrease.

The Role of DNA Barcodes in Understanding and Conservation of Mammal Diversity in Southeast Asia

PubMed Central

Francis, Charles M.; Borisenko, Alex V.; Ivanova, Natalia V.; Eger, Judith L.; Lim, Burton K.; Guillén-Servent, Antonio; Kruskop, Sergei V.; Mackie, Iain; Hebert, Paul D. N.

2010-01-01

Background Southeast Asia is recognized as a region of very high biodiversity, much of which is currently at risk due to habitat loss and other threats. However, many aspects of this diversity, even for relatively well-known groups such as mammals, are poorly known, limiting ability to develop conservation plans. This study examines the value of DNA barcodes, sequences of the mitochondrial COI gene, to enhance understanding of mammalian diversity in the region and hence to aid conservation planning. Methodology and Principal Findings DNA barcodes were obtained from nearly 1900 specimens representing 165 recognized species of bats. All morphologically or acoustically distinct species, based on classical taxonomy, could be discriminated with DNA barcodes except four closely allied species pairs. Many currently recognized species contained multiple barcode lineages, often with deep divergence suggesting unrecognized species. In addition, most widespread species showed substantial genetic differentiation across their distributions. Our results suggest that mammal species richness within the region may be underestimated by at least 50%, and there are higher levels of endemism and greater intra-specific population structure than previously recognized. Conclusions DNA barcodes can aid conservation and research by assisting field workers in identifying species, by helping taxonomists determine species groups needing more detailed analysis, and by facilitating the recognition of the appropriate units and scales for conservation planning. PMID:20838635

Molecular Barcoding of Aquatic Oligochaetes: Implications for Biomonitoring

PubMed Central

Vivien, Régis; Wyler, Sofia; Lafont, Michel; Pawlowski, Jan

2015-01-01

Aquatic oligochaetes are well recognized bioindicators of quality of sediments and water in watercourses and lakes. However, the difficult taxonomic determination based on morphological features compromises their more common use in eco-diagnostic analyses. To overcome this limitation, we investigated molecular barcodes as identification tool for broad range of taxa of aquatic oligochaetes. We report 185 COI and 52 ITS2 rDNA sequences for specimens collected in Switzerland and belonging to the families Naididae, Lumbriculidae, Enchytraeidae and Lumbricidae. Phylogenetic analyses allowed distinguishing 41 lineages separated by more than 10 % divergence in COI sequences. The lineage distinction was confirmed by Automatic Barcode Gap Discovery (ABGD) method and by ITS2 data. Our results showed that morphological identification underestimates the oligochaete diversity. Only 26 of the lineages could be assigned to morphospecies, of which seven were sequenced for the first time. Several cryptic species were detected within common morphospecies. Many juvenile specimens that could not be assigned morphologically have found their home after genetic analysis. Our study showed that COI barcodes performed very well as species identifiers in aquatic oligochaetes. Their easy amplification and good taxonomic resolution might help promoting aquatic oligochaetes as bioindicators for next generation environmental DNA biomonitoring of aquatic ecosystems. PMID:25856230

Benefits and Limitations of DNA Barcoding and Metabarcoding in Herbal Product Authentication.

PubMed

Raclariu, Ancuta Cristina; Heinrich, Michael; Ichim, Mihael Cristin; de Boer, Hugo

2018-03-01

Herbal medicines play an important role globally in the health care sector and in industrialised countries they are often considered as an alternative to mono-substance medicines. Current quality and authentication assessment methods rely mainly on morphology and analytical phytochemistry-based methods detailed in pharmacopoeias. Herbal products however are often highly processed with numerous ingredients, and even if these analytical methods are accurate for quality control of specific lead or marker compounds, they are of limited suitability for the authentication of biological ingredients. To review the benefits and limitations of DNA barcoding and metabarcoding in complementing current herbal product authentication. Recent literature relating to DNA based authentication of medicinal plants, herbal medicines and products are summarised to provide a basic understanding of how DNA barcoding and metabarcoding can be applied to this field. Different methods of quality control and authentication have varying resolution and usefulness along the value chain of these products. DNA barcoding can be used for authenticating products based on single herbal ingredients and DNA metabarcoding for assessment of species diversity in processed products, and both methods should be used in combination with appropriate hyphenated chemical methods for quality control. DNA barcoding and metabarcoding have potential in the context of quality control of both well and poorly regulated supply systems. Standardisation of protocols for DNA barcoding and DNA sequence-based identification are necessary before DNA-based biological methods can be implemented as routine analytical approaches and approved by the competent authorities for use in regulated procedures. © 2017 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd. © 2017 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd.

Filling Gaps in Biodiversity Knowledge for Macrofungi: Contributions and Assessment of an Herbarium Collection DNA Barcode Sequencing Project

PubMed Central

Osmundson, Todd W.; Robert, Vincent A.; Schoch, Conrad L.; Baker, Lydia J.; Smith, Amy; Robich, Giovanni; Mizzan, Luca; Garbelotto, Matteo M.

2013-01-01

Despite recent advances spearheaded by molecular approaches and novel technologies, species description and DNA sequence information are significantly lagging for fungi compared to many other groups of organisms. Large scale sequencing of vouchered herbarium material can aid in closing this gap. Here, we describe an effort to obtain broad ITS sequence coverage of the approximately 6000 macrofungal-species-rich herbarium of the Museum of Natural History in Venice, Italy. Our goals were to investigate issues related to large sequencing projects, develop heuristic methods for assessing the overall performance of such a project, and evaluate the prospects of such efforts to reduce the current gap in fungal biodiversity knowledge. The effort generated 1107 sequences submitted to GenBank, including 416 previously unrepresented taxa and 398 sequences exhibiting a best BLAST match to an unidentified environmental sequence. Specimen age and taxon affected sequencing success, and subsequent work on failed specimens showed that an ITS1 mini-barcode greatly increased sequencing success without greatly reducing the discriminating power of the barcode. Similarity comparisons and nonmetric multidimensional scaling ordinations based on pairwise distance matrices proved to be useful heuristic tools for validating the overall accuracy of specimen identifications, flagging potential misidentifications, and identifying taxa in need of additional species-level revision. Comparison of within- and among-species nucleotide variation showed a strong increase in species discriminating power at 1–2% dissimilarity, and identified potential barcoding issues (same sequence for different species and vice-versa). All sequences are linked to a vouchered specimen, and results from this study have already prompted revisions of species-sequence assignments in several taxa. PMID:23638077

Filling gaps in biodiversity knowledge for macrofungi: contributions and assessment of an herbarium collection DNA barcode sequencing project.

PubMed

Osmundson, Todd W; Robert, Vincent A; Schoch, Conrad L; Baker, Lydia J; Smith, Amy; Robich, Giovanni; Mizzan, Luca; Garbelotto, Matteo M

2013-01-01

Despite recent advances spearheaded by molecular approaches and novel technologies, species description and DNA sequence information are significantly lagging for fungi compared to many other groups of organisms. Large scale sequencing of vouchered herbarium material can aid in closing this gap. Here, we describe an effort to obtain broad ITS sequence coverage of the approximately 6000 macrofungal-species-rich herbarium of the Museum of Natural History in Venice, Italy. Our goals were to investigate issues related to large sequencing projects, develop heuristic methods for assessing the overall performance of such a project, and evaluate the prospects of such efforts to reduce the current gap in fungal biodiversity knowledge. The effort generated 1107 sequences submitted to GenBank, including 416 previously unrepresented taxa and 398 sequences exhibiting a best BLAST match to an unidentified environmental sequence. Specimen age and taxon affected sequencing success, and subsequent work on failed specimens showed that an ITS1 mini-barcode greatly increased sequencing success without greatly reducing the discriminating power of the barcode. Similarity comparisons and nonmetric multidimensional scaling ordinations based on pairwise distance matrices proved to be useful heuristic tools for validating the overall accuracy of specimen identifications, flagging potential misidentifications, and identifying taxa in need of additional species-level revision. Comparison of within- and among-species nucleotide variation showed a strong increase in species discriminating power at 1-2% dissimilarity, and identified potential barcoding issues (same sequence for different species and vice-versa). All sequences are linked to a vouchered specimen, and results from this study have already prompted revisions of species-sequence assignments in several taxa.

DNA barcoding of Clarias gariepinus, Coptodon zillii and Sarotherodon melanotheron from Southwestern Nigeria

PubMed Central

Falade, Mofolusho O.; Opene, Anthony J.; Benson, Otarigho

2016-01-01

DNA barcoding has been adopted as a gold standard rapid, precise and unifying identification system for animal species and provides a database of genetic sequences that can be used as a tool for universal species identification. In this study, we employed mitochondrial genes 16S rRNA (16S) and cytochrome oxidase subunit I (COI) for the identification of some Nigerian freshwater catfish and Tilapia species. Approximately 655 bp were amplified from the 5′ region of the mitochondrial cytochrome C oxidase subunit I (COI) gene whereas 570 bp were amplified for the 16S rRNA gene. Nucleotide divergences among sequences were estimated based on Kimura 2-parameter distances and the genetic relationships were assessed by constructing phylogenetic trees using the neighbour-joining (NJ) and maximum likelihood (ML) methods. Analyses of consensus barcode sequences for each species, and alignment of individual sequences from within a given species revealed highly consistent barcodes (99% similarity on average), which could be compared with deposited sequences in public databases. The nucleotide distance between species belonging to different genera based on COI ranged from 0.17% between Sarotherodon melanotheron and Coptodon zillii to 0.49% between Clarias gariepinus and C. zillii, indicating that S. melanotheron and C. zillii are closely related. Based on the data obtained, the utility of COI gene was confirmed in accurate identification of three fish species from Southwest Nigeria. PMID:27990256

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring.

PubMed

Rimet, Frédéric; Chaumeil, Philippe; Keck, François; Kermarrec, Lenaïg; Vasselon, Valentin; Kahlert, Maria; Franc, Alain; Bouchez, Agnès

2016-01-01

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/. © The Author(s) 2016. Published by Oxford University Press.

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring

PubMed Central

Rimet, Frédéric; Chaumeil, Philippe; Keck, François; Kermarrec, Lenaïg; Vasselon, Valentin; Kahlert, Maria; Franc, Alain; Bouchez, Agnès

2016-01-01

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/ PMID:26989149

Integrative taxonomy detects cryptic and overlooked fish species in a neotropical river basin.

PubMed

Gomes, Laís Carvalho; Pessali, Tiago Casarim; Sales, Naiara Guimarães; Pompeu, Paulo Santos; Carvalho, Daniel Cardoso

2015-10-01

The great freshwater fish diversity found in the neotropical region makes management and conservation actions challenging. Due to shortage of taxonomists and insufficient infrastructure to deal with such great biodiversity (i.e. taxonomic impediment), proposed remedies to accelerate species identification and descriptions include techniques that combine DNA-based identification and concise morphological description. The building of a DNA barcode reference database correlating meristic and genetic data was developed for 75 % of the Mucuri River basin's freshwater fish. We obtained a total of 141 DNA barcode sequences from 37 species belonging to 30 genera, 19 families, and 5 orders. Genetic distances within species, genera, and families were 0.74, 9.5, and 18.86 %, respectively. All species could be clearly identified by the DNA barcodes. Divergences between meristic morphological characteristics and DNA barcodes revealed two cryptic species among the Cyphocharax gilbert and Astyanax gr. bimaculatus specimens, and helped to identify two overlooked species within the Gymnotus and Astyanax taxa. Therefore, using a simplified model of neotropical biodiversity, we tested the efficiency of an integrative taxonomy approach for species discovery, identification of cryptic diversity, and accelerating biodiversity descriptions.

Review: DNA Barcoding and Chromatography Fingerprints for the Authentication of Botanicals in Herbal Medicinal Products

PubMed Central

2017-01-01

In the last two decades, there has been a tremendous increase in the global use of herbal medicinal products (HMPs) due to their claimed health benefits. This has led to increase in their demand and consequently, also, resulted in massive adulteration. This is due to the fact that most of the traditional methods cannot identify closely related species in a process product form. Therefore the urgent need for simple and rapid identification methods resulted in the discovery of a novel technique. DNA barcoding is a process that uses short DNA sequence from the standard genome for species identification. This technique is reliable and is not affected by external factors such as climates, age, or plant part. The difficulties in isolation of DNA of high quality in addition to other factors are among the challenges encountered using the DNA barcoding in the authentication of HMP. These limitations indicated that using DNA barcoding alone may ineffectively authenticate the HMP. Therefore, the combination of DNA barcoding with chromatographic fingerprint, a popular and generally accepted technique for the assessment and quality control of HMP, will offer an efficient solution to effectively evaluate the authenticity and quality consistency of HMP. Detailed and quality information about the main composition of the HMPs will help to ascertain their efficacy and safety as these are very important for quality control. PMID:28536641

Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

PubMed

Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

2018-01-01

We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region.

PubMed

Ma, Xin-Ye; Xie, Cai-Xiang; Liu, Chang; Song, Jing-Yuan; Yao, Hui; Luo, Kun; Zhu, Ying-Jie; Gao, Ting; Pang, Xiao-Hui; Qian, Jun; Chen, Shi-Lin

2010-01-01

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.

A regional approach to plant DNA barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the Canadian Arctic Archipelago.

PubMed

Clerc-Blain, Jessica L E; Starr, Julian R; Bull, Roger D; Saarela, Jeffery M

2010-01-01

Previous research on barcoding sedges (Carex) suggested that basic searches within a global barcoding database would probably not resolve more than 60% of the world's some 2000 species. In this study, we take an alternative approach and explore the performance of plant DNA barcoding in the Carex lineage from an explicitly regional perspective. We characterize the utility of a subset of the proposed protein-coding and noncoding plastid barcoding regions (matK, rpoB, rpoC1, rbcL, atpF-atpH, psbK-psbI) for distinguishing species of Carex and Kobresia in the Canadian Arctic Archipelago, a clearly defined eco-geographical region representing 1% of the Earth's landmass. Our results show that matK resolves the greatest number of species of any single-locus (95%), and when combined in a two-locus barcode, it provides 100% species resolution in all but one combination (matK + atpFH) during unweighted pair-group method with arithmetic mean averages (UPGMA) analyses. Noncoding regions were equally or more variable than matK, but as single markers they resolve substantially fewer taxa than matK alone. When difficulties with sequencing and alignment due to microstructural variation in noncoding regions are also considered, our results support other studies in suggesting that protein-coding regions are more practical as barcoding markers. Plastid DNA barcodes are an effective identification tool for species of Carex and Kobresia in the Canadian Arctic Archipelago, a region where the number of co-existing closely related species is limited. We suggest that if a regional approach to plant DNA barcoding was applied on a global scale, it could provide a solution to the generally poor species resolution seen in previous barcoding studies. © 2009 Blackwell Publishing Ltd.

Identification of maca (Lepidium meyenii Walp.) and its adulterants by a DNA-barcoding approach based on the ITS sequence.

PubMed

Chen, Jin-Jin; Zhao, Qing-Sheng; Liu, Yi-Lan; Zha, Sheng-Hua; Zhao, Bing

2015-09-01

Maca (Lepidium meyenii) is an herbaceous plant that grows in high plateaus and has been used as both food and folk medicine for centuries because of its benefits to human health. In the present study, ITS (internal transcribed spacer) sequences of forty-three maca samples, collected from different regions or vendors, were amplified and analyzed. The ITS sequences of nineteen potential adulterants of maca were also collected and analyzed. The results indicated that the ITS sequence of maca was consistent in all samples and unique when compared with its adulterants. Therefore, this DNA-barcoding approach based on the ITS sequence can be used for the molecular identification of maca and its adulterants. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Integrating DNA barcodes and morphology for species delimitation in the Corynoneura group (Diptera: Chironomidae: Orthocladiinae).

PubMed

Silva, F L; Wiedenbrug, S

2014-02-01

In this study, we use DNA barcodes for species delimitation to solve taxonomic conflicts in 86 specimens of 14 species belonging to the Corynoneura group (Diptera: Chironomidae: Orthocladiinae), from the Atlantic Forest, Brazil. Molecular analysis of cytochrome c-oxidase subunit I (COI) gene sequences supported 14 cohesive species groups, of which two similar groups were subsequently associated with morphological variation at the pupal stage. Eleven species previously described based on morphological criteria were linked to DNA markers. Furthermore, there is the possibility that there may be cryptic species within the Corynoneura group, since one group of species presented internal grouping, although no morphological divergence was observed. Our results support DNA-barcoding as an excellent tool for species delimitation in groups where taxonomy by means of morphology is difficult or even impossible.

DNA barcode reference data for the Korean herpetofauna and their applications.

PubMed

Jeong, Tae Jin; Jun, Jumin; Han, Sanghoon; Kim, Hyun Tae; Oh, Kyunghee; Kwak, Myounghai

2013-11-01

Recently, amphibians and reptiles have drawn attention because of declines in species and populations caused mainly by habitat loss, overexploitation and climate change. This study constructed a DNA barcode database for the Korean herpetofauna, including all the recorded amphibians and 68% of the recorded reptiles, to provide a useful, standardized tool for species identification in monitoring and management. A total of 103 individuals from 18 amphibian and 17 reptile species were used to generate barcode sequences using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and to compare it with other suggested barcode loci. Comparing 16S rRNA, cytochrome b (Cytb) and COI for amphibians and 12S rRNA, Cytb and COI for reptiles, our results revealed that COI is better than the other markers in terms of a high level of sequence variation without length variation and moderate amplification success. Although the COI marker had no clear barcoding gap because of the high level of intraspecific variation, all of the analysed individuals from the same species clustered together in a neighbour-joining tree. High intraspecific variation suggests the possibility of cryptic species. Finally, using this database, confiscated snakes were identified as Elaphe schrenckii, designated as endangered in Korea and a food contaminant was identified as the lizard Takydromus amurensis. © 2013 John Wiley & Sons Ltd.

Molecular identification and phylogenetic study of Demodex caprae.

PubMed

Zhao, Ya-E; Cheng, Juan; Hu, Li; Ma, Jun-Xian

2014-10-01

The DNA barcode has been widely used in species identification and phylogenetic analysis since 2003, but there have been no reports in Demodex. In this study, to obtain an appropriate DNA barcode for Demodex, molecular identification of Demodex caprae based on mitochondrial cox1 was conducted. Firstly, individual adults and eggs of D. caprae were obtained for genomic DNA (gDNA) extraction; Secondly, mitochondrial cox1 fragment was amplified, cloned, and sequenced; Thirdly, cox1 fragments of D. caprae were aligned with those of other Demodex retrieved from GenBank; Finally, the intra- and inter-specific divergences were computed and the phylogenetic trees were reconstructed to analyze phylogenetic relationship in Demodex. Results obtained from seven 429-bp fragments of D. caprae showed that sequence identities were above 99.1% among three adults and four eggs. The intraspecific divergences in D. caprae, Demodex folliculorum, Demodex brevis, and Demodex canis were 0.0-0.9, 0.5-0.9, 0.0-0.2, and 0.0-0.5%, respectively, while the interspecific divergences between D. caprae and D. folliculorum, D. canis, and D. brevis were 20.3-20.9, 21.8-23.0, and 25.0-25.3, respectively. The interspecific divergences were 10 times higher than intraspecific ones, indicating considerable barcoding gap. Furthermore, the phylogenetic trees showed that four Demodex species gathered separately, representing independent species; and Demodex folliculorum gathered with canine Demodex, D. caprae, and D. brevis in sequence. In conclusion, the selected 429-bp mitochondrial cox1 gene is an appropriate DNA barcode for molecular classification, identification, and phylogenetic analysis of Demodex. D. caprae is an independent species and D. folliculorum is closer to D. canis than to D. caprae or D. brevis.

Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence.

PubMed

Lavinia, Pablo D; Núñez Bustos, Ezequiel O; Kopuchian, Cecilia; Lijtmaer, Darío A; García, Natalia C; Hebert, Paul D N; Tubaro, Pablo L

2017-01-01

Because the tropical regions of America harbor the highest concentration of butterfly species, its fauna has attracted considerable attention. Much less is known about the butterflies of southern South America, particularly Argentina, where over 1,200 species occur. To advance understanding of this fauna, we assembled a DNA barcode reference library for 417 butterfly species of Argentina, focusing on the Atlantic Forest, a biodiversity hotspot. We tested the efficacy of this library for specimen identification, used it to assess the frequency of cryptic species, and examined geographic patterns of genetic variation, making this study the first large-scale genetic assessment of the butterflies of southern South America. The average sequence divergence to the nearest neighbor (i.e. minimum interspecific distance) was 6.91%, ten times larger than the mean distance to the furthest conspecific (0.69%), with a clear barcode gap present in all but four of the species represented by two or more specimens. As a consequence, the DNA barcode library was extremely effective in the discrimination of these species, allowing a correct identification in more than 95% of the cases. Singletons (i.e. species represented by a single sequence) were also distinguishable in the gene trees since they all had unique DNA barcodes, divergent from those of the closest non-conspecific. The clustering algorithms implemented recognized from 416 to 444 barcode clusters, suggesting that the actual diversity of butterflies in Argentina is 3%-9% higher than currently recognized. Furthermore, our survey added three new records of butterflies for the country (Eurema agave, Mithras hannelore, Melanis hillapana). In summary, this study not only supported the utility of DNA barcoding for the identification of the butterfly species of Argentina, but also highlighted several cases of both deep intraspecific and shallow interspecific divergence that should be studied in more detail.

Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence

PubMed Central

Núñez Bustos, Ezequiel O.; Kopuchian, Cecilia; Lijtmaer, Darío A.; García, Natalia C.; Hebert, Paul D. N.; Tubaro, Pablo L.

2017-01-01

Because the tropical regions of America harbor the highest concentration of butterfly species, its fauna has attracted considerable attention. Much less is known about the butterflies of southern South America, particularly Argentina, where over 1,200 species occur. To advance understanding of this fauna, we assembled a DNA barcode reference library for 417 butterfly species of Argentina, focusing on the Atlantic Forest, a biodiversity hotspot. We tested the efficacy of this library for specimen identification, used it to assess the frequency of cryptic species, and examined geographic patterns of genetic variation, making this study the first large-scale genetic assessment of the butterflies of southern South America. The average sequence divergence to the nearest neighbor (i.e. minimum interspecific distance) was 6.91%, ten times larger than the mean distance to the furthest conspecific (0.69%), with a clear barcode gap present in all but four of the species represented by two or more specimens. As a consequence, the DNA barcode library was extremely effective in the discrimination of these species, allowing a correct identification in more than 95% of the cases. Singletons (i.e. species represented by a single sequence) were also distinguishable in the gene trees since they all had unique DNA barcodes, divergent from those of the closest non-conspecific. The clustering algorithms implemented recognized from 416 to 444 barcode clusters, suggesting that the actual diversity of butterflies in Argentina is 3%–9% higher than currently recognized. Furthermore, our survey added three new records of butterflies for the country (Eurema agave, Mithras hannelore, Melanis hillapana). In summary, this study not only supported the utility of DNA barcoding for the identification of the butterfly species of Argentina, but also highlighted several cases of both deep intraspecific and shallow interspecific divergence that should be studied in more detail. PMID:29049373

Identification and reassessment of the specific status of some tropical freshwater midges (Diptera: Chironomidae) using DNA barcode data.

PubMed

Pramual, Pairot; Simwisat, Kusumart; Martin, Jon

2016-01-28

Chironomidae are a highly diverse group of insects. Members of this family are often included in programs monitoring the health of freshwater ecosystems. However, a difficulty in morphological identification, particularly of larval stages is the major obstacle to this application. In this study, we tested the efficiency of mitochondrial cytochrome c oxidase I (COI) sequences as the DNA barcoding region for species identification of Chironomidae in Thailand. The results revealed 14 species with a high success rate (>90%) for the correct species identification, which suggests the potential usefulness of the technique. However, some morphological species possess high (>3%) intraspecific genetic divergence that suggests these species could be species complexes and need further morphological or cytological examination. Sequence-based species delimitation analyses indicated that most specimens identified as Chironomus kiiensis, Tokunaga 1936, in Japan are conspecific with C. striatipennis, Kieffer 1912, although a small number form a separate cluster. A review of the descriptions of Kiefferulus tainanus (Kieffer 1912) and its junior synonym, K. biroi (Kieffer 1918), following our results, suggests that this synonymy is probably not correct and that K. tainanus occurs in Japan, China and Singapore, while K. biroi occurs in India and Thailand. Our results therefore revealed the usefulness of DNA barcoding for correct species identification of Chironomidae, particularly the immature stages. In addition, DNA barcodes could also uncover hidden diversity that can guide further taxonomic study, and offer a more efficient way to identify species than morphological analysis where large numbers of specimens are involved, provided the identifications of DNA barcodes in the databases are correct. Our studies indicate that this is not the case, and we identify cases of misidentifications for C. flaviplumus, Tokunaga 1940 and K. tainanus.

Closely-related taxa influence woody species discrimination via DNA barcoding: evidence from global forest dynamics plots.

PubMed

Pei, Nancai; Erickson, David L; Chen, Bufeng; Ge, Xuejun; Mi, Xiangcheng; Swenson, Nathan G; Zhang, Jin-Long; Jones, Frank A; Huang, Chun-Lin; Ye, Wanhui; Hao, Zhanqing; Hsieh, Chang-Fu; Lum, Shawn; Bourg, Norman A; Parker, John D; Zimmerman, Jess K; McShea, William J; Lopez, Ida C; Sun, I-Fang; Davies, Stuart J; Ma, Keping; Kress, W John

2015-10-12

To determine how well DNA barcodes from the chloroplast region perform in forest dynamics plots (FDPs) from global CTFS-ForestGEO network, we analyzed DNA barcoding sequences of 1277 plant species from a wide phylogenetic range (3 FDPs in tropics, 5 in subtropics and 5 in temperate zone) and compared the rates of species discrimination (RSD). We quantified RSD by two DNA barcode combinations (rbcL + matK and rbcL + matK + trnH-psbA) using a monophyly-based method (GARLI). We defined two indexes of closely-related taxa (Gm/Gt and S/G ratios) and correlated these ratios with RSD. The combination of rbcL + matK averagely discriminated 88.65%, 83.84% and 72.51% at the local, regional and global scales, respectively. An additional locus trnH-psbA increased RSD by 2.87%, 1.49% and 3.58% correspondingly. RSD varied along a latitudinal gradient and were negatively correlated with ratios of closely-related taxa. Successes of species discrimination generally depend on scales in global FDPs. We suggested that the combination of rbcL + matK + trnH-psbA is currently applicable for DNA barcoding-based phylogenetic studies on forest communities.

DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar

PubMed Central

Smith, M. Alex; Fisher, Brian L; Hebert, Paul D.N

2005-01-01

The role of DNA barcoding as a tool to accelerate the inventory and analysis of diversity for hyperdiverse arthropods is tested using ants in Madagascar. We demonstrate how DNA barcoding helps address the failure of current inventory methods to rapidly respond to pressing biodiversity needs, specifically in the assessment of richness and turnover across landscapes with hyperdiverse taxa. In a comparison of inventories at four localities in northern Madagascar, patterns of richness were not significantly different when richness was determined using morphological taxonomy (morphospecies) or sequence divergence thresholds (Molecular Operational Taxonomic Unit(s); MOTU). However, sequence-based methods tended to yield greater richness and significantly lower indices of similarity than morphological taxonomy. MOTU determined using our molecular technique were a remarkably local phenomenon—indicative of highly restricted dispersal and/or long-term isolation. In cases where molecular and morphological methods differed in their assignment of individuals to categories, the morphological estimate was always more conservative than the molecular estimate. In those cases where morphospecies descriptions collapsed distinct molecular groups, sequence divergences of 16% (on average) were contained within the same morphospecies. Such high divergences highlight taxa for further detailed genetic, morphological, life history, and behavioral studies. PMID:16214741

Spiders (Araneae) of Churchill, Manitoba: DNA barcodes and morphology reveal high species diversity and new Canadian records.

PubMed

Blagoev, Gergin A; Nikolova, Nadya I; Sobel, Crystal N; Hebert, Paul D N; Adamowicz, Sarah J

2013-11-26

Arctic ecosystems, especially those near transition zones, are expected to be strongly impacted by climate change. Because it is positioned on the ecotone between tundra and boreal forest, the Churchill area is a strategic locality for the analysis of shifts in faunal composition. This fact has motivated the effort to develop a comprehensive biodiversity inventory for the Churchill region by coupling DNA barcoding with morphological studies. The present study represents one element of this effort; it focuses on analysis of the spider fauna at Churchill. 198 species were detected among 2704 spiders analyzed, tripling the count for the Churchill region. Estimates of overall diversity suggest that another 10-20 species await detection. Most species displayed little intraspecific sequence variation (maximum <1%) in the barcode region of the cytochrome c oxidase subunit I (COI) gene, but four species showed considerably higher values (maximum = 4.1-6.2%), suggesting cryptic species. All recognized species possessed a distinct haplotype array at COI with nearest-neighbour interspecific distances averaging 8.57%. Three species new to Canada were detected: Robertus lyrifer (Theridiidae), Baryphyma trifrons (Linyphiidae), and Satilatlas monticola (Linyphiidae). The first two species may represent human-mediated introductions linked to the port in Churchill, but the other species represents a range extension from the USA. The first description of the female of S. monticola was also presented. As well, one probable new species of Alopecosa (Lycosidae) was recognized. This study provides the first comprehensive DNA barcode reference library for the spider fauna of any region. Few cryptic species of spiders were detected, a result contrasting with the prevalence of undescribed species in several other terrestrial arthropod groups at Churchill. Because most (97.5%) sequence clusters at COI corresponded with a named taxon, DNA barcoding reliably identifies spiders in the Churchill fauna. The capacity of DNA barcoding to enable the identification of otherwise taxonomically ambiguous specimens (juveniles, females) also represents a major advance for future monitoring efforts on this group.

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions

PubMed Central

Raupach, Michael J.; Barco, Andrea; Steinke, Dirk; Beermann, Jan; Laakmann, Silke; Mohrbeck, Inga; Neumann, Hermann; Kihara, Terue C.; Pointner, Karin; Radulovici, Adriana; Segelken-Voigt, Alexandra; Wesse, Christina; Knebelsberger, Thomas

2015-01-01

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences. PMID:26417993

DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya-Hengduan Mountains.

PubMed

Yan, Li-Jun; Liu, Jie; Möller, Michael; Zhang, Lin; Zhang, Xue-Mei; Li, De-Zhu; Gao, Lian-Ming

2015-07-01

The Himalaya-Hengduan Mountains encompass two global biodiversity hotspots with high levels of biodiversity and endemism. This area is one of the diversification centres of the genus Rhododendron, which is recognized as one of the most taxonomically challenging plant taxa due to recent adaptive radiations and rampant hybridization. In this study, four DNA barcodes were evaluated on 531 samples representing 173 species of seven sections of four subgenera in Rhododendron, with a high sampling density from the Himalaya-Hengduan Mountains employing three analytical methods. The varied approaches (nj, pwg and blast) had different species identification powers with blast performing best. With the pwg analysis, the discrimination rates for single barcodes varied from 12.21% to 25.19% with ITS < rbcL < matK < psbA-trnH. Combinations of ITS + psbA-trnH + matK and the four barcodes showed the highest discrimination ability (both 41.98%) among all possible combinations. As a single barcode, psbA-trnH performed best with a relatively high performance (25.19%). Overall, the three-marker combination of ITS + psbA-trnH + matK was found to be the best DNA barcode for identifying Rhododendron species. The relatively low discriminative efficiency of DNA barcoding in this genus (~42%) may possibly be attributable to too low sequence divergences as a result of a long generation time of Rhododendron and complex speciation patterns involving recent radiations and hybridizations. Taking the morphology, distribution range and habitat of the species into account, DNA barcoding provided additional information for species identification and delivered a preliminary assessment of biodiversity for the large genus Rhododendron in the biodiversity hotspots of the Himalaya-Hengduan Mountains. © 2014 John Wiley & Sons Ltd.

The Trichoptera barcode initiative: a strategy for generating a species-level Tree of Life

PubMed Central

Frandsen, Paul B.; Holzenthal, Ralph W.; Beet, Clare R.; Bennett, Kristi R.; Blahnik, Roger J.; Bonada, Núria; Cartwright, David; Chuluunbat, Suvdtsetseg; Cocks, Graeme V.; Collins, Gemma E.; deWaard, Jeremy; Dean, John; Flint, Oliver S.; Hausmann, Axel; Hendrich, Lars; Hess, Monika; Hogg, Ian D.; Kondratieff, Boris C.; Malicky, Hans; Milton, Megan A.; Morinière, Jérôme; Morse, John C.; Mwangi, François Ngera; Pauls, Steffen U.; Gonzalez, María Razo; Rinne, Aki; Robinson, Jason L.; Salokannel, Juha; Shackleton, Michael; Smith, Brian; Stamatakis, Alexandros; StClair, Ros; Thomas, Jessica A.; Zamora-Muñoz, Carmen; Ziesmann, Tanja

2016-01-01

DNA barcoding was intended as a means to provide species-level identifications through associating DNA sequences from unknown specimens to those from curated reference specimens. Although barcodes were not designed for phylogenetics, they can be beneficial to the completion of the Tree of Life. The barcode database for Trichoptera is relatively comprehensive, with data from every family, approximately two-thirds of the genera, and one-third of the described species. Most Trichoptera, as with most of life's species, have never been subjected to any formal phylogenetic analysis. Here, we present a phylogeny with over 16 000 unique haplotypes as a working hypothesis that can be updated as our estimates improve. We suggest a strategy of implementing constrained tree searches, which allow larger datasets to dictate the backbone phylogeny, while the barcode data fill out the tips of the tree. We also discuss how this phylogeny could be used to focus taxonomic attention on ambiguous species boundaries and hidden biodiversity. We suggest that systematists continue to differentiate between ‘Barcode Index Numbers’ (BINs) and ‘species’ that have been formally described. Each has utility, but they are not synonyms. We highlight examples of integrative taxonomy, using both barcodes and morphology for species description. This article is part of the themed issue ‘From DNA barcodes to biomes’. PMID:27481793

A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

PubMed Central

Bruni, Ilaria; Scaccabarozzi, Daniela; Sandionigi, Anna; Barbuto, Michela; Casiraghi, Maurizio; Labra, Massimo

2014-01-01

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands. PMID:25296114

Fungal pathogen (mis-) identifications: a case study with DNA barcodes on Melampsora rusts of aspen and white poplar.

PubMed

Feau, Nicolas; Vialle, Agathe; Allaire, Mathieu; Tanguay, Philippe; Joly, David L; Frey, Pascal; Callan, Brenda E; Hamelin, Richard C

2009-01-01

Wide variation and overlap in morphological characters have led to confusion in species identification within the fungal rust genus Melampsora. The Melampsora species with uredinial-telial stages on white poplar and aspens are especially prone to misidentification. This group includes the Melampsora populnea species complex and the highly destructive pine twisting rust, Melampsora pinitorqua, which alternates between hosts in Populus section Populus and Pinus. Our objective was to compare morphologically based identification to genetic material extracted from Melampsora species pathogenic to aspen and white poplar. We compared morphometric traits and DNA barcodes obtained from internal transcribed spacer (ITS), large ribosomal RNA subunit (28S), and mitochondrial cytochrome oxidase 1 (CO1) sequences to delimit within this taxonomically difficult group. Eight different Melampsora species were initially defined based on host specificity and morphometric data. DNA barcodes were then overlaid on these initial species definitions. The DNA barcodes, specifically those defined on ITS and 28S sequences, provided a highly accurate means of identifying and resolving Melampsora taxa. We highlighted species misidentification in specimens from Canadian herbaria related to either Melampsora medusae f. sp. tremuloidae or Melampsora aecidioides. Finally, we evidenced that the north-American species found on Populus alba, M. aecidioides is closely related but distinct from the four species of the M. populnea complex (Melampsora larici-tremulae, Melampsora magnusiana, Melampsora pinitorqua, and Melampsora rostrupii) found in Eurasia.

Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

PubMed Central

Walch, Georg; Knapp, Maria; Rainer, Georg; Peintner, Ursula

2016-01-01

Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera Cladosporium, Geomyces, Fusarium, and Mortierella could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. Absidia, Mucor, Trichoderma, and Penicillium isolates had noticeably lower PCR success. PMID:29376929

Implications of Hybridization, NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

PubMed Central

Ermakov, Oleg A.; Simonov, Evgeniy; Surin, Vadim L.; Titov, Sergey V.; Brandler, Oleg V.; Ivanova, Natalia V.; Borisenko, Alex V.

2015-01-01

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’. PMID:25617768

Cytochrome c oxidase subunit I barcoding of the green bee-eater (Merops orientalis).

PubMed

Arif, I A; Khan, H A; Shobrak, M; Williams, J

2011-10-21

DNA barcoding using mitochondrial cytochrome c oxidase subunit I (COI) is regarded as a standard method for species identification. Recent reports have also shown extended applications of COI gene analysis in phylogeny and molecular diversity studies. The bee-eaters are a group of near passerine birds in the family Meropidae. There are 26 species worldwide; five of them are found in Saudi Arabia. Until now, GenBank included a COI barcode for only one species of bee-eater, the European bee-eater (Merops apiaster). We sequenced the 694-bp segment of the COI gene of the green bee-eater M. orientalis and compared the sequences with those of M. apiaster. Pairwise sequence comparison showed 66 variable sites across all the eight sequences from both species, with an interspecific genetic distance of 0.0362. Two and one within-species variable sites were found, with genetic distances of 0.0005 and 0.0003 for M. apiaster and M. orientalis, respectively. This is the first study reporting barcodes for M. orientalis.

Building-up of a DNA barcode library for true bugs (insecta: hemiptera: heteroptera) of Germany reveals taxonomic uncertainties and surprises.

PubMed

Raupach, Michael J; Hendrich, Lars; Küchler, Stefan M; Deister, Fabian; Morinière, Jérome; Gossner, Martin M

2014-01-01

During the last few years, DNA barcoding has become an efficient method for the identification of species. In the case of insects, most published DNA barcoding studies focus on species of the Ephemeroptera, Trichoptera, Hymenoptera and especially Lepidoptera. In this study we test the efficiency of DNA barcoding for true bugs (Hemiptera: Heteroptera), an ecological and economical highly important as well as morphologically diverse insect taxon. As part of our study we analyzed DNA barcodes for 1742 specimens of 457 species, comprising 39 families of the Heteroptera. We found low nucleotide distances with a minimum pairwise K2P distance <2.2% within 21 species pairs (39 species). For ten of these species pairs (18 species), minimum pairwise distances were zero. In contrast to this, deep intraspecific sequence divergences with maximum pairwise distances >2.2% were detected for 16 traditionally recognized and valid species. With a successful identification rate of 91.5% (418 species) our study emphasizes the use of DNA barcodes for the identification of true bugs and represents an important step in building-up a comprehensive barcode library for true bugs in Germany and Central Europe as well. Our study also highlights the urgent necessity of taxonomic revisions for various taxa of the Heteroptera, with a special focus on various species of the Miridae. In this context we found evidence for on-going hybridization events within various taxonomically challenging genera (e.g. Nabis Latreille, 1802 (Nabidae), Lygus Hahn, 1833 (Miridae), Phytocoris Fallén, 1814 (Miridae)) as well as the putative existence of cryptic species (e.g. Aneurus avenius (Duffour, 1833) (Aradidae) or Orius niger (Wolff, 1811) (Anthocoridae)).

Building-Up of a DNA Barcode Library for True Bugs (Insecta: Hemiptera: Heteroptera) of Germany Reveals Taxonomic Uncertainties and Surprises

PubMed Central

Raupach, Michael J.; Hendrich, Lars; Küchler, Stefan M.; Deister, Fabian; Morinière, Jérome; Gossner, Martin M.

2014-01-01

During the last few years, DNA barcoding has become an efficient method for the identification of species. In the case of insects, most published DNA barcoding studies focus on species of the Ephemeroptera, Trichoptera, Hymenoptera and especially Lepidoptera. In this study we test the efficiency of DNA barcoding for true bugs (Hemiptera: Heteroptera), an ecological and economical highly important as well as morphologically diverse insect taxon. As part of our study we analyzed DNA barcodes for 1742 specimens of 457 species, comprising 39 families of the Heteroptera. We found low nucleotide distances with a minimum pairwise K2P distance <2.2% within 21 species pairs (39 species). For ten of these species pairs (18 species), minimum pairwise distances were zero. In contrast to this, deep intraspecific sequence divergences with maximum pairwise distances >2.2% were detected for 16 traditionally recognized and valid species. With a successful identification rate of 91.5% (418 species) our study emphasizes the use of DNA barcodes for the identification of true bugs and represents an important step in building-up a comprehensive barcode library for true bugs in Germany and Central Europe as well. Our study also highlights the urgent necessity of taxonomic revisions for various taxa of the Heteroptera, with a special focus on various species of the Miridae. In this context we found evidence for on-going hybridization events within various taxonomically challenging genera (e.g. Nabis Latreille, 1802 (Nabidae), Lygus Hahn, 1833 (Miridae), Phytocoris Fallén, 1814 (Miridae)) as well as the putative existence of cryptic species (e.g. Aneurus avenius (Duffour, 1833) (Aradidae) or Orius niger (Wolff, 1811) (Anthocoridae)). PMID:25203616

Multilocus inference of species trees and DNA barcoding.

PubMed

Mallo, Diego; Posada, David

2016-09-05

The unprecedented amount of data resulting from next-generation sequencing has opened a new era in phylogenetic estimation. Although large datasets should, in theory, increase phylogenetic resolution, massive, multilocus datasets have uncovered a great deal of phylogenetic incongruence among different genomic regions, due both to stochastic error and to the action of different evolutionary process such as incomplete lineage sorting, gene duplication and loss and horizontal gene transfer. This incongruence violates one of the fundamental assumptions of the DNA barcoding approach, which assumes that gene history and species history are identical. In this review, we explain some of the most important challenges we will have to face to reconstruct the history of species, and the advantages and disadvantages of different strategies for the phylogenetic analysis of multilocus data. In particular, we describe the evolutionary events that can generate species tree-gene tree discordance, compare the most popular methods for species tree reconstruction, highlight the challenges we need to face when using them and discuss their potential utility in barcoding. Current barcoding methods sacrifice a great amount of statistical power by only considering one locus, and a transition to multilocus barcodes would not only improve current barcoding methods, but also facilitate an eventual transition to species-tree-based barcoding strategies, which could better accommodate scenarios where the barcode gap is too small or inexistent.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

DNA barcode assessment of Ceramiales (Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

NASA Astrophysics Data System (ADS)

Du, Guoying; Wu, Feifei; Guo, Hao; Xue, Hongfan; Mao, Yunxiang

2015-05-01

A total of 142 specimens of Ceramiales (Rhodophyta) were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species, 14 genera, and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers, namely, partial large subunit rRNA gene (LSU), universal plastid amplicon (UPA), and partial mitochondrial cytochrome c oxidase subunit I gene (COI). No intraspecific divergence was found in our collection for these markers, except for a 1-3 bp divergence in the COI of Ceramium kondoi, Symphyocladia latiuscula, and Neosiphonia japonica. Because short DNA markers were used, the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in GenBank or BOLD (Barcode of Life Data) Systems. Three specimens were presumed as Heterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification, which was the first record in the investigated area, implying that it might be a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers, Heterosiphonia japonica converged with Dasya spp. and was distant from the other Heterosiphonia spp., implying that H. japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species, but the COI had a higher ability to discriminate between species.

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes.

PubMed

Geiger, M F; Herder, F; Monaghan, M T; Almada, V; Barbieri, R; Bariche, M; Berrebi, P; Bohlen, J; Casal-Lopez, M; Delmastro, G B; Denys, G P J; Dettai, A; Doadrio, I; Kalogianni, E; Kärst, H; Kottelat, M; Kovačić, M; Laporte, M; Lorenzoni, M; Marčić, Z; Özuluğ, M; Perdices, A; Perea, S; Persat, H; Porcelotti, S; Puzzi, C; Robalo, J; Šanda, R; Schneider, M; Šlechtová, V; Stoumboudi, M; Walter, S; Freyhof, J

2014-11-01

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule-coalescent (GMYC) model-based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA-barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large-scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities. © 2014 John Wiley & Sons Ltd.

Identification of a Herbal Powder by Deoxyribonucleic Acid Barcoding and Structural Analyses.

PubMed

Sheth, Bhavisha P; Thaker, Vrinda S

2015-10-01

Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. To identify a herbal powder obtained from a herbalist in the local vicinity of Rajkot, Gujarat, using deoxyribonucleic acid (DNA) barcoding and molecular tools. The DNA was extracted from a herbal powder and selected Cassia species, followed by the polymerase chain reaction (PCR) and sequencing of the rbcL barcode locus. Thereafter the sequences were subjected to National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis, followed by the protein three-dimension structure determination of the rbcL protein from the herbal powder and Cassia species namely Cassia fistula, Cassia tora and Cassia javanica (sequences obtained in the present study), Cassia Roxburghii, and Cassia abbreviata (sequences retrieved from Genbank). Further, the multiple and pairwise structural alignment were carried out in order to identify the herbal powder. The nucleotide sequences obtained from the selected species of Cassia were submitted to Genbank (Accession No. JX141397, JX141405, JX141420). The NCBI BLAST analysis of the rbcL protein from the herbal powder showed an equal sequence similarity (with reference to different parameters like E value, maximum identity, total score, query coverage) to C. javanica and C. roxburghii. In order to solve the ambiguities of the BLAST result, a protein structural approach was implemented. The protein homology models obtained in the present study were submitted to the protein model database (PM0079748-PM0079753). The pairwise structural alignment of the herbal powder (as template) and C. javanica and C. roxburghii (as targets individually) revealed a close similarity of the herbal powder with C. javanica. A strategy as used here, incorporating the integrated use of DNA barcoding and protein structural analyses could be adopted, as a novel rapid and economic procedure, especially in cases when protein coding loci are considered. Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. A herbal powder was obtained from a herbalist in the local vicinity of Rajkot, Gujarat. An integrated approach using DNA barcoding and structural analyses was carried out to identify the herbal powder. The herbal powder was identified as Cassia javanica L.

Mansonella ozzardi mitogenome and pseudogene characterisation provides new perspectives on filarial parasite systematics and CO-1 barcoding.

PubMed

Crainey, James Lee; Marín, Michel Abanto; Silva, Túllio Romão Ribeiro da; de Medeiros, Jansen Fernandes; Pessoa, Felipe Arley Costa; Santos, Yago Vinícius; Vicente, Ana Carolina Paulo; Luz, Sérgio Luiz Bessa

2018-04-18

Despite the broad distribution of M. ozzardi in Latin America and the Caribbean, there is still very little DNA sequence data available to study this neglected parasite's epidemiology. Mitochondrial DNA (mtDNA) sequences, especially the cytochrome oxidase (CO1) gene's barcoding region, have been targeted successfully for filarial diagnostics and for epidemiological, ecological and evolutionary studies. MtDNA-based studies can, however, be compromised by unrecognised mitochondrial pseudogenes, such as Numts. Here, we have used shot-gun Illumina-HiSeq sequencing to recover the first complete Mansonella genus mitogenome and to identify several mitochondrial-origin pseudogenes. Mitogenome phylogenetic analysis placed M. ozzardi in the Onchocercidae "ONC5" clade and suggested that Mansonella parasites are more closely related to Wuchereria and Brugia genera parasites than they are to Loa genus parasites. DNA sequence alignments, BLAST searches and conceptual translations have been used to compliment phylogenetic analysis showing that M. ozzardi from the Amazon and Caribbean regions are near-identical and that previously reported Peruvian M. ozzardi CO1 reference sequences are probably of pseudogene origin. In addition to adding a much-needed resource to the Mansonella genus's molecular tool-kit and providing evidence that some M. ozzardi CO1 sequence deposits are pseudogenes, our results suggest that all Neotropical M. ozzardi parasites are closely related.

DNA barcodes reveal microevolutionary signals in fire response trait in two legume genera

PubMed Central

Bello, Abubakar; Daru, Barnabas H.; Stirton, Charles H.; Chimphango, Samson B. M.; van der Bank, Michelle; Maurin, Olivier; Muasya, A. Muthama

2015-01-01

Large-scale DNA barcoding provides a new technique for species identification and evaluation of relationships across various levels (populations and species) and may reveal fundamental processes in recently diverged species. Here, we analysed DNA sequence variation in the recently diverged legumes from the Psoraleeae (Fabaceae) occurring in the Cape Floristic Region (CFR) of southern Africa to test the utility of DNA barcodes in species identification and discrimination. We further explored the phylogenetic signal on fire response trait (reseeding and resprouting) at species and generic levels. We showed that Psoraleoid legumes of the CFR exhibit a barcoding gap yielding the combination of matK and rbcLa (matK + rbcLa) data set as a better barcode than single regions. We found a high score (100 %) of correct identification of individuals to their respective genera but a very low score (<50 %) in identifying them to species. We found a considerable match (54 %) between genetic species and morphologically delimited species. We also found that different lineages showed a weak but significant phylogenetic conservatism in their response to fire as reseeders or resprouters, with more clustering of resprouters than would be expected by chance. These novel microevolutionary patterns might be acting continuously over time to produce multi-scale regularities of biodiversity. This study provides the first insight into the DNA barcoding campaign of land plants in species identification and detection of the phylogenetic signal in recently diverged lineages of the CFR. PMID:26507570

Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae).

PubMed

Ashfaq, Muhammad; Asif, Muhammad; Anjum, Zahid Iqbal; Zafar, Yusuf

2013-07-01

Although two plastid regions have been adopted as the standard markers for plant DNA barcoding, their limited resolution has provoked the consideration of other gene regions, especially in taxonomically diverse genera. The genus Gossypium (cotton) includes eight diploid genome groups (A-G, and K) and five allotetraploid species which are difficult to discriminate morphologically. In this study, we tested the effectiveness of three widely used markers (matK, rbcL, and ITS2) in the discrimination of 20 diploid and five tetraploid species of cotton. Sequences were analysed locus-wise and in combinations to determine the most effective strategy for species identification. Sequence recovery was high, ranging from 92% to 100% with mean pairwise interspecific distance highest for ITS2 (3.68%) and lowest for rbcL (0.43%). At a 0.5% threshold, the combination of matK+ITS2 produced the greatest number of species clusters. Based on 'best match' analysis, the combination of matK+ITS2 was best, while based on 'all species barcodes' analysis, ITS2 gave the highest percentage of correct species identifications (98.93%). The combination of sequences for all three markers produced the best resolved tree. The disparity index test based on matK+rbcL+ITS2 was significant (P < 0.05) for a higher number of species pairs than the individual gene sequences. Although all three barcodes separated the species with respect to their genome type, no single combination of barcodes could differentiate all the Gossypium species, and tetraploid species were particularly difficult. © 2013 John Wiley & Sons Ltd.

Who's there? - First morphological and DNA barcoding catalogue of the shallow Hawai'ian sponge fauna.

PubMed

Núñez Pons, Laura; Calcinai, Barbara; Gates, Ruth D

2017-01-01

The sponge fauna has been largely overlooked in the Archipelago of Hawai'i, notwithstanding the paramount role of this taxon in marine ecosystems. The lack of knowledge about Porifera populations inhabiting the Hawai'ian reefs limits the development of ecological studies aimed at understanding the functioning of these marine systems. Consequently, this project addresses this gap by describing the most representative sponge species in the shallow waters of the enigmatic bay of Kane'ohe Bay, in O'ahu Island. A total of 30 species (28 demosponges and two calcareous sponges) living associated to the reef structures are here reported. Six of these species are new records to the Hawai'ian Porifera catalogue and are suspected to be recent introductions to these islands. Morphological descriptions of the voucher specimens are provided, along with sequencing data of two partitions involving the mitochondrial cytochrome oxidase subunit 1 (COI) marker and a fragment covering partial (18S and 28S) and full (ITS-1, 5.8S and ITS-2) nuclear ribosomal genes. Species delimitations based on genetic distances were calculated to valitate how taxonomic assignments from DNA barcoding aligned with morphological identifications. Of the 60 sequences submitted to GenBank ~88% are the first sequencing records for the corresponding species and genetic marker. This work compiles the first catalogue combining morphological characters with DNA barcoding of Hawai'ian sponges, and contributes to the repository of public databases through the Sponge Barcoding Project initiative.

Who’s there? – First morphological and DNA barcoding catalogue of the shallow Hawai’ian sponge fauna

PubMed Central

Gates, Ruth D.

2017-01-01

The sponge fauna has been largely overlooked in the Archipelago of Hawai’i, notwithstanding the paramount role of this taxon in marine ecosystems. The lack of knowledge about Porifera populations inhabiting the Hawai’ian reefs limits the development of ecological studies aimed at understanding the functioning of these marine systems. Consequently, this project addresses this gap by describing the most representative sponge species in the shallow waters of the enigmatic bay of Kane’ohe Bay, in O’ahu Island. A total of 30 species (28 demosponges and two calcareous sponges) living associated to the reef structures are here reported. Six of these species are new records to the Hawai’ian Porifera catalogue and are suspected to be recent introductions to these islands. Morphological descriptions of the voucher specimens are provided, along with sequencing data of two partitions involving the mitochondrial cytochrome oxidase subunit 1 (COI) marker and a fragment covering partial (18S and 28S) and full (ITS-1, 5.8S and ITS-2) nuclear ribosomal genes. Species delimitations based on genetic distances were calculated to valitate how taxonomic assignments from DNA barcoding aligned with morphological identifications. Of the 60 sequences submitted to GenBank ~88% are the first sequencing records for the corresponding species and genetic marker. This work compiles the first catalogue combining morphological characters with DNA barcoding of Hawai’ian sponges, and contributes to the repository of public databases through the Sponge Barcoding Project initiative. PMID:29267311

Phylogenetic Reconstruction and DNA Barcoding for Closely Related Pine Moth Species (Dendrolimus) in China with Multiple Gene Markers

PubMed Central

Dai, Qing-Yan; Gao, Qiang; Wu, Chun-Sheng; Chesters, Douglas; Zhu, Chao-Dong; Zhang, Ai-Bing

2012-01-01

Unlike distinct species, closely related species offer a great challenge for phylogeny reconstruction and species identification with DNA barcoding due to their often overlapping genetic variation. We tested a sibling species group of pine moth pests in China with a standard cytochrome c oxidase subunit I (COI) gene and two alternative internal transcribed spacer (ITS) genes (ITS1 and ITS2). Five different phylogenetic/DNA barcoding analysis methods (Maximum likelihood (ML)/Neighbor-joining (NJ), “best close match” (BCM), Minimum distance (MD), and BP-based method (BP)), representing commonly used methodology (tree-based and non-tree based) in the field, were applied to both single-gene and multiple-gene analyses. Our results demonstrated clear reciprocal species monophyly for three relatively distant related species, Dendrolimus superans, D. houi, D. kikuchii, as recovered by both single and multiple genes while the phylogenetic relationship of three closely related species, D. punctatus, D. tabulaeformis, D. spectabilis, could not be resolved with the traditional tree-building methods. Additionally, we find the standard COI barcode outperforms two nuclear ITS genes, whatever the methods used. On average, the COI barcode achieved a success rate of 94.10–97.40%, while ITS1 and ITS2 obtained a success rate of 64.70–81.60%, indicating ITS genes are less suitable for species identification in this case. We propose the use of an overall success rate of species identification that takes both sequencing success and assignation success into account, since species identification success rates with multiple-gene barcoding system were generally overestimated, especially by tree-based methods, where only successfully sequenced DNA sequences were used to construct a phylogenetic tree. Non-tree based methods, such as MD, BCM, and BP approaches, presented advantages over tree-based methods by reporting the overall success rates with statistical significance. In addition, our results indicate that the most closely related species D. punctatus, D. tabulaeformis, and D. spectabilis, may be still in the process of incomplete lineage sorting, with occasional hybridizations occurring among them. PMID:22509245

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

PubMed

Chen, Juan; Zhao, Jietang; Erickson, David L; Xia, Nianhe; Kress, W John

2015-03-01

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. © 2014 John Wiley & Sons Ltd.

Barcode ITS2: a useful tool for identifying Trachelospermum jasminoides and a good monitor for medicine market.

PubMed

Yu, Ning; Wei, Yu-Long; Zhang, Xin; Zhu, Ning; Wang, Yan-Li; Zhu, Yue; Zhang, Hai-Ping; Li, Fen-Mei; Yang, Lan; Sun, Jia-Qi; Sun, Ai-Dong

2017-07-11

Trachelospermum jasminoides is commonly used in traditional Chinese medicine. However, the use of the plant's local alternatives is frequent, causing potential clinical problems. The T. jasminoides sold in the medicine market is commonly dried and sliced, making traditional identification methods difficult. In this study, the ITS2 region was evaluated on 127 sequences representing T. jasminoides and its local alternatives according to PCR and sequencing rates, intra- and inter-specific divergences, secondary structure, and discrimination capacity. Results indicated the 100% success rates of PCR and sequencing and the obvious presence of a barcoding gap. Results of BLAST 1, nearest distance and neighbor-joining tree methods showed that barcode ITS2 could successfully identify all the texted samples. The secondary structures of the ITS2 region provided another dimensionality for species identification. Two-dimensional images were obtained for better and easier identification. Previous studies on DNA barcoding concentrated more on the same family, genus, or species. However, an ideal barcode should be variable enough to identify closely related species. Meanwhile, the barcodes should also be conservative in identifying distantly related species. This study highlights the application of barcode ITS2 in solving practical problems in the distantly related local alternatives of medical plants.

DNA barcode variability and host plant usage of fruit flies (Diptera: Tephritidae) in Thailand.

PubMed

Kunprom, Chonticha; Pramual, Pairot

2016-10-01

The objectives of this study were to examine the genetic variation in fruit flies (Diptera: Tephritidae) in Thailand and to test the efficiency of the mitochondrial cytochrome c oxidase subunit I (COI) barcoding region for species-level identification. Twelve fruit fly species were collected from 24 host plant species of 13 families. The number of host plant species for each fruit fly species ranged between 1 and 11, with Bactrocera correcta found in the most diverse host plants. A total of 123 COI sequences were obtained from these fruit fly species. Sequences from the NCBI database were also included, for a total of 17 species analyzed. DNA barcoding identification analysis based on the best close match method revealed a good performance, with 94.4% of specimens correctly identified. However, many specimens (3.6%) had ambiguous identification, mostly due to intra- and interspecific overlap between members of the B. dorsalis complex. A phylogenetic tree based on the mitochondrial barcode sequences indicated that all species, except for the members of the B. dorsalis complex, were monophyletic with strong support. Our work supports recent calls for synonymization of these species. Divergent lineages were observed within B. correcta and B. tuberculata, and this suggested that these species need further taxonomic reexamination.

Barcoding and Border Biosecurity: Identifying Cyprinid Fishes in the Aquarium Trade

PubMed Central

Collins, Rupert A.; Armstrong, Karen F.; Meier, Rudolf; Yi, Youguang; Brown, Samuel D. J.; Cruickshank, Robert H.; Keeling, Suzanne; Johnston, Colin

2012-01-01

Background Poorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species. Methodology/Principal Findings We sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90–99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii. Conclusions/Significance We demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade. PMID:22276096

The Species Dilemma of Northeast Indian Mahseer (Actinopterygii: Cyprinidae): DNA Barcoding in Clarifying the Riddle

PubMed Central

Laskar, Boni A.; Bhattacharjee, Maloyjo J.; Dhar, Bishal; Mahadani, Pradosh; Kundu, Shantanu; Ghosh, Sankar K.

2013-01-01

Background The taxonomic validity of Northeast Indian endemic Mahseer species, Tor progeneius and Neolissochilus hexastichus, has been argued repeatedly. This is mainly due to disagreements in recognizing the species based on morphological characters. Consequently, both the species have been concealed for many decades. DNA barcoding has become a promising and an independent technique for accurate species level identification. Therefore, utilization of such technique in association with the traditional morphotaxonomic description can resolve the species dilemma of this important group of sport fishes. Methodology/Principal Findings Altogether, 28 mahseer specimens including paratypes were studied from different locations in Northeast India, and 24 morphometric characters were measured invariably. The Principal Component Analysis with morphometric data revealed five distinct groups of sample that were taxonomically categorized into 4 species, viz., Tor putitora, T. progeneius, Neolissochilus hexagonolepis and N. hexastichus. Analysis with a dataset of 76 DNA barcode sequences of different mahseer species exhibited that the queries of T. putitora and N. hexagonolepis clustered cohesively with the respective conspecific database sequences maintaining 0.8% maximum K2P divergence. The closest congeneric divergence was 3 times higher than the mean conspecific divergence and was considered as barcode gap. The maximum divergence among the samples of T. progeneius and T. putitora was 0.8% that was much below the barcode gap, indicating them being synonymous. The query sequences of N. hexastichus invariably formed a discrete and a congeneric clade with the database sequences and maintained the interspecific divergence that supported its distinct species status. Notably, N. hexastichus was encountered in a single site and seemed to be under threat. Conclusion This study substantiated the identification of N. hexastichus to be a true species, and tentatively regarded T. progeneius to be a synonym of T. putitora. It would guide the conservationists to initiate priority conservation of N. hexastichus and T. putitora. PMID:23341979

Sliding Window Analyses for Optimal Selection of Mini-Barcodes, and Application to 454-Pyrosequencing for Specimen Identification from Degraded DNA

PubMed Central

Boyer, Stephane; Brown, Samuel D. J.; Collins, Rupert A.; Cruickshank, Robert H.; Lefort, Marie-Caroline; Malumbres-Olarte, Jagoba; Wratten, Stephen D.

2012-01-01

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential ‘mini-barcodes’ for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation. PMID:22666489

Potential of DNA sequences to identify zoanthids (Cnidaria: Zoantharia).

PubMed

Sinniger, Frederic; Reimer, James D; Pawlowski, Jan

2008-12-01

The order Zoantharia is known for its chaotic taxonomy and difficult morphological identification. One method that potentially could help for examining such troublesome taxa is DNA barcoding, which identifies species using standard molecular markers. The mitochondrial cytochrome oxidase subunit I (COI) has been utilized to great success in groups such as birds and insects; however, its applicability in many other groups is controversial. Recently, some studies have suggested that barcoding is not applicable to anthozoans. Here, we examine the use of COI and mitochondrial 16S ribosomal DNA for zoanthid identification. Despite the absence of a clear barcoding gap, our results show that for most of 54 zoanthid samples, both markers could separate samples to the species, or species group, level, particularly when easily accessible ecological or distributional data were included. Additionally, we have used the short V5 region of mt 16S rDNA to identify eight old (13 to 50 years old) museum samples. We discuss advantages and disadvantages of COI and mt 16S rDNA as barcodes for Zoantharia, and recommend that either one or both of these markers be considered for zoanthid identification in the future.

Indigenous species barcode database improves the identification of zooplankton

PubMed Central

Yang, Jianghua; Zhang, Wanwan; Sun, Jingying; Xie, Yuwei; Zhang, Yimin; Burton, G. Allen; Yu, Hongxia

2017-01-01

Incompleteness and inaccuracy of DNA barcode databases is considered an important hindrance to the use of metabarcoding in biodiversity analysis of zooplankton at the species-level. Species barcoding by Sanger sequencing is inefficient for organisms with small body sizes, such as zooplankton. Here mitochondrial cytochrome c oxidase I (COI) fragment barcodes from 910 freshwater zooplankton specimens (87 morphospecies) were recovered by a high-throughput sequencing platform, Ion Torrent PGM. Intraspecific divergence of most zooplanktons was < 5%, except Branchionus leydign (Rotifer, 14.3%), Trichocerca elongate (Rotifer, 11.5%), Lecane bulla (Rotifer, 15.9%), Synchaeta oblonga (Rotifer, 5.95%) and Schmackeria forbesi (Copepod, 6.5%). Metabarcoding data of 28 environmental samples from Lake Tai were annotated by both an indigenous database and NCBI Genbank database. The indigenous database improved the taxonomic assignment of metabarcoding of zooplankton. Most zooplankton (81%) with barcode sequences in the indigenous database were identified by metabarcoding monitoring. Furthermore, the frequency and distribution of zooplankton were also consistent between metabarcoding and morphology identification. Overall, the indigenous database improved the taxonomic assignment of zooplankton. PMID:28977035

DNA barcoding and molecular systematics of the benthic and demersal organisms of the CEAMARC survey

NASA Astrophysics Data System (ADS)

Dettai, Agnes; Adamowizc, Sarah J.; Allcock, Louise; Arango, Claudia P.; Barnes, David K. A.; Barratt, Iain; Chenuil, Anne; Couloux, Arnaud; Cruaud, Corinne; David, Bruno; Denis, Françoise; Denys, Gael; Díaz, Angie; Eléaume, Marc; Féral, Jean-Pierre; Froger, Aurélie; Gallut, Cyril; Grant, Rachel; Griffiths, Huw J.; Held, Christoph; Hemery, Lenaïg G.; Hosie, Graham; Kuklinski, Piotr; Lecointre, Guillaume; Linse, Katrin; Lozouet, Pierre; Mah, Christopher; Monniot, Françoise; Norman, Mark D.; O'Hara, Timothy; Ozouf-Costaz, Catherine; Piedallu, Claire; Pierrat, Benjamin; Poulin, Elie; Puillandre, Nicolas; Riddle, Martin; Samadi, Sarah; Saucède, Thomas; Schubart, Christoph; Smith, Peter J.; Stevens, Darren W.; Steinke, Dirk; Strugnell, Jan M.; Tarnowska, K.; Wadley, Victoria; Ameziane, Nadia

2011-08-01

The Dumont d’Urville Sea (East Antarctic region) has been less investigated for DNA barcoding and molecular taxonomy than other parts of the Southern Ocean, such as the Ross Sea and the Antarctic Peninsula. The Collaborative East Antarctic MARine Census (CEAMARC) took place in this area during the austral summer of 2007-2008. The Australian vessel RSV Aurora Australis collected very diverse samples of demersal and benthic organisms. The specimens were sorted centrally, and then distributed to taxonomic experts for molecular and morphological taxonomy and identification, especially barcoding. The COI sequences generated from CEAMARC material provide a sizeable proportion of the Census of Antarctic Marine Life barcodes although the studies are still ongoing, and represent the only source of sequences for a number of species. Barcoding appears to be a valuable method for identification within most groups, despite low divergences and haplotype sharing in a few species, and it is also useful as a preliminary taxonomic exploration method. Several new species are being described. CEAMARC samples have already provided new material for phylogeographic and phylogenetic studies in cephalopods, pycnogonids, teleost fish, crinoids and sea urchins, helping these studies to provide a better insight in the patterns of evolution in the Southern Ocean.

mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences.

PubMed

Links, Matthew G; Chaban, Bonnie; Hemmingsen, Sean M; Muirhead, Kevin; Hill, Janet E

2013-08-15

Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database. Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure. mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.

Improved protocols to accelerate the assembly of DNA barcode reference libraries for freshwater zooplankton.

PubMed

Elías-Gutiérrez, Manuel; Valdez-Moreno, Martha; Topan, Janet; Young, Monica R; Cohuo-Colli, José Angel

2018-03-01

Currently, freshwater zooplankton sampling and identification methodologies have remained virtually unchanged since they were first established in the beginning of the XX century. One major contributing factor to this slow progress is the limited success of modern genetic methodologies, such as DNA barcoding, in several of the main groups. This study demonstrates improved protocols which enable the rapid assessment of most animal taxa inhabiting any freshwater system by combining the use of light traps, careful fixation at low temperatures using ethanol, and zooplankton-specific primers. We DNA-barcoded 2,136 specimens from a diverse array of taxonomic assemblages (rotifers, mollusks, mites, crustaceans, insects, and fishes) from several Canadian and Mexican lakes with an average sequence success rate of 85.3%. In total, 325 Barcode Index Numbers (BINs) were detected with only three BINs (two cladocerans and one copepod) shared between Canada and Mexico, suggesting a much narrower distribution range of freshwater zooplankton than previously thought. This study is the first to broadly explore the metazoan biodiversity of freshwater systems with DNA barcodes to construct a reference library that represents the first step for future programs which aim to monitor ecosystem health, track invasive species, or improve knowledge of the ecology and distribution of freshwater zooplankton.

[DNA barcoding and its utility in commonly-used medicinal snakes].

PubMed

Huang, Yong; Zhang, Yue-yun; Zhao, Cheng-jian; Xu, Yong-li; Gu, Ying-le; Huang, Wen-qi; Lin, Kui; Li, Li

2015-03-01

Identification accuracy of traditional Chinese medicine is crucial for the traditional Chinese medicine research, production and application. DNA barcoding based on the mitochondrial gene coding for cytochrome c oxidase subunit I (COI), are more and more used for identification of traditional Chinese medicine. Using universal barcoding primers to sequence, we discussed the feasibility of DNA barcoding method for identification commonly-used medicinal snakes (a total of 109 samples belonging to 19 species 15 genera 6 families). The phylogenetic trees using Neighbor-joining were constructed. The results indicated that the mean content of G + C(46.5%) was lower than that of A + T (53.5%). As calculated by Kimera-2-parameter model, the mean intraspecies genetic distance of Trimeresurus albolabris, Ptyas dhumnades and Lycodon rufozonatus was greater than 2%. Further phylogenetic relationship results suggested that identification of one sample of T. albolabris was erroneous. The identification of some samples of P. dhumnades was also not correct, namely originally P. korros was identified as P. dhumnades. Factors influence on intraspecific genetic distance difference of L. rufozonatus need to be studied further. Therefore, DNA barcoding for identification of medicinal snakes is feasible, and greatly complements the morphological classification method. It is necessary to further study in identification of traditional Chinese medicine.

DNA barcoding as a tool for coral reef conservation

NASA Astrophysics Data System (ADS)

Neigel, J.; Domingo, A.; Stake, J.

2007-09-01

DNA Barcoding (DBC) is a method for taxonomic identification of animals that is based entirely on the 5' portion of the mitochondrial gene, cytochrome oxidase subunit I ( COI-5). It can be especially useful for identification of larval forms or incomplete specimens lacking diagnostic morphological characters. DBC can also facilitate the discovery of species and in defining “molecular taxonomic units” in problematic groups. However, DBC is not a panacea for coral reef taxonomy. In two of the most ecologically important groups on coral reefs, the Anthozoa and Porifera, COI-5 sequences have diverged too little to be diagnostic for all species. Other problems for DBC include paraphyly in mitochondrial gene trees and lack of differentiation between hybrids and their maternal ancestors. DBC also depends on the availability of databases of COI-5 sequences, which are still in early stages of development. A global effort to barcode all fish species has demonstrated the importance of large-scale coordination and is yielding promising results. Whether or not COI-5 by itself is sufficient for species assignments has become a contentious question; it is generally advantageous to use sequences from multiple loci.

Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast.

PubMed

Lobo, Jorge; Teixeira, Marcos A L; Borges, Luisa M S; Ferreira, Maria S G; Hollatz, Claudia; Gomes, Pedro T; Sousa, Ronaldo; Ravara, Ascensão; Costa, Maria H; Costa, Filipe O

2016-01-01

Annelid polychaetes have been seldom the focus of dedicated DNA barcoding studies, despite their ecological relevance and often dominance, particularly in soft-bottom estuarine and coastal marine ecosystems. Here, we report the first assessment of the performance of DNA barcodes in the discrimination of shallow water polychaete species from the southern European Atlantic coast, focusing on specimens collected in estuaries and coastal ecosystems of Portugal. We analysed cytochrome oxidase I DNA barcodes (COI-5P) from 164 specimens, which were assigned to 51 morphospecies. To our data set from Portugal, we added available published sequences selected from the same species, genus or family, to inspect for taxonomic congruence among studies and collection location. The final data set comprised 290 specimens and 79 morphospecies, which generated 99 Barcode Index Numbers (BINs) within Barcode of Life Data Systems (BOLD). Among these, 22 BINs were singletons, 47 other BINs were concordant, confirming the initial identification based on morphological characters, and 30 were discordant, most of which consisted on multiple BINs found for the same morphospecies. Some of the most prominent cases in the latter category include Hediste diversicolor (O.F. Müller, 1776) (7), Eulalia viridis (Linnaeus, 1767) (2) and Owenia fusiformis (delle Chiaje, 1844) (5), all of them reported from Portugal and frequently used in ecological studies as environmental quality indicators. Our results for these species showed discordance between molecular lineages and morphospecies, or added additional relatively divergent lineages. The potential inaccuracies in environmental assessments, where underpinning polychaete species diversity is poorly resolved or clarified, demand additional and extensive investigation of the DNA barcode diversity in this group, in parallel with alpha taxonomy efforts. © 2015 John Wiley & Sons Ltd.

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes

PubMed Central

YUAN, QING-JUN; ZHANG, BIN; JIANG, DAN; ZHANG, WEN-JING; LIN, TSAI-YUN; WANG, NIAN-HE; CHIOU, SHU-JIAU; HUANG, LU-QI

2015-01-01

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia-recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH-psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH-psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes. PMID:24961287

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes.

PubMed

Yuan, Qing-Jun; Zhang, Bin; Jiang, Dan; Zhang, Wen-Jing; Lin, Tsai-Yun; Wang, Nian-He; Chiou, Shu-Jiau; Huang, Lu-Qi

2015-03-01

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia-recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH-psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH-psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes. © 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe.

PubMed

Chen, Bo-Ruei; Hale, Devin C; Ciolek, Peter J; Runge, Kurt W

2012-05-03

Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Advances in the use of DNA barcodes to build a community phylogeny for tropical trees in a Puerto Rican forest dynamics plot.

PubMed

Kress, W John; Erickson, David L; Swenson, Nathan G; Thompson, Jill; Uriarte, Maria; Zimmerman, Jess K

2010-11-09

Species number, functional traits, and phylogenetic history all contribute to characterizing the biological diversity in plant communities. The phylogenetic component of diversity has been particularly difficult to quantify in species-rich tropical tree assemblages. The compilation of previously published (and often incomplete) data on evolutionary relationships of species into a composite phylogeny of the taxa in a forest, through such programs as Phylomatic, has proven useful in building community phylogenies although often of limited resolution. Recently, DNA barcodes have been used to construct a robust community phylogeny for nearly 300 tree species in a forest dynamics plot in Panama using a supermatrix method. In that study sequence data from three barcode loci were used to generate a well-resolved species-level phylogeny. Here we expand upon this earlier investigation and present results on the use of a phylogenetic constraint tree to generate a community phylogeny for a diverse, tropical forest dynamics plot in Puerto Rico. This enhanced method of phylogenetic reconstruction insures the congruence of the barcode phylogeny with broadly accepted hypotheses on the phylogeny of flowering plants (i.e., APG III) regardless of the number and taxonomic breadth of the taxa sampled. We also compare maximum parsimony versus maximum likelihood estimates of community phylogenetic relationships as well as evaluate the effectiveness of one- versus two- versus three-gene barcodes in resolving community evolutionary history. As first demonstrated in the Panamanian forest dynamics plot, the results for the Puerto Rican plot illustrate that highly resolved phylogenies derived from DNA barcode sequence data combined with a constraint tree based on APG III are particularly useful in comparative analysis of phylogenetic diversity and will enhance research on the interface between community ecology and evolution.

DNA Barcoding Survey of Anurans across the Eastern Cordillera of Colombia and the Impact of the Andes on Cryptic Diversity

PubMed Central

Guarnizo, Carlos E.; Paz, Andrea; Muñoz-Ortiz, Astrid; Flechas, Sandra V.; Méndez-Narváez, Javier; Crawford, Andrew J.

2015-01-01

Colombia hosts the second highest amphibian species diversity on Earth, yet its fauna remains poorly studied, especially using molecular genetic techniques. We present the results of the first wide-scale DNA barcoding survey of anurans of Colombia, focusing on a transect across the Eastern Cordillera. We surveyed 10 sites between the Magdalena Valley to the west and the eastern foothills of the Eastern Cordillera, sequencing portions of the mitochondrial 16S ribosomal RNA and cytochrome oxidase subunit 1 (CO1) genes for 235 individuals from 52 nominal species. We applied two barcode algorithms, Automatic Barcode Gap Discovery and Refined Single Linkage Analysis, to estimate the number of clusters or “unconfirmed candidate species” supported by DNA barcode data. Our survey included ~7% of the anuran species known from Colombia. While barcoding algorithms differed slightly in the number of clusters identified, between three and ten nominal species may be obscuring candidate species (in some cases, more than one cryptic species per nominal species). Our data suggest that the high elevations of the Eastern Cordillera and the low elevations of the Chicamocha canyon acted as geographic barriers in at least seven nominal species, promoting strong genetic divergences between populations associated with the Eastern Cordillera. PMID:26000447

DNA barcodes identify Central Asian Colias butterflies (Lepidoptera, Pieridae).

PubMed

Laiho, Juha; Ståhls, Gunilla

2013-12-30

A majority of the known Colias species (Lepidoptera: Pieridae, Coliadinae) occur in the mountainous regions of Central-Asia, vast areas that are hard to access, rendering the knowledge of many species limited due to the lack of extensive sampling. Two gene regions, the mitochondrial COI 'barcode' region and the nuclear ribosomal protein RpS2 gene region were used for exploring the utility of these DNA markers for species identification. A comprehensive sampling of COI barcodes for Central Asian Colias butterflies showed that the barcodes facilitated identification of most of the included species. Phylogenetic reconstruction based on parsimony and Neighbour-Joining recovered most species as monophyletic entities. For the RpS2 gene region species-specific sequences were registered for some of the included Colias spp. Nevertheless, this gene region was not deemed useful as additional molecular 'barcode'. A parsimony analysis of the combined COI and RpS2 data did not support the current subgeneric classification based on morphological characteristics.

Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia

PubMed Central

Mishra, Priyanka; Kumar, Amit; Nagireddy, Akshitha; Shukla, Ashutosh K.

2017-01-01

DNA barcoding is used as a universal tool for delimiting species boundaries in taxonomically challenging groups, with different plastid and nuclear regions (rbcL, matK, ITS and psbA-trnH) being recommended as primary DNA barcodes for plants. We evaluated the feasibility of using these regions in the species-rich genus Terminalia, which exhibits various overlapping morphotypes with pantropical distribution, owing to its complex taxonomy. Terminalia bellerica and T. chebula are ingredients of the famous Ayurvedic Rasayana formulation Triphala, used for detoxification and rejuvenation. High demand for extracted phytochemicals as well as the high trade value of several species renders mandatory the need for the correct identification of traded plant material. Three different analytical methods with single and multilocus barcoding regions were tested to develop a DNA barcode reference library from 222 individuals representing 41 Terminalia species. All the single barcodes tested had a lower discriminatory power than the multilocus regions, and the combination of matK+ITS had the highest resolution rate (94.44%). The average intra-specific variations (0.0188±0.0019) were less than the distance to the nearest neighbour (0.106±0.009) with matK and ITS. Distance-based Neighbour Joining analysis outperformed the character-based Maximum Parsimony method in the identification of traded species such as T. arjuna, T. chebula and T. tomentosa, which are prone to adulteration. rbcL was shown to be a highly conservative region with only 3.45% variability between all of the sequences. The recommended barcode combination, rbcL+matK, failed to perform in the genus Terminalia. Considering the complexity of resolution observed with single regions, the present study proposes the combination of matK+ITS as the most successful barcode in Terminalia. PMID:28829803

Challenges and progress in making DNA-based AIS early ...

EPA Pesticide Factsheets

The ability of DNA barcoding to find additional species in hard-to-sample locations or hard-to-identify samples is well established. Nevertheless, adoption of DNA barcoding into regular monitoring programs has been slow, in part due to issues of standardization and interpretation that need resolving. In this presentation, we describe our progress towards incorporating DNA-based identification into broad-spectrum aquatic invasive species early-detection monitoring in the Laurentian Great Lakes. Our work uses community biodiversity information as the basis for evaluating survey performance for various taxonomic groups. Issues we are tackling in bringing DNA-based data to bear on AIS monitoring design include: 1) Standardizing methodology and work flow from field collection and sample handling through bioinformatics post-processing; 2) Determining detection sensitivity and accounting for inter-species differences in DNA amplification and primer affinity; 3) Differentiating sequencing and barcoding errors from legitimate new finds when range and natural history information is limited; and 4) Accounting for the different nature of morphology- vs. DNA-based biodiversity information in subsequent analysis (e.g., via species accumulation curves, multi-metric indices). not applicable

PCR cycles above routine numbers do not compromise high-throughput DNA barcoding results.

PubMed

Vierna, J; Doña, J; Vizcaíno, A; Serrano, D; Jovani, R

2017-10-01

High-throughput DNA barcoding has become essential in ecology and evolution, but some technical questions still remain. Increasing the number of PCR cycles above the routine 20-30 cycles is a common practice when working with old-type specimens, which provide little amounts of DNA, or when facing annealing issues with the primers. However, increasing the number of cycles can raise the number of artificial mutations due to polymerase errors. In this work, we sequenced 20 COI libraries in the Illumina MiSeq platform. Libraries were prepared with 40, 45, 50, 55, and 60 PCR cycles from four individuals belonging to four species of four genera of cephalopods. We found no relationship between the number of PCR cycles and the number of mutations despite using a nonproofreading polymerase. Moreover, even when using a high number of PCR cycles, the resulting number of mutations was low enough not to be an issue in the context of high-throughput DNA barcoding (but may still remain an issue in DNA metabarcoding due to chimera formation). We conclude that the common practice of increasing the number of PCR cycles should not negatively impact the outcome of a high-throughput DNA barcoding study in terms of the occurrence of point mutations.

Cross-shore and Vertical Distributions of Invertebrate Larvae Using Autonomous Sampling Coupled with Genetic Analysis

NASA Astrophysics Data System (ADS)

Govindarajan, A.; Pineda, J.; Purcell, M.; Tradd, K.; Packard, G.; Girard, A.; Dennett, M.; Breier, J. A., Jr.

2016-02-01

We present a new method to estimate the distribution of invertebrate larvae relative to environmental variables such as temperature, salinity, and circulation. A large volume in situ filtering system developed for discrete biogeochemical sampling in the deep-sea (the Suspended Particulate Rosette "SUPR" multisampler) was mounted to the autonomous underwater vehicle REMUS 600 for coastal larval and environmental sampling. We describe the results of SUPR-REMUS deployments conducted in Buzzards Bay, Massachusetts (2014) and west of Martha's Vineyard, Massachusetts (2015). We collected discrete samples cross-shore and from surface, middle, and bottom layers of the water column. Samples were preserved for DNA analysis. Our Buzzards Bay deployment targeted barnacle larvae, which are abundant in late winter and early spring. For these samples, we used morphological analysis and DNA barcodes generated by Sanger sequencing to obtain stage and species-specific cross-shore and vertical distributions. We targeted bivalve larvae in our 2015 deployments, and genetic analysis of larvae from these samples is underway. For these samples, we are comparing species barcode data derived from traditional Sanger sequencing of individuals to those obtained from next generation sequencing (NGS) of bulk plankton samples. Our results demonstrate the utility of autonomous sampling combined with DNA barcoding for studying larval distributions and transport dynamics.

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

Nanofluidic Device with Embedded Nanopore

NASA Astrophysics Data System (ADS)

Zhang, Yuning; Reisner, Walter

2014-03-01

Nanofluidic based devices are robust methods for biomolecular sensing and single DNA manipulation. Nanopore-based DNA sensing has attractive features that make it a leading candidate as a single-molecule DNA sequencing technology. Nanochannel based extension of DNA, combined with enzymatic or denaturation-based barcoding schemes, is already a powerful approach for genome analysis. We believe that there is revolutionary potential in devices that combine nanochannels with nanpore detectors. In particular, due to the fast translocation of a DNA molecule through a standard nanopore configuration, there is an unfavorable trade-off between signal and sequence resolution. With a combined nanochannel-nanopore device, based on embedding a nanopore inside a nanochannel, we can in principle gain independent control over both DNA translocation speed and sensing signal, solving the key draw-back of the standard nanopore configuration. We demonstrate that we can detect - using fluorescent microscopy - successful translocation of DNA from the nanochannel out through the nanopore, a possible method to 'select' a given barcode for further analysis. We also show that in equilibrium DNA will not escape through an embedded sub-persistence length nanopore until a certain voltage bias is added.

Assessment of mangroves from Goa, west coast India using DNA barcode.

PubMed

Saddhe, Ankush Ashok; Jamdade, Rahul Arvind; Kumar, Kundan

2016-01-01

Mangroves are salt-tolerant forest ecosystems of tropical and subtropical intertidal regions. They are among most productive, diverse, biologically important ecosystem and inclined toward threatened system. Identification of mangrove species is of critical importance in conserving and utilizing biodiversity, which apparently hindered by a lack of taxonomic expertise. In recent years, DNA barcoding using plastid markers rbcL and matK has been suggested as an effective method to enrich traditional taxonomic expertise for rapid species identification and biodiversity inventories. In the present study, we performed assessment of available 14 mangrove species of Goa, west coast India based on core DNA barcode markers, rbcL and matK. 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 rbcL (97.7 %) and matK (95.5 %) region. The two candidate chloroplast barcoding regions (rbcL, matK) yielded barcode gaps. Our results clearly demonstrated that matK locus assigned highest correct identification rates (72.09 %) based on TaxonDNA Best Match criteria. The concatenated rbcL + matK loci were able to adequately discriminate all mangrove genera and species to some extent except those in Rhizophora, Sonneratia and Avicennia. Our study provides the first endorsement of the species resolution among mangroves using plastid genes with few exceptions. Our future work will be focused on evaluation of other barcode markers to delineate complete resolution of mangrove species and identification of putative hybrids.

Existence of species complex largely reduced barcoding success for invasive species of Tephritidae: a case study in Bactrocera spp.

PubMed

Jiang, F; Jin, Q; Liang, L; Zhang, A B; Li, Z H

2014-11-01

Fruit flies in the family Tephritidae are the economically important pests that have many species complexes. DNA barcoding has gradually been verified as an effective tool for identifying species in a wide range of taxonomic groups, and there are several publications on rapid and accurate identification of fruit flies based on this technique; however, comprehensive analyses of large and new taxa for the effectiveness of DNA barcoding for fruit flies identification have been rare. In this study, we evaluated the COI barcode sequences for the diagnosis of fruit flies using 1426 sequences for 73 species of Bactrocera distributed worldwide. Tree-based [neighbour-joining (NJ)]; distance-based, such as Best Match (BM), Best Close Match (BCM) and Minimum Distance (MD); and character-based methods were used to evaluate the barcoding success rates obtained with maintaining the species complex in the data set, treating a species complex as a single taxon unit, and removing the species complex. Our results indicate that the average divergence between species was 14.04% (0.00-25.16%), whereas within a species this was 0.81% (0.00-9.71%); the existence of species complexes largely reduced the barcoding success for Tephritidae, for example relatively low success rates (74.4% based on BM and BCM and 84.8% based on MD) were obtained when the sequences from species complexes were included in the analysis, whereas significantly higher success rates were achieved if the species complexes were treated as a single taxon or removed from the data set - BM (98.9%), BCM (98.5%) and MD (97.5%), or BM (98.1%), BCM (97.4%) and MD (98.2%). © 2014 John Wiley & Sons Ltd.

Testing DNA barcode performance in 1000 species of European lepidoptera: large geographic distances have small genetic impacts.

PubMed

Huemer, Peter; Mutanen, Marko; Sefc, Kristina M; Hebert, Paul D N

2014-01-01

This study examines the performance of DNA barcodes (mt cytochrome c oxidase 1 gene) in the identification of 1004 species of Lepidoptera shared by two localities (Finland, Austria) that are 1600 km apart. Maximum intraspecific distances for the pooled data were less than 2% for 880 species (87.6%), while deeper divergence was detected in 124 species. Despite such variation, the overall DNA barcode library possessed diagnostic COI sequences for 98.8% of the taxa. Because a reference library based on Finnish specimens was highly effective in identifying specimens from Austria, we conclude that barcode libraries based on regional sampling can often be effective for a much larger area. Moreover, dispersal ability (poor, good) and distribution patterns (disjunct, fragmented, continuous, migratory) had little impact on levels of intraspecific geographic divergence. Furthermore, the present study revealed that, despite the intensity of past taxonomic work on European Lepidoptera, nearly 20% of the species shared by Austria and Finland require further work to clarify their status. Particularly discordant BIN (Barcode Index Number) cases should be checked to ascertain possible explanatory factors such as incorrect taxonomy, hybridization, introgression, and Wolbachia infections.

Flexbar 3.0 - SIMD and multicore parallelization.

PubMed

Roehr, Johannes T; Dieterich, Christoph; Reinert, Knut

2017-09-15

High-throughput sequencing machines can process many samples in a single run. For Illumina systems, sequencing reads are barcoded with an additional DNA tag that is contained in the respective sequencing adapters. The recognition of barcode and adapter sequences is hence commonly needed for the analysis of next-generation sequencing data. Flexbar performs demultiplexing based on barcodes and adapter trimming for such data. The massive amounts of data generated on modern sequencing machines demand that this preprocessing is done as efficiently as possible. We present Flexbar 3.0, the successor of the popular program Flexbar. It employs now twofold parallelism: multi-threading and additionally SIMD vectorization. Both types of parallelism are used to speed-up the computation of pair-wise sequence alignments, which are used for the detection of barcodes and adapters. Furthermore, new features were included to cover a wide range of applications. We evaluated the performance of Flexbar based on a simulated sequencing dataset. Our program outcompetes other tools in terms of speed and is among the best tools in the presented quality benchmark. https://github.com/seqan/flexbar. johannes.roehr@fu-berlin.de or knut.reinert@fu-berlin.de. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

The first initiative of DNA barcoding of ornamental plants from Egypt and potential applications in horticulture industry

PubMed Central

Ashfaq, Muhammad; Ali, Hayssam M.; Yessoufou, Kowiyou

2017-01-01

DNA barcoding relies on short and standardized gene regions to identify species. The agricultural and horticultural applications of barcoding such as for marketplace regulation and copyright protection remain poorly explored. This study examines the effectiveness of the standard plant barcode markers (matK and rbcL) for the identification of plant species in private and public nurseries in northern Egypt. These two markers were sequenced from 225 specimens of 161 species and 62 plant families of horticultural importance. The sequence recovery was similar for rbcL (96.4%) and matK (84%), but the number of specimens assigned correctly to the respective genera and species was lower for rbcL (75% and 29%) than matK (85% and 40%). The combination of rbcL and matK brought the number of correct generic and species assignments to 83.4% and 40%, respectively. Individually, the efficiency of both markers varied among different plant families; for example, all palm specimens (Arecaceae) were correctly assigned to species while only one individual of Asteraceae was correctly assigned to species. Further, barcodes reliably assigned ornamental horticultural and medicinal plants correctly to genus while they showed a lower or no success in assigning these plants to species and cultivars. For future, we recommend the combination of a complementary barcode (e.g. ITS or trnH-psbA) with rbcL + matK to increase the performance of taxa identification. By aiding species identification of horticultural crops and ornamental palms, the analysis of the barcode regions will have large impact on horticultural industry. PMID:28199378

The first initiative of DNA barcoding of ornamental plants from Egypt and potential applications in horticulture industry.

PubMed

O Elansary, Hosam; Ashfaq, Muhammad; Ali, Hayssam M; Yessoufou, Kowiyou

2017-01-01

DNA barcoding relies on short and standardized gene regions to identify species. The agricultural and horticultural applications of barcoding such as for marketplace regulation and copyright protection remain poorly explored. This study examines the effectiveness of the standard plant barcode markers (matK and rbcL) for the identification of plant species in private and public nurseries in northern Egypt. These two markers were sequenced from 225 specimens of 161 species and 62 plant families of horticultural importance. The sequence recovery was similar for rbcL (96.4%) and matK (84%), but the number of specimens assigned correctly to the respective genera and species was lower for rbcL (75% and 29%) than matK (85% and 40%). The combination of rbcL and matK brought the number of correct generic and species assignments to 83.4% and 40%, respectively. Individually, the efficiency of both markers varied among different plant families; for example, all palm specimens (Arecaceae) were correctly assigned to species while only one individual of Asteraceae was correctly assigned to species. Further, barcodes reliably assigned ornamental horticultural and medicinal plants correctly to genus while they showed a lower or no success in assigning these plants to species and cultivars. For future, we recommend the combination of a complementary barcode (e.g. ITS or trnH-psbA) with rbcL + matK to increase the performance of taxa identification. By aiding species identification of horticultural crops and ornamental palms, the analysis of the barcode regions will have large impact on horticultural industry.

DNA Barcoding of Malagasy Rosewoods: Towards a Molecular Identification of CITES-Listed Dalbergia Species.

PubMed

Hassold, Sonja; Lowry, Porter P; Bauert, Martin R; Razafintsalama, Annick; Ramamonjisoa, Lolona; Widmer, Alex

2016-01-01

Illegal selective logging of tropical timber is of increasing concern worldwide. Madagascar is a biodiversity hotspot and home to some of the world's most sought after tropical timber species. Malagasy rosewoods belong to the genus Dalbergia (Fabaceae), which is highly diverse and has a pantropical distribution, but these timber species are among the most threatened as a consequence of intensive illegal selective logging and deforestation. Reliable identification of Dalbergia species from Madagascar is important for law enforcement but is almost impossible without fertile plant material, which is often unavailable during forest inventories or when attempting to identify logged trees of cut wood. DNA barcoding has been promoted as a promising tool for species identification in such cases. In this study we tested whether DNA barcoding with partial sequences of three plastid markers (matK, rbcL and trnL (UAA)) can distinguish between Dalbergia from Madagascar and from other areas of its distributional range, and whether Malagasy species can be distinguished from one another. Phylogenetic analyses revealed that the Malagasy Dalbergia species studied form two monophyletic groups, each containing two subgroups, only one of which corresponds to a single species. We characterized diagnostic polymorphisms in the three DNA barcoding markers that allow rapid discrimination between Dalbergia from Madagascar and from other areas of its distribution range. Species identification success based on individual barcoding markers or combinations was poor, whereas subgroup identification success was much higher (up to 98%), revealing both the value and limitations of a DNA barcoding approach for the identification of closely related Malagasy rosewoods.

Discriminatory power of rbcL barcode locus for authentication of some of United Arab Emirates (UAE) native plants.

PubMed

Maloukh, Lina; Kumarappan, Alagappan; Jarrar, Mohammad; Salehi, Jawad; El-Wakil, Houssam; Rajya Lakshmi, T V

2017-06-01

DNA barcoding of United Arab Emirates (UAE) native plants is of high practical and scientific value as the plants adapt to very harsh environmental conditions that challenge their identification. Fifty-one plant species belonged to 22 families, 2 monocots, and 20 eudicots; a maximum number of species being legumes and grasses were collected. To authenticate the morphological identification of the wild plant taxa, rbcL and matK regions were used in the study. The primer universality and discriminatory power of rbcL is 100%, while it is 35% for matK locus for these plant species. The sequences were submitted to GenBank; accession numbers were obtained for all the rbcL sequences and for 6 of matK sequences. We suggest rbcL as a promising barcode locus for the tested group of 51 plants. In the present study, an inexpensive, simple method of identification of rare desert plant taxa through rbcL barcode is being reported.

Using high-throughput barcode sequencing to efficiently map connectomes.

PubMed

Peikon, Ian D; Kebschull, Justus M; Vagin, Vasily V; Ravens, Diana I; Sun, Yu-Chi; Brouzes, Eric; Corrêa, Ivan R; Bressan, Dario; Zador, Anthony M

2017-07-07

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular Identification of Commercialized Medicinal Plants in Southern Morocco

PubMed Central

Krüger, Åsa; Rydberg, Anders; Abbad, Abdelaziz; Björk, Lars; Martin, Gary

2012-01-01

Background Medicinal plant trade is important for local livelihoods. However, many medicinal plants are difficult to identify when they are sold as roots, powders or bark. DNA barcoding involves using a short, agreed-upon region of a genome as a unique identifier for species– ideally, as a global standard. Research Question What is the functionality, efficacy and accuracy of the use of barcoding for identifying root material, using medicinal plant roots sold by herbalists in Marrakech, Morocco, as a test dataset. Methodology In total, 111 root samples were sequenced for four proposed barcode regions rpoC1, psbA-trnH, matK and ITS. Sequences were searched against a tailored reference database of Moroccan medicinal plants and their closest relatives using BLAST and Blastclust, and through inference of RAxML phylograms of the aligned market and reference samples. Principal Findings Sequencing success was high for rpoC1, psbA-trnH, and ITS, but low for matK. Searches using rpoC1 alone resulted in a number of ambiguous identifications, indicating insufficient DNA variation for accurate species-level identification. Combining rpoC1, psbA-trnH and ITS allowed the majority of the market samples to be identified to genus level. For a minority of the market samples, the barcoding identification differed significantly from previous hypotheses based on the vernacular names. Conclusions/Significance Endemic plant species are commercialized in Marrakech. Adulteration is common and this may indicate that the products are becoming locally endangered. Nevertheless the majority of the traded roots belong to species that are common and not known to be endangered. A significant conclusion from our results is that unknown samples are more difficult to identify than earlier suggested, especially if the reference sequences were obtained from different populations. A global barcoding database should therefore contain sequences from different populations of the same species to assure the reference sequences characterize the species throughout its distributional range. PMID:22761800

A new method for species identification via protein-coding and non-coding DNA barcodes by combining machine learning with bioinformatic methods.

PubMed

Zhang, Ai-bing; Feng, Jie; Ward, Robert D; Wan, Ping; Gao, Qiang; Wu, Jun; Zhao, Wei-zhong

2012-01-01

Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI) region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS) genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF) to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish) and two representing non-coding ITS barcodes (rust fungi and brown algae). Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ) and Maximum likelihood (ML) methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI) of 99.75-100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62-98.40%) for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60-99.37%) for 1094 brown algae queries, both using ITS barcodes.

Identification of Forensically Important Calliphoridae and Sarcophagidae Species Collected in Korea Using SNaPshot Multiplex System Targeting the Cytochrome c Oxidase Subunit I Gene

PubMed Central

Park, Ji Hye

2018-01-01

Estimation of postmortem interval (PMI) is paramount in modern forensic investigation. After the disappearance of the early postmortem phenomena conventionally used to estimate PMI, entomologic evidence provides important indicators for PMI estimation. The age of the oldest fly larvae or pupae can be estimated to pinpoint the time of oviposition, which is considered the minimum PMI (PMImin). The development rate of insects is usually temperature dependent and species specific. Therefore, species identification is mandatory for PMImin estimation using entomological evidence. The classical morphological identification method cannot be applied when specimens are damaged or have not yet matured. To overcome this limitation, some investigators employ molecular identification using mitochondrial cytochrome c oxidase subunit I (COI) nucleotide sequences. The molecular identification method commonly uses Sanger's nucleotide sequencing and molecular phylogeny, which are complex and time consuming and constitute another obstacle for forensic investigators. In this study, instead of using conventional Sanger's nucleotide sequencing, single-nucleotide polymorphisms (SNPs) in the COI gene region, which are unique between fly species, were selected and targeted for single-base extension (SBE) technology. These SNPs were genotyped using a SNaPshot® kit. Eleven Calliphoridae and seven Sarcophagidae species were covered. To validate this genotyping, fly DNA samples (103 adults, 84 larvae, and 4 pupae) previously confirmed by DNA barcoding were used. This method worked quickly with minimal DNA, providing a potential alternative to conventional DNA barcoding. Consisting of only a few simple electropherogram peaks, the results were more straightforward compared with those of the conventional DNA barcoding produced by Sanger's nucleotide sequencing. PMID:29682531

Towards a global barcode library for Lymantria (Lepidoptera: Lymantriinae) tussock moths of biosecurity concern.

PubMed

deWaard, Jeremy R; Mitchell, Andrew; Keena, Melody A; Gopurenko, David; Boykin, Laura M; Armstrong, Karen F; Pogue, Michael G; Lima, Joao; Floyd, Robin; Hanner, Robert H; Humble, Leland M

2010-12-09

Detecting and controlling the movements of invasive species, such as insect pests, relies upon rapid and accurate species identification in order to initiate containment procedures by the appropriate authorities. Many species in the tussock moth genus Lymantria are significant forestry pests, including the gypsy moth Lymantria dispar L., and consequently have been a focus for the development of molecular diagnostic tools to assist in identifying species and source populations. In this study we expand the taxonomic and geographic coverage of the DNA barcode reference library, and further test the utility of this diagnostic method, both for species/subspecies assignment and for determination of geographic provenance of populations. Cytochrome oxidase I (COI) barcodes were obtained from 518 individuals and 36 species of Lymantria, including sequences assembled and generated from previous studies, vouchered material in public collections, and intercepted specimens obtained from surveillance programs in Canada. A maximum likelihood tree was constructed, revealing high bootstrap support for 90% of species clusters. Bayesian species assignment was also tested, and resulted in correct assignment to species and subspecies in all instances. The performance of barcoding was also compared against the commonly employed NB restriction digest system (also based on COI); while the latter is informative for discriminating gypsy moth subspecies, COI barcode sequences provide greater resolution and generality by encompassing a greater number of haplotypes across all Lymantria species, none shared between species. This study demonstrates the efficacy of DNA barcodes for diagnosing species of Lymantria and reinforces the view that the approach is an under-utilized resource with substantial potential for biosecurity and surveillance. Biomonitoring agencies currently employing the NB restriction digest system would gather more information by transitioning to the use of DNA barcoding, a change which could be made relatively seamlessly as the same gene region underlies both protocols.

DNA Barcodes for Species Identification in the Hyperdiverse Ant Genus Pheidole (Formicidae: Myrmicinae)

PubMed Central

Ng'endo, R.N.; Osiemo, Z.B.; Brandl, R.

2013-01-01

DNA sequencing is increasingly being used to assist in species identification in order to overcome taxonomic impediment. However, few studies attempt to compare the results of these molecular studies with a more traditional species delineation approach based on morphological characters. Mitochondrial DNA Cytochrome oxidase subunit 1 (CO1) gene was sequenced, measuring 636 base pairs, from 47 ants of the genus Pheidole (Formicidae: Myrmicinae) collected in the Brazilian Atlantic Forest to test whether the morphology-based assignment of individuals into species is supported by DNA-based species delimitation. Twenty morphospecies were identified, whereas the barcoding analysis identified 19 Molecular Operational Taxonomic Units (MOTUs). Fifteen out of the 19 DNA-based clusters allocated, using sequence divergence thresholds of 2% and 3%, matched with morphospecies. Both thresholds yielded the same number of MOTUs. Only one MOTU was successfully identified to species level using the CO1 sequences of Pheidole species already in the Genbank. The average pairwise sequence divergence for all 47 sequences was 19%, ranging between 0–25%. In some cases, however, morphology and molecular based methods differed in their assignment of individuals to morphospecies or MOTUs. The occurrence of distinct mitochondrial lineages within morphological species highlights groups for further detailed genetic and morphological studies, and therefore a pluralistic approach using several methods to understand the taxonomy of difficult lineages is advocated. PMID:23902257

DNA Barcoding the Medusozoa using mtCOI

NASA Astrophysics Data System (ADS)

Ortman, Brian D.; Bucklin, Ann; Pagès, Francesc; Youngbluth, Marsh

2010-12-01

The Medusozoa are a clade within the Cnidaria comprising the classes Hydrozoa, Scyphozoa, and Cubozoa. Identification of medusozoan species is challenging, even for taxonomic experts, due to their fragile forms and complex, morphologically-distinct life history stages. In this study 231 sequences for a portion of the mitochondrial Cytochrome Oxidase I (mtCOI) gene were obtained from 95 species of Medusozoans including; 84 hydrozoans (61 siphonophores, eight anthomedusae, four leptomedusae, seven trachymedusae, and four narcomedusae), 10 scyphozoans (three coronatae, four semaeostomae, two rhizostomae, and one stauromedusae), and one cubozoan. This region of mtCOI has been used as a DNA barcode (i.e., a molecular character for species recognition and discrimination) for a diverse array of taxa, including some Cnidaria. Kimura 2-parameter (K2P) genetic distances between sequence variants within species ranged from 0 to 0.057 (mean 0.013). Within the 13 genera for which multiple species were available, K2P distance between congeneric species ranged from 0.056 to 0.381. A cluster diagram generated by Neighbor Joining (NJ) using K2P distances reliably clustered all barcodes of the same species with ≥99% bootstrap support, ensuring accurate identification of species. Intra- and inter-specific variation of the mtCOI gene for the Medusozoa are appropriate for this gene to be used as a DNA barcode for species-level identification, but not for phylogenetic analysis or taxonomic classification of unknown sequences at higher taxonomic levels. This study provides a set of molecular tools that can be used to address questions of speciation, biodiversity, life-history, and population boundaries in the Medusozoa.

DNA Barcodes for the FIshes of the Narmada, One of India’s Longest Rivers

PubMed Central

Khedkar, Gulab Dattarao; Jamdade, Rahul; Naik, Suresh; David, Lior; Haymer, David

2014-01-01

This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura’s 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River. PMID:24991801

DNA barcodes for the fishes of the Narmada, one of India's longest rivers.

PubMed

Khedkar, Gulab Dattarao; Jamdade, Rahul; Naik, Suresh; David, Lior; Haymer, David

2014-01-01

This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura's 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River.

Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

PubMed Central

Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

2016-01-01

Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

Spiders (Araneae) of Churchill, Manitoba: DNA barcodes and morphology reveal high species diversity and new Canadian records

PubMed Central

2013-01-01

Background Arctic ecosystems, especially those near transition zones, are expected to be strongly impacted by climate change. Because it is positioned on the ecotone between tundra and boreal forest, the Churchill area is a strategic locality for the analysis of shifts in faunal composition. This fact has motivated the effort to develop a comprehensive biodiversity inventory for the Churchill region by coupling DNA barcoding with morphological studies. The present study represents one element of this effort; it focuses on analysis of the spider fauna at Churchill. Results 198 species were detected among 2704 spiders analyzed, tripling the count for the Churchill region. Estimates of overall diversity suggest that another 10–20 species await detection. Most species displayed little intraspecific sequence variation (maximum <1%) in the barcode region of the cytochrome c oxidase subunit I (COI) gene, but four species showed considerably higher values (maximum = 4.1-6.2%), suggesting cryptic species. All recognized species possessed a distinct haplotype array at COI with nearest-neighbour interspecific distances averaging 8.57%. Three species new to Canada were detected: Robertus lyrifer (Theridiidae), Baryphyma trifrons (Linyphiidae), and Satilatlas monticola (Linyphiidae). The first two species may represent human-mediated introductions linked to the port in Churchill, but the other species represents a range extension from the USA. The first description of the female of S. monticola was also presented. As well, one probable new species of Alopecosa (Lycosidae) was recognized. Conclusions This study provides the first comprehensive DNA barcode reference library for the spider fauna of any region. Few cryptic species of spiders were detected, a result contrasting with the prevalence of undescribed species in several other terrestrial arthropod groups at Churchill. Because most (97.5%) sequence clusters at COI corresponded with a named taxon, DNA barcoding reliably identifies spiders in the Churchill fauna. The capacity of DNA barcoding to enable the identification of otherwise taxonomically ambiguous specimens (juveniles, females) also represents a major advance for future monitoring efforts on this group. PMID:24279427

Building a DNA barcode reference library for the true butterflies (Lepidoptera) of Peninsula Malaysia: what about the subspecies?

PubMed

Wilson, John-James; Sing, Kong-Wah; Sofian-Azirun, Mohd

2013-01-01

The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity.

Building a DNA Barcode Reference Library for the True Butterflies (Lepidoptera) of Peninsula Malaysia: What about the Subspecies?

PubMed Central

Wilson, John-James; Sing, Kong-Wah; Sofian-Azirun, Mohd

2013-01-01

The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity. PMID:24282514

DNA barcoding for species identification in deep-sea clams (Mollusca: Bivalvia: Vesicomyidae).

PubMed

Liu, Jun; Zhang, Haibin

2018-01-15

Deep-sea clams (Bivalvia: Vesicomyidae) have been found in reduced environments over the world oceans, but taxonomy of this group remains confusing at species and supraspecific levels due to their high-morphological similarity and plasticity. In the present study, we collected mitochondrial COI sequences to evaluate the utility of DNA barcoding on identifying vesicomyid species. COI dataset identified 56 well-supported putative species/operational taxonomic units (OTUs), approximately covering half of the extant vesicomyid species. One species (OTU2) was first detected, and may represent a new species. Average distances between species ranged from 1.65 to 29.64%, generally higher than average intraspecific distances (0-1.41%) when excluding Pliocardia sp.10 cf. venusta (average intraspecific distance 1.91%). Local barcoding gap existed in 33 of the 35 species when comparing distances of maximum interspecific and minimum interspecific distances with two exceptions (Abyssogena southwardae and Calyptogena rectimargo-starobogatovi). The barcode index number (BIN) system determined 41 of the 56 species/OTUs, each with a unique BIN, indicating their validity. Three species were found to have two BINs, together with their high level of intraspecific variation, implying cryptic diversity within them. Although fewer 16 S sequences were collected, similar results were obtained. Nineteen putative species were determined and no overlap observed between intra- and inter-specific variation. Implications of DNA barcoding for the Vesicomyidae taxonomy were then discussed. Findings of this study will provide important evidence for taxonomic revision in this problematic clam group, and accelerate the discovery of new vesicomyid species in the future.

Untangling taxonomy: a DNA barcode reference library for Canadian spiders.

PubMed

Blagoev, Gergin A; deWaard, Jeremy R; Ratnasingham, Sujeevan; deWaard, Stephanie L; Lu, Liuqiong; Robertson, James; Telfer, Angela C; Hebert, Paul D N

2016-01-01

Approximately 1460 species of spiders have been reported from Canada, 3% of the global fauna. This study provides a DNA barcode reference library for 1018 of these species based upon the analysis of more than 30,000 specimens. The sequence results show a clear barcode gap in most cases with a mean intraspecific divergence of 0.78% vs. a minimum nearest-neighbour (NN) distance averaging 7.85%. The sequences were assigned to 1359 Barcode index numbers (BINs) with 1344 of these BINs composed of specimens belonging to a single currently recognized species. There was a perfect correspondence between BIN membership and a known species in 795 cases, while another 197 species were assigned to two or more BINs (556 in total). A few other species (26) were involved in BIN merges or in a combination of merges and splits. There was only a weak relationship between the number of specimens analysed for a species and its BIN count. However, three species were clear outliers with their specimens being placed in 11-22 BINs. Although all BIN splits need further study to clarify the taxonomic status of the entities involved, DNA barcodes discriminated 98% of the 1018 species. The present survey conservatively revealed 16 species new to science, 52 species new to Canada and major range extensions for 426 species. However, if most BIN splits detected in this study reflect cryptic taxa, the true species count for Canadian spiders could be 30-50% higher than currently recognized. © 2015 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Detection of plant-based adulterants in turmeric powder using DNA barcoding.

PubMed

Parvathy, V A; Swetha, V P; Sheeja, T E; Sasikumar, B

2015-01-01

In its powdered form, turmeric [Curcuma longa L. (Zingiberaceae)], a spice of medical importance, is often adulterated lowering its quality. The study sought to detect plant-based adulterants in traded turmeric powder using DNA barcoding. Accessions of Curcuma longa L., Curcuma zedoaria Rosc. (Zingiberaceae), and cassava starch served as reference samples. Three barcoding loci, namely ITS, rbcL, and matK, were used for PCR amplification of the reference samples and commercial samples representing 10 different companies. PCR success rate, sequencing efficiency, occurrence of SNPs, and BLAST analysis were used to assess the potential of the barcoding loci in authenticating the traded samples of turmeric. The PCR and sequencing success of the loci rbcL and ITS were found to be 100%, whereas matK showed no amplification. ITS proved to be the ideal locus because it showed greater variability than rbcL in discriminating the Curcuma species. The presence of C. zedoaria could be detected in one of the samples whereas cassava starch, wheat, barley, and rye in other two samples although the label claimed nothing other than turmeric powder in the samples. Unlabeled materials in turmeric powder are considered as adulterants or fillers, added to increase the bulk weight and starch content of the commodity for economic gains. These adulterants pose potential health hazards to consumers who are allergic to these plants, lowering the product's medicinal value and belying the claim that the product is gluten free. The study proved DNA barcoding as an efficient tool for testing the integrity and the authenticity of commercial products of turmeric.

DNA barcoding for molecular identification of Demodex based on mitochondrial genes.

PubMed

Hu, Li; Yang, YuanJun; Zhao, YaE; Niu, DongLing; Yang, Rui; Wang, RuiLing; Lu, Zhaohui; Li, XiaoQi

2017-12-01

There has been no widely accepted DNA barcode for species identification of Demodex. In this study, we attempted to solve this issue. First, mitochondrial cox1-5' and 12S gene fragments of Demodex folloculorum, D. brevis, D. canis, and D. caprae were amplified, cloned, and sequenced for the first time; intra/interspecific divergences were computed and phylogenetic trees were reconstructed. Then, divergence frequency distribution plots of those two gene fragments were drawn together with mtDNA cox1-middle region and 16S obtained in previous studies. Finally, their identification efficiency was evaluated by comparing barcoding gap. Results indicated that 12S had the higher identification efficiency. Specifically, for cox1-5' region of the four Demodex species, intraspecific divergences were less than 2.0%, and interspecific divergences were 21.1-31.0%; for 12S, intraspecific divergences were less than 1.4%, and interspecific divergences were 20.8-26.9%. The phylogenetic trees demonstrated that the four Demodex species clustered separately, and divergence frequency distribution plot showed that the largest intraspecific divergence of 12S (1.4%) was less than cox1-5' region (2.0%), cox1-middle region (3.1%), and 16S (2.8%). The barcoding gap of 12S was 19.4%, larger than cox1-5' region (19.1%), cox1-middle region (11.3%), and 16S (13.0%); the interspecific divergence span of 12S was 6.2%, smaller than cox1-5' region (10.0%), cox1-middle region (14.1%), and 16S (11.4%). Moreover, 12S has a moderate length (517 bp) for sequencing at once. Therefore, we proposed mtDNA 12S was more suitable than cox1 and 16S to be a DNA barcode for classification and identification of Demodex at lower category level.

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures.

PubMed

Olivar, Jay Edneil C; Alaba, Joanner Paulus Erik P; Atienza, Jose Francisco M; Tan, Jerick Jeffrey S; Umali, Maximo T; Alejandro, Grecebio Jonathan D

2016-05-01

The majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.

[Identification of original species of Mantidis Oötheca (Sangpiaoxiao) based on DNA barcoding].

PubMed

Wang, Xi; Hou, Fei-xia; Wang, Yi-xuan; Wang, Yu-xian; Li, Jun-de; Yuan, Yuan; Peng, Cheng; Guo, Jin-lin

2015-10-01

Both market research and literature reports both found that the ootheca of mantodea was all used as medicine. However, Chinese Pharmacopoeia only records the ootheca of three mantis species. The clinical use of ootheca unrecorded in Chinese Pharmacopoeia, will pose potential risks to drug safety. It's urgent to identify the origin of Mantidis Oötheca. The current researches about original animal in Mantidis Oötheca are based on morphology and unanimous. DNA barcoding fill gaps of the traditional morphological identification, which is widely used in animal classification studies. This study first use DNA barcoding to analyze genetic distance among different Mantidis Oötheca types, align COI sequences between mantis and Mantidis Oötheca and construct the phylogeny tree. The result confirmed that Tenodera sinensis and Hierodula patellifera were the origin insects of Tuanpiaoxiao and Heipiaoxiao, respectively, and Statilia maculate and Mantis religiosa were the origin insects of Changpiaoxiao.

DNA Barcodes Confirm the Taxonomic and Conservation Status of a Species of Tree on the Brink of Extinction in the Pacific.

PubMed

Costion, Craig M; Kress, W John; Crayn, Darren M

2016-01-01

The taxonomic status of a single island, narrow range endemic plant species from Palau, Micronesia (Timonius salsedoi) was assessed using DNA barcode markers, additional plastid loci, and morphology in order to verify its conservation status. DNA barcode loci distinguished T. salsedoi from all other Timonius species sampled from Palau, and were supported by sequence data from the atpB-rbcL intergenic spacer region. Timonius salsedoi was only known from two mature individual trees in 2012. Due to its extremely narrow range and population size, it had previously been recommended to be listed as Critically Endangered Status under three separate IUCN Criteria. In 2014 a second survey of the population following a typhoon revealed that the only two known trees had died suggesting that this species may now be extinct. Comprehensive follow up surveys of suitable habitat for this species are urgently required.

A reliable DNA barcode reference library for the identification of the North European shelf fish fauna.

PubMed

Knebelsberger, Thomas; Landi, Monica; Neumann, Hermann; Kloppmann, Matthias; Sell, Anne F; Campbell, Patrick D; Laakmann, Silke; Raupach, Michael J; Carvalho, Gary R; Costa, Filipe O

2014-09-01

Valid fish species identification is an essential step both for fundamental science and fisheries management. The traditional identification is mainly based on external morphological diagnostic characters, leading to inconsistent results in many cases. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for a valid identification of 93 North Atlantic fish species originating from the North Sea and adjacent waters, including many commercially exploited species. Neighbour-joining analysis based on K2P genetic distances formed nonoverlapping clusters for all species with a ≥99% bootstrap support each. Identification was successful for 100% of the species as the minimum genetic distance to the nearest neighbour always exceeded the maximum intraspecific distance. A barcoding gap was apparent for the whole data set. Within-species distances ranged from 0 to 2.35%, while interspecific distances varied between 3.15 and 28.09%. Distances between congeners were on average 51-fold higher than those within species. The validation of the sequence library by applying BOLDs barcode index number (BIN) analysis tool and a ranking system demonstrated high taxonomic reliability of the DNA barcodes for 85% of the investigated fish species. Thus, the sequence library presented here can be confidently used as a benchmark for identification of at least two-thirds of the typical fish species recorded for the North Sea. © 2014 John Wiley & Sons Ltd.

Specific PCR Identification between Peucedanum praeruptorum and Angelica decursiva and Identification between Them and Adulterant Using DNA Barcode.

PubMed

Han, Bang-Xing; Yuan, Yuan; Huang, Lu-Qi; Zhao, Qun; Tan, Ling-Ling; Song, Xiang-Wen; He, Xiao-Mei; Xu, Tao; Liu, Feng; Wang, Jian

2017-01-01

The traditional Chinese medicine (TCM) Qianhu and Zihuaqianhu are the dried roots of Peucedanum praeruptorum and Angelica decursiva , respectively. Since the plant sources of Qianhu and Zihuaqianhu are more complex, the chemical compositions of P. praeruptorum and A. decursiva are significantly different, and many adulterants exist because of the differences in traditional understanding and medication habits. Therefore, the rapid and accurate identification methods are required. The aim was to study the feasibility of using DNA barcoding to distinguish between Traditional Chinese medicine Qianhu ( Peucedanum praeruptorum ), Zihuaqianhu ( Angelica decursiva ), and common adulterants, based on internal transcribed spacer (ITS) sequences, as well as specific PCR identification between P. praeruptorum and A. decursiva . The ITS sequences of P. praeruptorum , A. decursiva , and adulterant were studied, and a phylogenetic tree was constructed. Based on the ITS barcode, the specific PCR primer pairs QH-CP19s/QH-CP19a and ZHQH-CP3s/ZHQH-CP3a were designed for P. praeruptorum and A. decursiva , respectively. The amplification conditions were optimized, and specific PCR products were obtained. The results showed that the phylogenetic trees constructed using the BI and MP methods were consistent, and P. praeruptorum and A. decursiva sequence haplotypes formed their own monophyly. The experimental results showed that in PCR products, the target bands appeared in the genuine drug and not in the adulterant, which suggests the high specificity of the two primer pairs. The ITS sequence was ideal DNA barcode to identify P. praeruptorum , A. decursiva , and adulterant. The specific PCR is a quick and effective method to distinguish between P. praeruptorum and A. decursiva . Peucedanum praeruptorum and Angelica decursiva sequence haplotypes formed their own monophyly.The ITS sequence was ideal DNA barcode to identify P. praeruptorum , A. decursiva , and adulterant.Specific PCR is a quick and effective method to distinguish between P. praeruptorum and A. decursiva . Abbreviations used: TCM: The traditional Chinese medicine, P.: Peucedanum , A.: Angelica , ITS: The internal transcribed spacer, PCR: Polymerase chain reaction, NCBI: National Center for Biotechnology Information, NI: Number of individuals, HN: Haplotype number; GAN: Gen Bank accession numbers, L.: Ligusticum , O.: Ostericum , A.: Angelica , P.: Pimpinella , BI: Bayesian inference, MP: Maximum parsimony, AIC: Akaike Information Criterion, MCMC: Markov Chains Monte Carlo, TBR: Tree bisection-reconnection, LPP: Length of PCR product, PRP: PCR reaction procedure, SNP: Single nucleotide polymorphisms, PP: Posterior probability, BS: Bootstrap.Qun Zhao.

Biodiversity inventories in high gear: DNA barcoding facilitates a rapid biotic survey of a temperate nature reserve.

PubMed

Telfer, Angela C; Young, Monica R; Quinn, Jenna; Perez, Kate; Sobel, Crystal N; Sones, Jayme E; Levesque-Beaudin, Valerie; Derbyshire, Rachael; Fernandez-Triana, Jose; Rougerie, Rodolphe; Thevanayagam, Abinah; Boskovic, Adrian; Borisenko, Alex V; Cadel, Alex; Brown, Allison; Pages, Anais; Castillo, Anibal H; Nicolai, Annegret; Glenn Mockford, Barb Mockford; Bukowski, Belén; Wilson, Bill; Trojahn, Brock; Lacroix, Carole Ann; Brimblecombe, Chris; Hay, Christoper; Ho, Christmas; Steinke, Claudia; Warne, Connor P; Garrido Cortes, Cristina; Engelking, Daniel; Wright, Danielle; Lijtmaer, Dario A; Gascoigne, David; Hernandez Martich, David; Morningstar, Derek; Neumann, Dirk; Steinke, Dirk; Marco DeBruin, Donna DeBruin; Dobias, Dylan; Sears, Elizabeth; Richard, Ellen; Damstra, Emily; Zakharov, Evgeny V; Laberge, Frederic; Collins, Gemma E; Blagoev, Gergin A; Grainge, Gerrie; Ansell, Graham; Meredith, Greg; Hogg, Ian; McKeown, Jaclyn; Topan, Janet; Bracey, Jason; Guenther, Jerry; Sills-Gilligan, Jesse; Addesi, Joseph; Persi, Joshua; Layton, Kara K S; D'Souza, Kareina; Dorji, Kencho; Grundy, Kevin; Nghidinwa, Kirsti; Ronnenberg, Kylee; Lee, Kyung Min; Xie, Linxi; Lu, Liuqiong; Penev, Lyubomir; Gonzalez, Mailyn; Rosati, Margaret E; Kekkonen, Mari; Kuzmina, Maria; Iskandar, Marianne; Mutanen, Marko; Fatahi, Maryam; Pentinsaari, Mikko; Bauman, Miriam; Nikolova, Nadya; Ivanova, Natalia V; Jones, Nathaniel; Weerasuriya, Nimalka; Monkhouse, Norman; Lavinia, Pablo D; Jannetta, Paul; Hanisch, Priscila E; McMullin, R Troy; Ojeda Flores, Rafael; Mouttet, Raphaëlle; Vender, Reid; Labbee, Renee N; Forsyth, Robert; Lauder, Rob; Dickson, Ross; Kroft, Ruth; Miller, Scott E; MacDonald, Shannon; Panthi, Sishir; Pedersen, Stephanie; Sobek-Swant, Stephanie; Naik, Suresh; Lipinskaya, Tatsiana; Eagalle, Thanushi; Decaëns, Thibaud; Kosuth, Thibault; Braukmann, Thomas; Woodcock, Tom; Roslin, Tomas; Zammit, Tony; Campbell, Victoria; Dinca, Vlad; Peneva, Vlada; Hebert, Paul D N; deWaard, Jeremy R

2015-01-01

Comprehensive biotic surveys, or 'all taxon biodiversity inventories' (ATBI), have traditionally been limited in scale or scope due to the complications surrounding specimen sorting and species identification. To circumvent these issues, several ATBI projects have successfully integrated DNA barcoding into their identification procedures and witnessed acceleration in their surveys and subsequent increase in project scope and scale. The Biodiversity Institute of Ontario partnered with the rare Charitable Research Reserve and delegates of the 6th International Barcode of Life Conference to complete its own rapid, barcode-assisted ATBI of an established land trust in Cambridge, Ontario, Canada. The existing species inventory for the rare Charitable Research Reserve was rapidly expanded by integrating a DNA barcoding workflow with two surveying strategies - a comprehensive sampling scheme over four months, followed by a one-day bioblitz involving international taxonomic experts. The two surveys resulted in 25,287 and 3,502 specimens barcoded, respectively, as well as 127 human observations. This barcoded material, all vouchered at the Biodiversity Institute of Ontario collection, covers 14 phyla, 29 classes, 117 orders, and 531 families of animals, plants, fungi, and lichens. Overall, the ATBI documented 1,102 new species records for the nature reserve, expanding the existing long-term inventory by 49%. In addition, 2,793 distinct Barcode Index Numbers (BINs) were assigned to genus or higher level taxonomy, and represent additional species that will be added once their taxonomy is resolved. For the 3,502 specimens, the collection, sequence analysis, taxonomic assignment, data release and manuscript submission by 100+ co-authors all occurred in less than one week. This demonstrates the speed at which barcode-assisted inventories can be completed and the utility that barcoding provides in minimizing and guiding valuable taxonomic specialist time. The final product is more than a comprehensive biotic inventory - it is also a rich dataset of fine-scale occurrence and sequence data, all archived and cross-linked in the major biodiversity data repositories. This model of rapid generation and dissemination of essential biodiversity data could be followed to conduct regional assessments of biodiversity status and change, and potentially be employed for evaluating progress towards the Aichi Targets of the Strategic Plan for Biodiversity 2011-2020.

Biodiversity inventories in high gear: DNA barcoding facilitates a rapid biotic survey of a temperate nature reserve

PubMed Central

Young, Monica R; Quinn, Jenna; Perez, Kate; Sobel, Crystal N; Sones, Jayme E; Levesque-Beaudin, Valerie; Derbyshire, Rachael; Fernandez-Triana, Jose; Rougerie, Rodolphe; Thevanayagam, Abinah; Boskovic, Adrian; Borisenko, Alex V; Cadel, Alex; Brown, Allison; Pages, Anais; Castillo, Anibal H; Nicolai, Annegret; Glenn Mockford, Barb Mockford; Bukowski, Belén; Wilson, Bill; Trojahn, Brock; Lacroix, Carole Ann; Brimblecombe, Chris; Hay, Christoper; Ho, Christmas; Steinke, Claudia; Warne, Connor P; Garrido Cortes, Cristina; Engelking, Daniel; Wright, Danielle; Lijtmaer, Dario A; Gascoigne, David; Hernandez Martich, David; Morningstar, Derek; Neumann, Dirk; Steinke, Dirk; Marco DeBruin, Donna DeBruin; Dobias, Dylan; Sears, Elizabeth; Richard, Ellen; Damstra, Emily; Zakharov, Evgeny V; Laberge, Frederic; Collins, Gemma E; Blagoev, Gergin A; Grainge, Gerrie; Ansell, Graham; Meredith, Greg; Hogg, Ian; McKeown, Jaclyn; Topan, Janet; Bracey, Jason; Guenther, Jerry; Sills-Gilligan, Jesse; Addesi, Joseph; Persi, Joshua; Layton, Kara K S; D'Souza, Kareina; Dorji, Kencho; Grundy, Kevin; Nghidinwa, Kirsti; Ronnenberg, Kylee; Lee, Kyung Min; Xie, Linxi; Lu, Liuqiong; Penev, Lyubomir; Gonzalez, Mailyn; Rosati, Margaret E; Kekkonen, Mari; Kuzmina, Maria; Iskandar, Marianne; Mutanen, Marko; Fatahi, Maryam; Pentinsaari, Mikko; Bauman, Miriam; Nikolova, Nadya; Ivanova, Natalia V; Jones, Nathaniel; Weerasuriya, Nimalka; Monkhouse, Norman; Lavinia, Pablo D; Jannetta, Paul; Hanisch, Priscila E; McMullin, R. Troy; Ojeda Flores, Rafael; Mouttet, Raphaëlle; Vender, Reid; Labbee, Renee N; Forsyth, Robert; Lauder, Rob; Dickson, Ross; Kroft, Ruth; Miller, Scott E; MacDonald, Shannon; Panthi, Sishir; Pedersen, Stephanie; Sobek-Swant, Stephanie; Naik, Suresh; Lipinskaya, Tatsiana; Eagalle, Thanushi; Decaëns, Thibaud; Kosuth, Thibault; Braukmann, Thomas; Woodcock, Tom; Roslin, Tomas; Zammit, Tony; Campbell, Victoria; Dinca, Vlad; Peneva, Vlada; Hebert, Paul D N

2015-01-01

Abstract Background Comprehensive biotic surveys, or ‘all taxon biodiversity inventories’ (ATBI), have traditionally been limited in scale or scope due to the complications surrounding specimen sorting and species identification. To circumvent these issues, several ATBI projects have successfully integrated DNA barcoding into their identification procedures and witnessed acceleration in their surveys and subsequent increase in project scope and scale. The Biodiversity Institute of Ontario partnered with the rare Charitable Research Reserve and delegates of the 6th International Barcode of Life Conference to complete its own rapid, barcode-assisted ATBI of an established land trust in Cambridge, Ontario, Canada. New information The existing species inventory for the rare Charitable Research Reserve was rapidly expanded by integrating a DNA barcoding workflow with two surveying strategies – a comprehensive sampling scheme over four months, followed by a one-day bioblitz involving international taxonomic experts. The two surveys resulted in 25,287 and 3,502 specimens barcoded, respectively, as well as 127 human observations. This barcoded material, all vouchered at the Biodiversity Institute of Ontario collection, covers 14 phyla, 29 classes, 117 orders, and 531 families of animals, plants, fungi, and lichens. Overall, the ATBI documented 1,102 new species records for the nature reserve, expanding the existing long-term inventory by 49%. In addition, 2,793 distinct Barcode Index Numbers (BINs) were assigned to genus or higher level taxonomy, and represent additional species that will be added once their taxonomy is resolved. For the 3,502 specimens, the collection, sequence analysis, taxonomic assignment, data release and manuscript submission by 100+ co-authors all occurred in less than one week. This demonstrates the speed at which barcode-assisted inventories can be completed and the utility that barcoding provides in minimizing and guiding valuable taxonomic specialist time. The final product is more than a comprehensive biotic inventory – it is also a rich dataset of fine-scale occurrence and sequence data, all archived and cross-linked in the major biodiversity data repositories. This model of rapid generation and dissemination of essential biodiversity data could be followed to conduct regional assessments of biodiversity status and change, and potentially be employed for evaluating progress towards the Aichi Targets of the Strategic Plan for Biodiversity 2011–2020. PMID:26379469

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

PubMed Central

Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

2012-01-01

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.

PubMed

Schoch, Conrad L; Seifert, Keith A; Huhndorf, Sabine; Robert, Vincent; Spouge, John L; Levesque, C André; Chen, Wen

2012-04-17

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

Molecular Identification of Dendrobium Species (Orchidaceae) Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study.

PubMed

Feng, Shangguo; Jiang, Yan; Wang, Shang; Jiang, Mengying; Chen, Zhe; Ying, Qicai; Wang, Huizhong

2015-09-11

The over-collection and habitat destruction of natural Dendrobium populations for their commercial medicinal value has led to these plants being under severe threat of extinction. In addition, many Dendrobium plants are similarly shaped and easily confused during the absence of flowering stages. In the present study, we examined the application of the ITS2 region in barcoding and phylogenetic analyses of Dendrobium species (Orchidaceae). For barcoding, ITS2 regions of 43 samples in Dendrobium were amplified. In combination with sequences from GenBank, the sequences were aligned using Clustal W and genetic distances were computed using MEGA V5.1. The success rate of PCR amplification and sequencing was 100%. There was a significant divergence between the inter- and intra-specific genetic distances of ITS2 regions, while the presence of a barcoding gap was obvious. Based on the BLAST1, nearest distance and TaxonGAP methods, our results showed that the ITS2 regions could successfully identify the species of most Dendrobium samples examined; Second, we used ITS2 as a DNA marker to infer phylogenetic relationships of 64 Dendrobium species. The results showed that cluster analysis using the ITS2 region mainly supported the relationship between the species of Dendrobium established by traditional morphological methods and many previous molecular analyses. To sum up, the ITS2 region can not only be used as an efficient barcode to identify Dendrobium species, but also has the potential to contribute to the phylogenetic analysis of the genus Dendrobium.

Molecular Identification of Dendrobium Species (Orchidaceae) Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study

PubMed Central

Feng, Shangguo; Jiang, Yan; Wang, Shang; Jiang, Mengying; Chen, Zhe; Ying, Qicai; Wang, Huizhong

2015-01-01

The over-collection and habitat destruction of natural Dendrobium populations for their commercial medicinal value has led to these plants being under severe threat of extinction. In addition, many Dendrobium plants are similarly shaped and easily confused during the absence of flowering stages. In the present study, we examined the application of the ITS2 region in barcoding and phylogenetic analyses of Dendrobium species (Orchidaceae). For barcoding, ITS2 regions of 43 samples in Dendrobium were amplified. In combination with sequences from GenBank, the sequences were aligned using Clustal W and genetic distances were computed using MEGA V5.1. The success rate of PCR amplification and sequencing was 100%. There was a significant divergence between the inter- and intra-specific genetic distances of ITS2 regions, while the presence of a barcoding gap was obvious. Based on the BLAST1, nearest distance and TaxonGAP methods, our results showed that the ITS2 regions could successfully identify the species of most Dendrobium samples examined; Second, we used ITS2 as a DNA marker to infer phylogenetic relationships of 64 Dendrobium species. The results showed that cluster analysis using the ITS2 region mainly supported the relationship between the species of Dendrobium established by traditional morphological methods and many previous molecular analyses. To sum up, the ITS2 region can not only be used as an efficient barcode to identify Dendrobium species, but also has the potential to contribute to the phylogenetic analysis of the genus Dendrobium. PMID:26378526

Evaluation of DNA barcode candidates for the discrimination of Artemisia L.

PubMed

Liu, Geyu; Ning, Huixia; Ayidaerhan, Nurbolati; Aisa, Haji Akber

2017-11-01

Because of the very similar morphologies and wide diversity of Artemisia L. varieties, they are difficult to identify, and there have been many arguments about the systematic classification Artemisia L., especially concerning the division of species. DNA barcode technology is used to rapidly identify species based on standard short DNA sequences. To evaluate seven candidate DNA barcodes (ITS, ITS2, psbA-trnH, rbcL, matK, rpoB, and rpoC1) regarding their ability to identify closely related species of the Artemisia genus in Xinjiang. The corresponding PCR amplification efficiency, detectable genetic divergence, identification efficiency and phylogenetic tree were assessed. We found that the internal transcribed spacer (ITS) region exhibited the highest interspecific divergence, which was significantly higher than the observed intraspecific variation and showed the highest identification efficiency, followed by ITS2, psbA-trnH and, finally, rpoB. matK, rbcL, and rpoC1 performed poorly in this evaluation. ITS, ITS2, and psbA-trnH were able to perfectly identify the tested species Artemisia annua, A. absinthium, A. rupestris, A. tonurnefortiana, A. austriaca, A. dracunculus, A. vulgaris, and A. macrocephala. Therefore, we propose the ITS, ITS2, and psbA-trnH regions as promising DNA barcodes for the closely related species of Artemisia L. in Xinjiang.

DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine.

PubMed

Yang, Fan; Ding, Fei; Chen, Hong; He, Mingqi; Zhu, Shixin; Ma, Xin; Jiang, Li; Li, Haifeng

2018-01-01

Animal-based traditional medicine not only plays a significant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difficult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding offers an accurate and efficient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. These techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identification and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine.

DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine

PubMed Central

Yang, Fan; Ding, Fei; Chen, Hong; He, Mingqi; Zhu, Shixin; Ma, Xin; Jiang, Li

2018-01-01

Animal-based traditional medicine not only plays a significant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difficult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding offers an accurate and efficient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. These techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identification and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine. PMID:29849709

Integrative taxonomy supports new candidate fish species in a poorly studied neotropical region: the Jequitinhonha River Basin.

PubMed

Pugedo, Marina Lages; de Andrade Neto, Francisco Ricardo; Pessali, Tiago Casarim; Birindelli, José Luís Olivan; Carvalho, Daniel Cardoso

2016-06-01

Molecular identification through DNA barcoding has been proposed as a way to standardize a global biodiversity identification system using a partial sequence of the mitochondrial COI gene. We applied an integrative approach using DNA barcoding and traditional morphology-based bioassessment to identify fish from a neotropical region possessing low taxonomic knowledge: the Jequitinhonha River Basin (Southeastern Brazil). The Jequitinhonha River Basin (JRB) has a high rate of endemism and is considered an area of high priority for fish conservation, with estimates indicating the presence of around 110 native and non-indigenous species. DNA barcodes were obtained from 260 individuals belonging to 52 species distributed among 35 genera, 21 families and 6 orders, including threatened and rare species such as Rhamdia jequitinhonha and Steindachneridion amblyurum. The mean Kimura two-parameter genetic distances within species, genera and families were: 0.44, 12.16 and 20.58 %, respectively. Mean intraspecific genetic variation ranged from 0 to 11.43 %, and high values (>2 %) were recovered for five species. Species with a deep intraspecific distance, possibly flagging overlooked taxa, were detected within the genus Pimelodella. Fifteen species, only identified to the genus level, had unique BINs, with a nearest neighbor distance over 2 % and therefore, potential new candidate species supported by DNA barcoding. The integrative taxonomy approach using DNA barcoding and traditional taxonomy may be a remedy to taxonomy impediment, accelerating species identification by flagging potential new candidate species and to adequately conserve the megadiverse neotropical ichthyofauna.

Probing planetary biodiversity with DNA barcodes: The Noctuoidea of North America

PubMed Central

Lafontaine, J. Donald; Schmidt, B. Christian; deWaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

This study reports the assembly of a DNA barcode reference library for species in the lepidopteran superfamily Noctuoidea from Canada and the USA. Based on the analysis of 69,378 specimens, the library provides coverage for 97.3% of the noctuoid fauna (3565 of 3664 species). In addition to verifying the strong performance of DNA barcodes in the discrimination of these species, the results indicate close congruence between the number of species analyzed (3565) and the number of sequence clusters (3816) recognized by the Barcode Index Number (BIN) system. Distributional patterns across 12 North American ecoregions are examined for the 3251 species that have GPS data while BIN analysis is used to quantify overlap between the noctuoid faunas of North America and other zoogeographic regions. This analysis reveals that 90% of North American noctuoids are endemic and that just 7.5% and 1.8% of BINs are shared with the Neotropics and with the Palearctic, respectively. One third (29) of the latter species are recent introductions and, as expected, they possess low intraspecific divergences. PMID:28570635

Aspergillus and Penicillium identification using DNA sequences: Barcode or MLST?

USDA-ARS?s Scientific Manuscript database

Current methods in DNA technology can detect single nucleotide polymorphisms with measurable accuracy using several different approaches appropriate for different uses. If there are even single nucleotide differences that are invariant markers of the species, we can accomplish identification through...

Plans and progress for building a Great Lakes fauna DNA barcode reference library

EPA Science Inventory

DNA reference libraries provide researchers with an important tool for assessing regional biodiversity by allowing unknown genetic sequences to be assigned identities, while also providing a means for taxonomists to validate identifications. Expanding the representation of Great...

Barcoding Tetrahymena: discriminating species and identifying unknowns using the cytochrome c oxidase subunit I (cox-1) barcode.

PubMed

Kher, Chandni P; Doerder, F Paul; Cooper, Jason; Ikonomi, Pranvera; Achilles-Day, Undine; Küpper, Frithjof C; Lynn, Denis H

2011-01-01

DNA barcoding using the mitochondrial cytochromecoxidase subunit I (cox-1) gene has recently gained popularity as a tool for species identification of a variety of taxa. The primary objective of our research was to explore the efficacy of using cox-1 barcoding for species identification within the genusTetrahymena. We first increased intraspecific sampling forTetrahymena canadensis, Tetrahymena hegewischi, Tetrahymena pyriformis, Tetrahymena rostrata, Tetrahymena thermophila, and Tetrahymena tropicalis. Increased sampling efforts show that intraspecific sequence divergence is typically less than 1%, though it may be more in some species. The barcoding also showed that some strains might be misidentified or mislabeled. We also used cox-1 barcodes to provide species identifications for 51 unidentified environmental isolates, with a success rate of 98%. Thus, cox-1 barcoding is an invaluable tool for protistologists, especially when used in conjunction with morphological studies. 2010 Elsevier GmbH. All rights reserved.

Use of DNA Barcodes to Identify Invasive Armyworm Spodoptera Species in Florida

PubMed Central

Nagoshi, Rodney N.; Brambila, Julieta; Meagher, Robert L.

2011-01-01

A critical component for sustaining adequate food production is the protection of local agriculture from invasive pest insects. Essential to this goal is the ability to accurately distinguish foreign from closely related domestic species, a process that has traditionally required identification using diagnostic morphological “keys” that can be both subtle and labor-intensive. This is the case for the Lepidopteran group of insects represented by Spodoptera, a genus of Noctuidae “armyworm” moths that includes several important agricultural pests. Two of the most destructive species, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) and S. litura (F.) are not yet established in North America. To facilitate the monitoring for these pests, the feasibility of using DNA barcoding methodology for distinguishing between domestic and foreign Spodoptera species was tested. A DNA barcoding database was derived for a subset of Spodoptera species native to Florida, with an emphasis on those attracted to pheromone blends developed for S. litura or S. littoralis. These were then compared to the barcode sequences of S. litura collected from Taiwan and S. littoralis from Portugal. Consistent discrimination of the different species was obtained with phenetic relationships produced that were generally in agreement with phylogenetic studies using morphological characteristics. The data presented here indicate that DNA barcoding has the potential to be an efficient and accurate supplement to morphological methods for the identification of invasive Spodoptera pests in North America. PMID:22239735

Use of DNA barcodes to identify invasive armyworm Spodoptera species in Florida.

PubMed

Nagoshi, Rodney N; Brambila, Julieta; Meagher, Robert L

2011-01-01

A critical component for sustaining adequate food production is the protection of local agriculture from invasive pest insects. Essential to this goal is the ability to accurately distinguish foreign from closely related domestic species, a process that has traditionally required identification using diagnostic morphological "keys" that can be both subtle and labor-intensive. This is the case for the Lepidopteran group of insects represented by Spodoptera, a genus of Noctuidae "armyworm" moths that includes several important agricultural pests. Two of the most destructive species, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) and S. litura (F.) are not yet established in North America. To facilitate the monitoring for these pests, the feasibility of using DNA barcoding methodology for distinguishing between domestic and foreign Spodoptera species was tested. A DNA barcoding database was derived for a subset of Spodoptera species native to Florida, with an emphasis on those attracted to pheromone blends developed for S. litura or S. littoralis. These were then compared to the barcode sequences of S. litura collected from Taiwan and S. littoralis from Portugal. Consistent discrimination of the different species was obtained with phenetic relationships produced that were generally in agreement with phylogenetic studies using morphological characteristics. The data presented here indicate that DNA barcoding has the potential to be an efficient and accurate supplement to morphological methods for the identification of invasive Spodoptera pests in North America.

Nucleotide Sequence Database Comparison for Routine Dermatophyte Identification by Internal Transcribed Spacer 2 Genetic Region DNA Barcoding.

PubMed

Normand, A C; Packeu, A; Cassagne, C; Hendrickx, M; Ranque, S; Piarroux, R

2018-05-01

Conventional dermatophyte identification is based on morphological features. However, recent studies have proposed to use the nucleotide sequences of the rRNA internal transcribed spacer (ITS) region as an identification barcode of all fungi, including dermatophytes. Several nucleotide databases are available to compare sequences and thus identify isolates; however, these databases often contain mislabeled sequences that impair sequence-based identification. We evaluated five of these databases on a clinical isolate panel. We selected 292 clinical dermatophyte strains that were prospectively subjected to an ITS2 nucleotide sequence analysis. Sequences were analyzed against the databases, and the results were compared to clusters obtained via DNA alignment of sequence segments. The DNA tree served as the identification standard throughout the study. According to the ITS2 sequence identification, the majority of strains (255/292) belonged to the genus Trichophyton , mainly T. rubrum complex ( n = 184), T. interdigitale ( n = 40), T. tonsurans ( n = 26), and T. benhamiae ( n = 5). Other genera included Microsporum (e.g., M. canis [ n = 21], M. audouinii [ n = 10], Nannizzia gypsea [ n = 3], and Epidermophyton [ n = 3]). Species-level identification of T. rubrum complex isolates was an issue. Overall, ITS DNA sequencing is a reliable tool to identify dermatophyte species given that a comprehensive and correctly labeled database is consulted. Since many inaccurate identification results exist in the DNA databases used for this study, reference databases must be verified frequently and amended in line with the current revisions of fungal taxonomy. Before describing a new species or adding a new DNA reference to the available databases, its position in the phylogenetic tree must be verified. Copyright © 2018 American Society for Microbiology.

DNA barcoding using skin exuviates can improve identification and biodiversity studies of snakes.

PubMed

Khedkar, Trupti; Sharma, Rashmi; Tiknaik, Anita; Khedkar, Gulab; Naikwade, Bhagwat S; Ron, Tetsuzan Benny; Haymer, David

2016-01-01

Snakes represent a taxonomically underdeveloped group of animals in India with a lack of experts and incomplete taxonomic descriptions being the main deterrents to advances in this area. Molecular taxonomic approaches using DNA barcoding could aid in snake identification as well as studies of biodiversity. Here a non-invasive sampling method using DNA barcoding is tested using skin exuviates. Taxonomically authenticated samples were collected and tested for validation and comparisons to unknown snake exuviate samples. This approach was also used to construct the first comprehensive study targeting the snake species from Maharashtra state in India. A total of 92 skin exuviate samples were collected and tested for this study. Of these, 81 samples were successfully DNA barcoded and compared with unknown samples for assignment of taxonomic identity. Good quality DNA was obtained irrespective of age and quality of the exuviate material, and all unknown samples were successfully identified. A total of 23 species of snakes were identified, six of which were in the list of Endangered species (Red Data Book). Intra- and inter-specific distance values were also calculated, and these were sufficient to allow discrimination among species and between species without ambiguity in most cases. Two samples were suspected to represent cryptic species based on deep K2P divergence values (>3%), and one sample could be identified to the genus level only. Eleven samples failed to amplify COI sequences, suggesting the need for alternative PCR primer pairs. This study clearly documents how snake skin exuviates can be used for DNA barcoding, estimates of diversity and population genetic structuring in a noninvasive manner.

Fast Census of Moth Diversity in the Neotropics: A Comparison of Field-Assigned Morphospecies and DNA Barcoding in Tiger Moths

PubMed Central

Zenker, Mauricio M.; Rougerie, Rodolphe; Teston, José A.; Laguerre, Michel; Pie, Marcio R.; Freitas, André V. L.

2016-01-01

The morphological species delimitations (i.e. morphospecies) have long been the best way to avoid the taxonomic impediment and compare insect taxa biodiversity in highly diverse tropical and subtropical regions. The development of DNA barcoding, however, has shown great potential to replace (or at least complement) the morphospecies approach, with the advantage of relying on automated methods implemented in computer programs or even online rather than in often subjective morphological features. We sampled moths extensively for two years using light traps in a patch of the highly endangered Atlantic Forest of Brazil to produce a nearly complete census of arctiines (Noctuoidea: Erebidae), whose species richness was compared using different morphological and molecular approaches (DNA barcoding). A total of 1,075 barcode sequences of 286 morphospecies were analyzed. Based on the clustering method Barcode Index Number (BIN) we found a taxonomic bias of approximately 30% in our initial morphological assessment. However, a morphological reassessment revealed that the correspondence between morphospecies and molecular operational taxonomic units (MOTUs) can be up to 94% if differences in genitalia morphology are evaluated in individuals of different MOTUs originated from the same morphospecies (putative cases of cryptic species), and by recording if individuals of different genders in different morphospecies merge together in the same MOTU (putative cases of sexual dimorphism). The results of two other clustering methods (i.e. Automatic Barcode Gap Discovery and 2% threshold) were very similar to those of the BIN approach. Using empirical data we have shown that DNA barcoding performed substantially better than the morphospecies approach, based on superficial morphology, to delimit species of a highly diverse moth taxon, and thus should be used in species inventories. PMID:26859488

Fast Census of Moth Diversity in the Neotropics: A Comparison of Field-Assigned Morphospecies and DNA Barcoding in Tiger Moths.

PubMed

Zenker, Mauricio M; Rougerie, Rodolphe; Teston, José A; Laguerre, Michel; Pie, Marcio R; Freitas, André V L

2016-01-01

The morphological species delimitations (i.e. morphospecies) have long been the best way to avoid the taxonomic impediment and compare insect taxa biodiversity in highly diverse tropical and subtropical regions. The development of DNA barcoding, however, has shown great potential to replace (or at least complement) the morphospecies approach, with the advantage of relying on automated methods implemented in computer programs or even online rather than in often subjective morphological features. We sampled moths extensively for two years using light traps in a patch of the highly endangered Atlantic Forest of Brazil to produce a nearly complete census of arctiines (Noctuoidea: Erebidae), whose species richness was compared using different morphological and molecular approaches (DNA barcoding). A total of 1,075 barcode sequences of 286 morphospecies were analyzed. Based on the clustering method Barcode Index Number (BIN) we found a taxonomic bias of approximately 30% in our initial morphological assessment. However, a morphological reassessment revealed that the correspondence between morphospecies and molecular operational taxonomic units (MOTUs) can be up to 94% if differences in genitalia morphology are evaluated in individuals of different MOTUs originated from the same morphospecies (putative cases of cryptic species), and by recording if individuals of different genders in different morphospecies merge together in the same MOTU (putative cases of sexual dimorphism). The results of two other clustering methods (i.e. Automatic Barcode Gap Discovery and 2% threshold) were very similar to those of the BIN approach. Using empirical data we have shown that DNA barcoding performed substantially better than the morphospecies approach, based on superficial morphology, to delimit species of a highly diverse moth taxon, and thus should be used in species inventories.

Oligonucleotide indexing of DNA barcodes: identification of tuna and other scombrid species in food products.

PubMed

Botti, Sara; Giuffra, Elisabetta

2010-08-23

DNA barcodes are a global standard for species identification and have countless applications in the medical, forensic and alimentary fields, but few barcoding methods work efficiently in samples in which DNA is degraded, e.g. foods and archival specimens. This limits the choice of target regions harbouring a sufficient number of diagnostic polymorphisms. The method described here uses existing PCR and sequencing methodologies to detect mitochondrial DNA polymorphisms in complex matrices such as foods. The reported application allowed the discrimination among 17 fish species of the Scombridae family with high commercial interest such as mackerels, bonitos and tunas which are often present in processed seafood. The approach can be easily upgraded with the release of new genetic diversity information to increase the range of detected species. Cocktail of primers are designed for PCR using publicly available sequences of the target sequence. They are composed of a fixed 5' region and of variable 3' cocktail portions that allow amplification of any member of a group of species of interest. The population of short amplicons is directly sequenced and indexed using primers containing a longer 5' region and the non polymorphic portion of the cocktail portion. A 226 bp region of CytB was selected as target after collection and screening of 148 online sequences; 85 SNPs were found, of which 75 were present in at least two sequences. Primers were also designed for two shorter sub-fragments that could be amplified from highly degraded samples. The test was used on 103 samples of seafood (canned tuna and scomber, tuna salad, tuna sauce) and could successfully detect the presence of different or additional species that were not identified on the labelling of canned tuna, tuna salad and sauce samples. The described method is largely independent of the degree of degradation of DNA source and can thus be applied to processed seafood. Moreover, the method is highly flexible: publicly available sequence information on mitochondrial genomes are rapidly increasing for most species, facilitating the choice of target sequences and the improvement of resolution of the test. This is particularly important for discrimination of marine and aquaculture species for which genome information is still limited.

In vivo generation of DNA sequence diversity for cellular barcoding

PubMed Central

Peikon, Ian D.; Gizatullina, Diana I.; Zador, Anthony M.

2014-01-01

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic ‘barcode’ that can be recovered by DNA sequencing. Our method is a two-component system comprised of a genetic barcode cassette whose fragments are shuffled by Rci, a site-specific DNA invertase. The system is highly scalable, with the potential to generate theoretical diversities in the billions. We demonstrate the feasibility of this technique in Escherichia coli. Currently, this method could be employed to track the dynamics of populations of microbes through various bottlenecks. Advances of this method should prove useful in tracking interactions of cells within a network, and/or heterogeneity within complex biological samples. PMID:25013177

FISH-BOL and seafood identification: geographically dispersed case studies reveal systemic market substitution across Canada.

PubMed

Hanner, Robert; Becker, Sven; Ivanova, Natalia V; Steinke, Dirk

2011-10-01

The Fish Barcode of Life campaign involves a broad international collaboration among scientists working to advance the identification of fishes using DNA barcodes. With over 25% of the world's known ichthyofauna currently profiled, forensic identification of seafood products is now feasible and is becoming routine. Driven by growing consumer interest in the food supply, investigative reporters from five different media establishments procured seafood samples (n = 254) from numerous retail establishments located among five Canadian metropolitan areas between 2008 and 2010. The specimens were sent to the Canadian Centre for DNA Barcoding for analysis. By integrating the results from these individual case studies in a summary analysis, we provide a broad perspective on seafood substitution across Canada. Barcodes were recovered from 93% of the samples (n = 236), and identified using the Barcode of Life Data Systems "species identification" engine ( www.barcodinglife.org ). A 99% sequence similarity threshold was employed as a conservative matching criterion for specimen identification to the species level. Comparing these results against the Canadian Food Inspection Agency's "Fish List" a guideline to interpreting "false, misleading or deceptive" names (as per s 27 of the Fish Inspection regulations) demonstrated that 41% of the samples were mislabeled. Most samples were readily identified; however, this was not true in all cases because some samples had no close match. Others were ambiguous due to limited barcode resolution (or imperfect taxonomy) observed within a few closely related species complexes. The latter cases did not significantly impact the results because even the partial resolution achieved was sufficient to demonstrate mislabeling. This work highlights the functional utility of barcoding for the identification of diverse market samples. It also demonstrates how barcoding serves as a bridge linking scientific nomenclature with approved market names, potentially empowering regulatory bodies to enforce labeling standards. By synchronizing taxonomic effort with sequencing effort and database curation, barcoding provides a molecular identification resource of service to applied forensics.

Incorporating DNA barcodes into a multi-year inventory of the fishes of the hyperdiverse Lower Congo River, with a multi-gene performance assessment of the genus Labeo as a case study.

PubMed

Lowenstein, Jacob H; Osmundson, Todd W; Becker, Sven; Hanner, Robert; Stiassny, Melanie L J

2011-10-01

Here we describe preliminary efforts to integrate DNA barcoding into an ongoing inventory of the Lower Congo River (LCR) ichthyofauna. The 350 km stretch of the LCR from Pool Malebo to Boma includes the world's largest river rapids. The LCR ichthyofauna is hyperdiverse and rich in endemism due to high habitat heterogeneity, numerous dispersal barriers, and its downstream location in the basin. We have documented 328 species from the LCR, 25% of which are thought to be endemic. In addition to detailing progress made to generate a reference sequence library of DNA barcodes for these fishes, we ask how DNA can be used at the current stage of the Fish Barcode of Life initiative, as a work in progress currently of limited utility to a wide audience. Two possibilities that we explore are the potential for DNA barcodes to generate discrete diagnostic characters for species, and to help resolve problematic taxa lacking clear morphologically diagnostic characters such as many species of the cyprinid genus Labeo, which we use as a case study. Our molecular analysis helped to clarify the validity of some species that were the subject of historical debate, and we were able to construct a molecular key for all monophyletic and morphologically recognizable species. Several species sampled from across the Congo Basin and widely distributed throughout Central and West Africa were recovered as paraphyletic based on our molecular data. Our study underscores the importance of generating reference barcodes for specimens collected from, or in close proximity to, type localities, particularly where species are poorly understood taxonomically and the extent of their geographical distributions have yet to be established.

Unexpected diversity of sandflies (Diptera: Psychodidae) in tourist caves in Northern Thailand.

PubMed

Sukantamala, Jedsada; Sing, Kong-Wah; Jaturas, Narong; Polseela, Raxsina; Wilson, John-James

2017-11-01

Certain species of Phlebotomine sandflies (Diptera: Psychodidae) are vectors of the protozoa which causes leishmaniasis. Sandflies are found breeding in enclosed places like caves. Thailand is a popular tourist destination, including for ecotourism activities like caving, which increases the risk of contact between tourists and sandflies. Surveillance of sandflies is important for monitoring this risk but identification of species based on morphology is challenged by phenotypic plasticity and cryptic diversity. DNA barcodes have been used for the identification of sandflies in Thailand. We collected sandflies using CDC light trap from four tourist caves in Northern Thailand. Female sandflies were provisionally sorted into 13 morphospecies and 19 unidentified specimens. DNA was extracted from the thorax and legs of sandflies and the DNA barcode region of cytochrome c oxidase I mtDNA amplified and sequenced. The specimens were sorted into 22 molecular operational taxonomic units (MOTU) based on the 145 DNA barcodes, which is significantly more than the morphospecies. Several of the taxa thought to be present in multiple caves, based on morphospecies sorting, split into cave-specific MOTU which likely represent cryptic species. Several MOTU reported in an earlier study from Wihan Cave, Thailand, were also found in these caves. This supports the use of DNA barcodes to investigate species diversity of sandflies and their useful role in surveillance of sandflies in Thailand.

Deep COI sequencing of standardized benthic samples unveils overlooked diversity of Jordanian coral reefs in the northern Red Sea.

PubMed

Al-Rshaidat, Mamoon M D; Snider, Allison; Rosebraugh, Sydney; Devine, Amanda M; Devine, Thomas D; Plaisance, Laetitia; Knowlton, Nancy; Leray, Matthieu

2016-09-01

High-throughput sequencing (HTS) of DNA barcodes (metabarcoding), particularly when combined with standardized sampling protocols, is one of the most promising approaches for censusing overlooked cryptic invertebrate communities. We present biodiversity estimates based on sequencing of the cytochrome c oxidase subunit 1 (COI) gene for coral reefs of the Gulf of Aqaba, a semi-enclosed system in the northern Red Sea. Samples were obtained from standardized sampling devices (Autonomous Reef Monitoring Structures (ARMS)) deployed for 18 months. DNA barcoding of non-sessile specimens >2 mm revealed 83 OTUs in six phyla, of which only 25% matched a reference sequence in public databases. Metabarcoding of the 2 mm - 500 μm and sessile bulk fractions revealed 1197 OTUs in 15 animal phyla, of which only 4.9% matched reference barcodes. These results highlight the scarcity of COI data for cryptobenthic organisms of the Red Sea. Compared with data obtained using similar methods, our results suggest that Gulf of Aqaba reefs are less diverse than two Pacific coral reefs but much more diverse than an Atlantic oyster reef at a similar latitude. The standardized approaches used here show promise for establishing baseline data on biodiversity, monitoring the impacts of environmental change, and quantifying patterns of diversity at regional and global scales.

DNA in a bottle-Rapid metabarcoding survey for early alerts of invasive species in ports.

PubMed

Borrell, Yaisel J; Miralles, Laura; Do Huu, Hoang; Mohammed-Geba, Khaled; Garcia-Vazquez, Eva

2017-01-01

Biota monitoring in ports is increasingly needed for biosecurity reasons and safeguarding marine biodiversity from biological invasion. Present and future international biosecurity directives can be accomplished only if the biota acquired by maritime traffic in ports is controlled. Methodologies for biota inventory are diverse and now rely principally on extensive and labor-intensive sampling along with taxonomic identification by experts. In this study, we employed an extremely simplified environmental DNA (eDNA) sampling methodology from only three 1-L bottles of water per port, followed by metabarcoding (high-throughput sequencing and DNA-based species identification) using 18S rDNA and Cytochrome oxidase I as genetic barcodes. Eight Bay of Biscay ports with available inventory of fouling invertebrates were employed as a case study. Despite minimal sampling efforts, three invasive invertebrates were detected: the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus and the polychaete Polydora triglanda. The same species have been previously found from visual and DNA barcoding (genetic identification of individuals) surveys in the same ports. The current costs of visual surveys, conventional DNA barcoding and this simplified metabarcoding protocol were compared. The results encourage the use of metabarcoding for early biosecurity alerts.

DNA in a bottle—Rapid metabarcoding survey for early alerts of invasive species in ports

PubMed Central

Miralles, Laura; Do Huu, Hoang; Mohammed-Geba, Khaled; Garcia-Vazquez, Eva

2017-01-01

Biota monitoring in ports is increasingly needed for biosecurity reasons and safeguarding marine biodiversity from biological invasion. Present and future international biosecurity directives can be accomplished only if the biota acquired by maritime traffic in ports is controlled. Methodologies for biota inventory are diverse and now rely principally on extensive and labor-intensive sampling along with taxonomic identification by experts. In this study, we employed an extremely simplified environmental DNA (eDNA) sampling methodology from only three 1-L bottles of water per port, followed by metabarcoding (high-throughput sequencing and DNA-based species identification) using 18S rDNA and Cytochrome oxidase I as genetic barcodes. Eight Bay of Biscay ports with available inventory of fouling invertebrates were employed as a case study. Despite minimal sampling efforts, three invasive invertebrates were detected: the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus and the polychaete Polydora triglanda. The same species have been previously found from visual and DNA barcoding (genetic identification of individuals) surveys in the same ports. The current costs of visual surveys, conventional DNA barcoding and this simplified metabarcoding protocol were compared. The results encourage the use of metabarcoding for early biosecurity alerts. PMID:28873426

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

PubMed Central

Moser, Lindsey A.; Ramirez-Carvajal, Lisbeth; Puri, Vinita; Pauszek, Steven J.; Matthews, Krystal; Dilley, Kari A.; Mullan, Clancy; McGraw, Jennifer; Khayat, Michael; Beeri, Karen; Yee, Anthony; Dugan, Vivien; Heise, Mark T.; Frieman, Matthew B.; Rodriguez, Luis L.; Bernard, Kristen A.; Wentworth, David E.

2016-01-01

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. PMID:27822536

DNA record of some traditional small millet landraces in India and Nepal.

PubMed

Ragupathy, Subramanyam; Dhivya, Shanmughanandhan; Patel, Kirit; Sritharan, Abiran; Sambandan, Kathirvelu; Gartaula, Hom; Sathishkumar, Ramalingam; Khadka, Kamal; Nirmala, Balasubramanian C; Kumari, A Nirmala; Newmaster, Steven G

2016-12-01

Despite the extensive use of small millet landraces as an important source of nutrition for people living in semi-arid regions, they are presently marginalized and their diversity and distribution are threatened at a global scale. Local farmers have developed ancient breeding programs entrenched in traditional knowledge (TK) that has sustained rural cultures for thousands of years. The convention on biological diversity seeks fair and equitable sharing of genetic resources arising from local knowledge and requires signatory nations to provide appropriate policy and legal framework to farmers' rights over plant genetic resources and associated TK. DNA barcoding employed in this study is proposed as a model for conservation of genetic diversity and an essential step towards documenting and protecting farmers' rights and TK. Our study focuses on 32 landraces of small millets that are still used by indigenous farmers located in the rain fed areas of rural India and Nepal. Traditional knowledge of traits and utility was gathered using participatory methods and semi-structured interviews with key informants. DNA was extracted and sequenced (rbcL, trnH-psbA and ITS2) from 160 samples. Both multivariate analysis of traits and phylogenetic analyses were used to assess diversity among small millet landraces. Our research revealed considerable variation in traits and DNA sequences among the 32 small millet landraces. We utilized a tiered approach using ITS2 DNA barcode to make 100 % accurate landrace (32 landraces) and species (six species) assignments for all 160 blind samples in our study. We have also recorded precious TK of nutritional value, ecological and agricultural traits used by local farmers for each of these traditional landraces. This research demonstrates the potential of DNA barcoding as a reliable identification tool and for use in evaluating and conserving genetic diversity of small millets. We suggest ways in which DNA barcodes could be used in the Protection of Plant Varieties and Farmers' Rights in India and Nepal.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

PubMed

Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

2017-03-14

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

NASA Astrophysics Data System (ADS)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

PubMed Central

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding.

PubMed

Lan, Freeman; Demaree, Benjamin; Ahmed, Noorsher; Abate, Adam R

2017-07-01

The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here we present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. We demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. We use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

PubMed Central

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding

PubMed Central

De Ley, Paul; De Ley, Irma Tandingan; Morris, Krystalynne; Abebe, Eyualem; Mundo-Ocampo, Manuel; Yoder, Melissa; Heras, Joseph; Waumann, Dora; Rocha-Olivares, Axayácatl; Jay Burr, A.H; Baldwin, James G; Thomas, W. Kelley

2005-01-01

Molecular surveys of meiofaunal diversity face some interesting methodological challenges when it comes to interstitial nematodes from soils and sediments. Morphology-based surveys are greatly limited in processing speed, while barcoding approaches for nematodes are hampered by difficulties of matching sequence data with traditional taxonomy. Intermediate technology is needed to bridge the gap between both approaches. An example of such technology is video capture and editing microscopy, which consists of the recording of taxonomically informative multifocal series of microscopy images as digital video clips. The integration of multifocal imaging with sequence analysis of the D2D3 region of large subunit (LSU) rDNA is illustrated here in the context of a combined morphological and barcode sequencing survey of marine nematodes from Baja California and California. The resulting video clips and sequence data are made available online in the database NemATOL (http://nematol.unh.edu/). Analyses of 37 barcoded nematodes suggest that these represent at least 32 species, none of which matches available D2D3 sequences in public databases. The recorded multifocal vouchers allowed us to identify most specimens to genus, and will be used to match specimens with subsequent species identifications and descriptions of preserved specimens. Like molecular barcodes, multifocal voucher archives are part of a wider effort at structuring and changing the process of biodiversity discovery. We argue that data-rich surveys and phylogenetic tools for analysis of barcode sequences are an essential component of the exploration of phyla with a high fraction of undiscovered species. Our methods are also directly applicable to other meiofauna such as for example gastrotrichs and tardigrades. PMID:16214752

High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing.

PubMed

Marsic, Damien; Méndez-Gómez, Héctor R; Zolotukhin, Sergei

2015-01-01

Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency.

DNA Barcoding through Quaternary LDPC Codes

PubMed Central

Tapia, Elizabeth; Spetale, Flavio; Krsticevic, Flavia; Angelone, Laura; Bulacio, Pilar

2015-01-01

For many parallel applications of Next-Generation Sequencing (NGS) technologies short barcodes able to accurately multiplex a large number of samples are demanded. To address these competitive requirements, the use of error-correcting codes is advised. Current barcoding systems are mostly built from short random error-correcting codes, a feature that strongly limits their multiplexing accuracy and experimental scalability. To overcome these problems on sequencing systems impaired by mismatch errors, the alternative use of binary BCH and pseudo-quaternary Hamming codes has been proposed. However, these codes either fail to provide a fine-scale with regard to size of barcodes (BCH) or have intrinsic poor error correcting abilities (Hamming). Here, the design of barcodes from shortened binary BCH codes and quaternary Low Density Parity Check (LDPC) codes is introduced. Simulation results show that although accurate barcoding systems of high multiplexing capacity can be obtained with any of these codes, using quaternary LDPC codes may be particularly advantageous due to the lower rates of read losses and undetected sample misidentification errors. Even at mismatch error rates of 10−2 per base, 24-nt LDPC barcodes can be used to multiplex roughly 2000 samples with a sample misidentification error rate in the order of 10−9 at the expense of a rate of read losses just in the order of 10−6. PMID:26492348

DNA Barcoding through Quaternary LDPC Codes.

PubMed

Tapia, Elizabeth; Spetale, Flavio; Krsticevic, Flavia; Angelone, Laura; Bulacio, Pilar

2015-01-01

For many parallel applications of Next-Generation Sequencing (NGS) technologies short barcodes able to accurately multiplex a large number of samples are demanded. To address these competitive requirements, the use of error-correcting codes is advised. Current barcoding systems are mostly built from short random error-correcting codes, a feature that strongly limits their multiplexing accuracy and experimental scalability. To overcome these problems on sequencing systems impaired by mismatch errors, the alternative use of binary BCH and pseudo-quaternary Hamming codes has been proposed. However, these codes either fail to provide a fine-scale with regard to size of barcodes (BCH) or have intrinsic poor error correcting abilities (Hamming). Here, the design of barcodes from shortened binary BCH codes and quaternary Low Density Parity Check (LDPC) codes is introduced. Simulation results show that although accurate barcoding systems of high multiplexing capacity can be obtained with any of these codes, using quaternary LDPC codes may be particularly advantageous due to the lower rates of read losses and undetected sample misidentification errors. Even at mismatch error rates of 10(-2) per base, 24-nt LDPC barcodes can be used to multiplex roughly 2000 samples with a sample misidentification error rate in the order of 10(-9) at the expense of a rate of read losses just in the order of 10(-6).

Two new and one newly recorded species of Polypedilum Kieffer 1912 with DNA barcodes from Oriental China (Chironomidae: Diptera).

PubMed

Yan, Chuncai; Song, Chao; Liu, Ting; Zhao, Guangjun; Hou, Ziyuan; Cao, Wei; Wang, Xinhua

2017-03-02

Polypedilum (Tripodura) enshiense Song & Wang sp. n. and P. (Tripodura) jianfengense Song & Wang sp. n. are described and illustrated as male imagines from China. P. (Uresipedilum) paraconvictum Yamamoto, Yamamoto & Hirowatari, 2016 is recorded from China for the first time. Cytochrome coxidase subunit I (COI) sequence of above species are uploaded to Barcode of Life Data Systems (BOLD).

Identification, Discrimination, and Discovery of Species of Marine Planktonic Ostracods Using DNA Barcodes.

PubMed

Nigro, Lisa M; Angel, Martin V; Blachowiak-Samolyk, Katarzyna; Hopcroft, Russell R; Bucklin, Ann

2016-01-01

The Ostracoda (Crustacea; Class Ostracoda) is a diverse, frequently abundant, and ecologically important component of the marine zooplankton assemblage. There are more than 200 described species of marine planktonic ostracods, many of which (especially conspecific species) can be identified only by microscopic examination and dissection of fragile morphological characters. Given the complexity of species identification and increasing lack of expert taxonomists, DNA barcodes (short DNA sequences for species discrimination and identification) are particularly useful and necessary. Results are reported from analysis of 210 specimens of 78 species of marine planktonic ostracods, including two novel species, and 51 species for which barcodes have not been previously published. Specimens were collected during 2006 to 2008 from the Atlantic, Indian, and Southern Oceans, Greenland Sea and Gulf of Alaska. Samples were collected from surface to 5,000 m using various collection devices. DNA sequence variation was analyzed for a 598 base-pair region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Kimura-2-Parameter (K2P) genetic distances within described species (mean = 0.010 ± 0.017 SD) were significantly smaller than between species (0.260 + 0.080), excluding eight taxa hypothesized to comprise cryptic species due to morphological variation (especially different size forms) and/or collection from different geographic regions. These taxa showed similar K2P distance values within (0.014 + 0.026) and between (0.221 ± 0.068) species. All K2P distances > 0.1 resulted from comparisons between identified or cryptic species, with no overlap between intra- and interspecific genetic distances. A Neighbor Joining tree resolved nearly all described species analyzed, with multiple sequences forming monophyletic clusters with high bootstrap values (typically 99%). Based on taxonomically and geographically extensive sampling and analysis (albeit with small sample sizes), the COI barcode region was shown to be a valuable character for discrimination, recognition, identification, and discovery of species of marine planktonic ostracods.

Identification, Discrimination, and Discovery of Species of Marine Planktonic Ostracods Using DNA Barcodes

PubMed Central

2016-01-01

The Ostracoda (Crustacea; Class Ostracoda) is a diverse, frequently abundant, and ecologically important component of the marine zooplankton assemblage. There are more than 200 described species of marine planktonic ostracods, many of which (especially conspecific species) can be identified only by microscopic examination and dissection of fragile morphological characters. Given the complexity of species identification and increasing lack of expert taxonomists, DNA barcodes (short DNA sequences for species discrimination and identification) are particularly useful and necessary. Results are reported from analysis of 210 specimens of 78 species of marine planktonic ostracods, including two novel species, and 51 species for which barcodes have not been previously published. Specimens were collected during 2006 to 2008 from the Atlantic, Indian, and Southern Oceans, Greenland Sea and Gulf of Alaska. Samples were collected from surface to 5,000 m using various collection devices. DNA sequence variation was analyzed for a 598 base-pair region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Kimura-2-Parameter (K2P) genetic distances within described species (mean = 0.010 ± 0.017 SD) were significantly smaller than between species (0.260 + 0.080), excluding eight taxa hypothesized to comprise cryptic species due to morphological variation (especially different size forms) and/or collection from different geographic regions. These taxa showed similar K2P distance values within (0.014 + 0.026) and between (0.221 ± 0.068) species. All K2P distances > 0.1 resulted from comparisons between identified or cryptic species, with no overlap between intra- and interspecific genetic distances. A Neighbor Joining tree resolved nearly all described species analyzed, with multiple sequences forming monophyletic clusters with high bootstrap values (typically 99%). Based on taxonomically and geographically extensive sampling and analysis (albeit with small sample sizes), the COI barcode region was shown to be a valuable character for discrimination, recognition, identification, and discovery of species of marine planktonic ostracods. PMID:26730595

Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.

PubMed

Li, De-Zhu; Gao, Lian-Ming; Li, Hong-Tao; Wang, Hong; Ge, Xue-Jun; Liu, Jian-Quan; Chen, Zhi-Duan; Zhou, Shi-Liang; Chen, Shi-Lin; Yang, Jun-Bo; Fu, Cheng-Xin; Zeng, Chun-Xia; Yan, Hai-Fei; Zhu, Ying-Jie; Sun, Yong-Shuai; Chen, Si-Yun; Zhao, Lei; Wang, Kun; Yang, Tuo; Duan, Guang-Wen

2011-12-06

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH-psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1-92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9-79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification

PubMed Central

2013-01-01

Background Next-generation-sequencing (NGS) technologies combined with a classic DNA barcoding approach have enabled fast and credible measurement for biodiversity of mixed environmental samples. However, the PCR amplification involved in nearly all existing NGS protocols inevitably introduces taxonomic biases. In the present study, we developed new Illumina pipelines without PCR amplifications to analyze terrestrial arthropod communities. Results Mitochondrial enrichment directly followed by Illumina shotgun sequencing, at an ultra-high sequence volume, enabled the recovery of Cytochrome c Oxidase subunit 1 (COI) barcode sequences, which allowed for the estimation of species composition at high fidelity for a terrestrial insect community. With 15.5 Gbp Illumina data, approximately 97% and 92% were detected out of the 37 input Operational Taxonomic Units (OTUs), whether the reference barcode library was used or not, respectively, while only 1 novel OTU was found for the latter. Additionally, relatively strong correlation between the sequencing volume and the total biomass was observed for species from the bulk sample, suggesting a potential solution to reveal relative abundance. Conclusions The ability of the new Illumina PCR-free pipeline for DNA metabarcoding to detect small arthropod specimens and its tendency to avoid most, if not all, false positives suggests its great potential in biodiversity-related surveillance, such as in biomonitoring programs. However, further improvement for mitochondrial enrichment is likely needed for the application of the new pipeline in analyzing arthropod communities at higher diversity. PMID:23587339

Highlighting Astyanax Species Diversity through DNA Barcoding

PubMed Central

Oliveira, Carlos Alexandre Miranda; de Melo, Filipe Augusto Gonçalves; Bertaco, Vinicius de Araújo; de Astarloa, Juan M. Díaz; Rosso, Juan J.; Foresti, Fausto; Oliveira, Claudio

2016-01-01

DNA barcoding has been used extensively to solve taxonomic questions and identify new species. Neotropical fishes are found in a wide variety of shapes and sizes, with a large number of species yet to be described, many of which are very difficult to identify. Characidae is the most species-rich family of the Characiformes, and many of its genera are affected by taxonomic uncertainties, including the widely-distributed, species-rich genus Astyanax. In this study, we present an extensive analysis of Astyanax covering almost its entire area of occurrence, based on DNA barcoding. The use of different approaches (ABGD, GMYC and BIN) to the clustering of the sequences revealed ample consistency in the results obtained by the initial cutoff value of 2% divergence for putative species in the Neighbor-Joining analysis using the Kimura-2-parameter model. The results indicate the existence of five Astyanax lineages. Some groups, such as that composed by the trans-Andean forms, are mostly composed of well-defined species, and in others a number of nominal species are clustered together, hampering the delimitation of species, which in many cases proved impossible. The results confirm the extreme complexity of the systematics of the genus Astyanax and show that DNA barcoding can be an useful tool to address these complexes questions. PMID:27992537

Evaluation of DNA barcoding and identification of new haplomorphs in Canadian deerflies and horseflies.

PubMed

Cywinska, A; Hannan, M A; Kevan, P G; Roughley, R E; Iranpour, M; Hunter, F F

2010-12-01

This paper reports the first tests of the suitability of the standardized mitochondrial cytochrome c oxidase subunit I (COI) barcoding system for the identification of Canadian deerflies and horseflies. Two additional mitochondrial molecular markers were used to determine whether unambiguous species recognition in tabanids can be achieved. Our 332 Canadian tabanid samples yielded 650 sequences from five genera and 42 species. Standard COI barcodes demonstrated a strong A + T bias (mean 68.1%), especially at third codon positions (mean 93.0%). Our preliminary test of this system showed that the standard COI barcode worked well for Canadian Tabanidae: the target DNA can be easily recovered from small amounts of insect tissue and aligned for all tabanid taxa. Each tabanid species possessed distinctive sets of COI haplotypes which discriminated well among species. Average conspecific Kimura two-parameter (K2P) divergence (0.49%) was 12 times lower than the average divergence within species. Both the neighbour-joining and the Bayesian methods produced trees with identical monophyletic species groups. Two species, Chrysops dawsoni Philip and Chrysops montanus Osten Sacken (Diptera: Tabanidae), showed relatively deep intraspecific sequence divergences (∼ 10 times the average) for all three mitochondrial gene regions analysed. We suggest provisional differentiation of Ch. montanus into two haplotypes, namely, Ch. montanus haplomorph 1 and Ch. montanus haplomorph 2, both defined by their molecular sequences and by newly discovered differences in structural features near their ocelli. © 2010 Brock University. Medical and Veterinary Entomology © 2010 The Royal Entomological Society.

DNA barcoding reveals a cryptic nemertean invasion in Atlantic and Mediterranean waters

NASA Astrophysics Data System (ADS)

Fernández-Álvarez, Fernando Ángel; Machordom, Annie

2013-09-01

For several groups, like nemerteans, morphology-based identification is a hard discipline, but DNA barcoding may help non-experts in the identification process. In this study, DNA barcoding is used to reveal the cryptic invasion of Pacific Cephalothrix cf. simula into Atlantic and Mediterranean coasts. Although DNA barcoding is a promising method for the identification of Nemertea, only 6 % of the known number of nemertean species is currently associated with a correct DNA barcode. Therefore, additional morphological and molecular studies are necessary to advance the utility of DNA barcoding in the characterisation of possible nemertean alien invasions.

Identification of Amazonian trees with DNA barcodes.

PubMed

Gonzalez, Mailyn Adriana; Baraloto, Christopher; Engel, Julien; Mori, Scott A; Pétronelli, Pascal; Riéra, Bernard; Roger, Aurélien; Thébaud, Christophe; Chave, Jérôme

2009-10-16

Large-scale plant diversity inventories are critical to develop informed conservation strategies. However, the workload required for classic taxonomic surveys remains high and is particularly problematic for megadiverse tropical forests. Based on a comprehensive census of all trees in two hectares of a tropical forest in French Guiana, we examined whether plant DNA barcoding could contribute to increasing the quality and the pace of tropical plant biodiversity surveys. Of the eight plant DNA markers we tested (rbcLa, rpoC1, rpoB, matK, ycf5, trnL, psbA-trnH, ITS), matK and ITS had a low rate of sequencing success. More critically, none of the plastid markers achieved a rate of correct plant identification greater than 70%, either alone or combined. The performance of all barcoding markers was noticeably low in few species-rich clades, such as the Laureae, and the Sapotaceae. A field test of the approach enabled us to detect 130 molecular operational taxonomic units in a sample of 252 juvenile trees. Including molecular markers increased the identification rate of juveniles from 72% (morphology alone) to 96% (morphology and molecular) of the individuals assigned to a known tree taxon. We conclude that while DNA barcoding is an invaluable tool for detecting errors in identifications and for identifying plants at juvenile stages, its limited ability to identify collections will constrain the practical implementation of DNA-based tropical plant biodiversity programs.

A new Heraclides swallowtail (Lepidoptera, Papilionidae) from North America is recognized by the pattern on its neck

PubMed Central

Shiraiwa, Kojiro; Cong, Qian; Grishin, Nick V.

2014-01-01

Abstract Heraclides rumiko Shiraiwa & Grishin, sp. n. is described from southwestern United States, Mexico, and Central America (type locality: USA, Texas, Duval County). It is closely allied to Heraclides cresphontes (Cramer, 1777) and the two species are sympatric in central Texas. The new species is diagnosed by male genitalia and exhibits a nearly 3% difference from Heraclides cresphontes in the COI DNA barcode sequence of mitochondrial DNA. The two Heraclides species can usually be told apart by the shape and size of yellow spots on the neck, by the wing shape, and the details of wing patterns. “Western Giant Swallowtail” is proposed as the English name for Heraclides rumiko. To stabilize nomenclature, neotype for Papilio cresphontes Cramer, 1777, an eastern United States species, is designated from Brooklyn, New York, USA; and lectotype for Papilio thoas Linnaeus, 1771 is designated from Suriname. We sequenced DNA barcodes and ID tags of nearly 400 Papilionini specimens completing coverage of all Heraclides species. Comparative analyses of DNA barcodes, genitalia, and facies suggest that Heraclides oviedo (Gundlach, 1866), reinstated status, is a species-level taxon rather than a subspecies of Heraclides thoas (Linnaeus, 1771); and Heraclides pallas (G. Gray, [1853]), reinstated status, with its subspecies Heraclides Papilio bajaensis (J. Brown & Faulkner, 1992), comb. n., and Heraclides anchicayaensis Constantino, Le Crom & Salazar, 2002, stat. n., are not conspecific with Heraclides astyalus (Godart, 1819). PMID:25610342

Tropical Plant–Herbivore Networks: Reconstructing Species Interactions Using DNA Barcodes

PubMed Central

García-Robledo, Carlos; Erickson, David L.; Staines, Charles L.; Erwin, Terry L.; Kress, W. John

2013-01-01

Plants and their associated insect herbivores, represent more than 50% of all known species on earth. The first step in understanding the mechanisms generating and maintaining this important component of biodiversity is to identify plant-herbivore associations. In this study we determined insect-host plant associations for an entire guild of insect herbivores using plant DNA extracted from insect gut contents. Over two years, in a tropical rain forest in Costa Rica (La Selva Biological Station), we recorded the full diet breadth of rolled-leaf beetles, a group of herbivores that feed on plants in the order Zingiberales. Field observations were used to determine the accuracy of diet identifications using a three-locus DNA barcode (rbcL, trnH-psbA and ITS2). Using extraction techniques for ancient DNA, we obtained high-quality sequences for two of these loci from gut contents (rbcL and ITS2). Sequences were then compared to a comprehensive DNA barcode library of the Zingiberales. The rbcL locus identified host plants to family (success/sequence = 58.8%) and genus (success/sequence = 47%). For all Zingiberales except Heliconiaceae, ITS2 successfully identified host plants to genus (success/sequence = 67.1%) and species (success/sequence = 61.6%). Kindt’s sampling estimates suggest that by collecting ca. four individuals representing each plant-herbivore interaction, 99% of all host associations included in this study can be identified to genus. For plants that amplified ITS2, 99% of the hosts can be identified to species after collecting at least four individuals representing each interaction. Our study demonstrates that host plant identifications at the species-level using DNA barcodes are feasible, cost-effective, and reliable, and that reconstructing plant-herbivore networks with these methods will become the standard for a detailed understanding of these interactions. PMID:23308128

What is in your cup of tea? DNA Verity Test to characterize black and green commercial teas

PubMed Central

Comparone, Maria; Di Maio, Antonietta; Del Guacchio, Emanuele; Menale, Bruno; Troisi, Jacopo; Aliberti, Francesco

2017-01-01

In this study, we used several molecular techniques to develop a fast and reliable protocol (DNA Verity Test, DVT) for the characterization and confirmation of the species or taxa present in herbal infusions. As a model plant for this protocol, Camellia sinensis, a traditional tea plant, was selected due to the following reasons: its historical popularity as a (healthy) beverage, its high selling value, the importation of barely recognizable raw product (i.e., crushed), and the scarcity of studies concerning adulterants or contamination. The DNA Verity Test includes both the sequencing of DNA barcoding markers and genotyping of labeled-PCR DNA barcoding fragments for each sample analyzed. This protocol (DVT) was successively applied to verify the authenticity of 32 commercial teas (simple or admixture), and the main results can be summarized as follows: (1) the DVT protocol is suitable to detect adulteration in tea matrices (contaminations or absence of certified ingredients), and the method can be exported for the study of other similar systems; (2) based on the BLAST analysis of the sequences of rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized; (3) rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta; (4) ITS2 is not an ideal DNA barcode for tea samples, reflecting potential incomplete lineage sorting and hybridization/introgression phenomena in C. sinensis taxa; (5) the genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates using the trnH(GUG)-psbA barcoding marker; (6) two herbal companies provided no authentic products with a contaminant or without some of the listed ingredients; and (7) the leaf matrices present in some teabags could be constituted using an admixture of different C. sinensis haplotypes and/or allied species (C. pubicosta). PMID:28542606

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

Barcode index number, taxonomic rank and modes of speciation: examples from fish.

PubMed

Kartavtsev, Yuri Phedorovich

2018-05-01

Species delimitation by DNA sequence data or DNA barcoding is successful, as confirmed by the vast BOLD data base. However, the theory that would explain this fact has not been developed yet. An approach based on Barcoding Index Number (BIN), suggested in the assignment, allows delimiting of taxa of three ranks (species, genera, and families) and statistical validation with a high precision of delimiting (over 80%), as well as shows for majority of Co-1-based single gene trees good correspondence between their topology and conventional taxa content for analyzed fish species (R 2  ≈ 0.84-0.98). Knowledge of deviations from these data can help to find out new taxa and improve biodiversity description. It is concluded that delimiting is successful for bulk of cases because the geographic mode of speciation prevails in nature. It takes a long time for new taxa to form in isolation, which allows accumulation of random mutations and many different nucleotide substitutions between them that can be detected by molecular markers and give unique DNA barcodes. The use of BIN approach, described here, can aid greatly in making this important question clearer especially under wider examination of other organisms.

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.

PubMed

Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

2013-01-01

The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use. © 2012 Blackwell Publishing Ltd.

Combining and Comparing Coalescent, Distance and Character-Based Approaches for Barcoding Microalgaes: A Test with Chlorella-Like Species (Chlorophyta).

PubMed

Zou, Shanmei; Fei, Cong; Song, Jiameng; Bao, Yachao; He, Meilin; Wang, Changhai

2016-01-01

Several different barcoding methods of distinguishing species have been advanced, but which method is the best is still controversial. Chlorella is becoming particularly promising in the development of second-generation biofuels. However, the taxonomy of Chlorella-like organisms is easily confused. Here we report a comprehensive barcoding analysis of Chlorella-like species from Chlorella, Chloroidium, Dictyosphaerium and Actinastrum based on rbcL, ITS, tufA and 16S sequences to test the efficiency of traditional barcoding, GMYC, ABGD, PTP, P ID and character-based barcoding methods. First of all, the barcoding results gave new insights into the taxonomic assessment of Chlorella-like organisms studied, including the clear species discrimination and resolution of potentially cryptic species complexes in C. sorokiniana, D. ehrenbergianum and C. Vulgaris. The tufA proved to be the most efficient barcoding locus, which thus could be as potential "specific barcode" for Chlorella-like species. The 16S failed in discriminating most closely related species. The resolution of GMYC, PTP, P ID, ABGD and character-based barcoding methods were variable among rbcL, ITS and tufA genes. The best resolution for species differentiation appeared in tufA analysis where GMYC, PTP, ABGD and character-based approaches produced consistent groups while the PTP method over-split the taxa. The character analysis of rbcL, ITS and tufA sequences could clearly distinguish all taxonomic groups respectively, including the potentially cryptic lineages, with many character attributes. Thus, the character-based barcoding provides an attractive complement to coalescent and distance-based barcoding. Our study represents the test that proves the efficiency of multiple DNA barcoding in species discrimination of microalgaes.

A DNA Barcoding Method to Discriminate between the Model Plant Brachypodium distachyon and Its Close Relatives B. stacei and B. hybridum (Poaceae)

PubMed Central

López-Alvarez, Diana; López-Herranz, Maria Luisa; Betekhtin, Alexander; Catalán, Pilar

2012-01-01

Background Brachypodium distachyon s. l. has been widely investigated across the world as a model plant for temperate cereals and biofuel grasses. However, this annual plant shows three cytotypes that have been recently recognized as three independent species, the diploids B. distachyon (2n = 10) and B. stacei (2n = 20) and their derived allotetraploid B. hybridum (2n = 30). Methodology/Principal Findings We propose a DNA barcoding approach that consists of a rapid, accurate and automatable species identification method using the standard DNA sequences of complementary plastid (trnLF) and nuclear (ITS, GI) loci. The highly homogenous but largely divergent B. distachyon and B. stacei diploids could be easily distinguished (100% identification success) using direct trnLF (2.4%), ITS (5.5%) or GI (3.8%) sequence divergence. By contrast, B. hybridum could only be unambiguously identified through the use of combined trnLF+ITS sequences (90% of identification success) or by cloned GI sequences (96.7%) that showed 5.4% (ITS) and 4% (GI) rate divergence between the two parental sequences found in the allopolyploid. Conclusion/Significance Our data provide an unbiased and effective barcode to differentiate these three closely-related species from one another. This procedure overcomes the taxonomic uncertainty generated from methods based on morphology or flow cytometry identifications that have resulted in some misclassifications of the model plant and its allies. Our study also demonstrates that the allotetraploid B. hybridum has resulted from bi-directional crosses of B. distachyon and B. stacei plants acting either as maternal or paternal parents. PMID:23240000

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

PubMed Central

2017-01-01

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. PMID:28199790

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

PubMed

MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

2017-03-13

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

Imagining Sisyphus happy: DNA barcoding and the unnamed majority.

PubMed

Blaxter, Mark

2016-09-05

The vast majority of life on the Earth is physically small, and is classifiable as micro- or meiobiota. These organisms are numerically dominant and it is likely that they are also abundantly speciose. By contrast, the vast majority of taxonomic effort has been expended on 'charismatic megabionts': larger organisms where a wealth of morphology has facilitated Linnaean species definition. The hugely successful Linnaean project is unlikely to be extensible to the totality of approximately 10 million species in a reasonable time frame and thus alternative toolkits and methodologies need to be developed. One such toolkit is DNA barcoding, particularly in its metabarcoding or metagenetics mode, where organisms are identified purely by the presence of a diagnostic DNA sequence in samples that are not processed for morphological identification. Building on secure Linnaean foundations, classification of unknown (and unseen) organisms to molecular operational taxonomic units (MOTUs) and deployment of these MOTUs in biodiversity science promises a rewarding resolution to the Sisyphean task of naming all the world's species.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

DNA barcoding of euryglossine bees and the description of new species of Euhesma Michener (Hymenoptera, Colletidae, Euryglossinae)

PubMed Central

Hogendoorn, Katja; Stevens, Mark; Leijs, Remko

2015-01-01

Abstract This paper launches an open access DNA barcoding project “AUSBS” under the Barcoding of Life Datasystems (BOLD). The aims of the project are to help scientists who lack the necessary morphological knowledge to identify known species using molecular markers, to aid native bee specialists with the recognition of species groups that morphologically are difficult to define, and, eventually, to assist with the recognition of new species among known species. Using integrative taxonomy, i.e. morphological comparison to type specimens in Australian museum collections combined with phylogenetic analysis of a fragment of the mitochondrial DNA cytochrome c oxidase subunit I (mtCOI) gene sequences led to the recognition of four new species of Euhesma Michener (Hymenoptera: Colletidae: Euryglossini) collected during intensive surveys in remote Australian conservation areas, which are described. The new species are Euhesma micans, Euhesma lyngouriae, and Euhesma aulaca in a species group associated with Eremophila flowers, and Euhesma albamala in the walkeriana species group. PMID:26448713

DNA barcoding of marine ornamental fishes from India.

PubMed

Bamaniya, Dhaval C; Pavan-Kumar, A; Gireesh-Babu, P; Sharma, Niti; Reang, Dhalongsaih; Krishna, Gopal; Lakra, W S

2016-09-01

India has rich marine ornamental fish diversity with 400 fish species distributed in Gulf of Munnar/Palk Bay, Gulf of Kutch, and in reefs around Andaman & Nicobar and Lakshadweep Islands. Marine ornamental fish identification at the field level is very difficult because of their high diversity and profound changes in appearance during their developmental stages and camouflage. To facilitate ornamental fish trading with ease and in compliance with the biodiversity act, DNA barcoding technique could be used to accurately identify species. In this study, DNA barcodes were generated for 31 species of commercially important marine ornamental fishes from India. The average genetic distance (K2P model) within species, genus, and family was 0.446, 13.08, and 20.09%, respectively. Intraspecific variation has increased several folds (15-20 times) after including conspecific sequences from different geographical locations. The presence of allopatric lineages/cryptic species was observed in the Indo-pacific region. The NJ tree constructed based on K2P values showed distinct clusters shared by congeneric species specific to populations.

Barcoded NS31/AML2 primers for sequencing of arbuscular mycorrhizal communities in environmental samples1

PubMed Central

Morgan, Benjamin S. T.; Egerton-Warburton, Louise M.

2017-01-01

Premise of the study: Arbuscular mycorrhizal fungi (AMF) are globally important root symbioses that enhance plant growth and nutrition and influence ecosystem structure and function. To better characterize levels of AMF diversity relevant to ecosystem function, deeper sequencing depth in environmental samples is needed. In this study, Illumina barcoded primers and a bioinformatics pipeline were developed and applied to study AMF diversity and community structure in environmental samples. Methods: Libraries of small subunit ribosomal RNA fragment amplicons were amplified from environmental DNA using a single-step PCR reaction with barcoded NS31/AML2 primers. Amplicons were sequenced on an Illumina MiSeq sequencer using version 2, 2 × 250-bp paired-end chemistry, and analyzed using QIIME and RDP Classifier. Results: Sequencing captured 196 to 6416 operational taxonomic units (OTUs; depending on clustering parameters) representing nine AMF genera. Regardless of clustering parameters, ∼20 OTUs dominated AMF communities (78–87% reads) with the remaining reads distributed among other OTUs. Analyses also showed significant biogeographic differences in AMF communities and that community composition could be linked to specific edaphic factors. Discussion: Barcoded NS31/AML2 primers and Illumina MiSeq sequencing provide a powerful approach to address AMF diversity and variations in fungal assemblages across host plants, ecosystems, and responses to environmental drivers including global change. PMID:28924511

DNA barcode based wildlife forensics for resolving the origin of claw samples using a novel primer cocktail.

PubMed

Khedkar, Gulab D; Abhayankar, Shil Bapurao; Nalage, Dinesh; Ahmed, Shaikh Nadeem; Khedkar, Chandraprakash D

2016-11-01

Excessive wildlife hunting for commercial purposes can have negative impacts on biodiversity and may result in species extinction. To ensure compliance with legal statutes, forensic identification approaches relying on molecular markers may be used to identify the species of origin of animal material from hairs, claw, blood, bone, or meat. Using this approach, DNA sequences from the COI "barcoding" gene have been used to identify material from a number of domesticated animal species. However, many wild species of carnivores still present great challenges in generating COI barcodes using standard "universal" primer pairs. In the work presented here, the mitochondrial COI gene was successfully amplified using a novel primer cocktail, and the products were sequenced to determine the species of twenty one unknown samples of claw material collected as part of forensic wildlife case investigations. Sixteen of the unknown samples were recognized to have originated from either Panthera leo or P. pardus individuals. The remaining five samples could be identified only to the family level due to the absence of reference animal sequences. This is the first report on the use of COI sequences for the identification of P. pardus and P. leo from claw samples as part of forensic investigations in India. The study also highlights the need for adequate reference material to aid in the resolution of suspected cases of illegal wildlife harvesting.

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

PubMed

An, Jeung Hee; Kim, Tae-Hyung; Oh, Byung-Keun; Choi, Jeong Woo

2012-01-01

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

Discriminating plants using the DNA barcode rbcLb: an appraisal based on a large data set.

PubMed

Dong, Wenpan; Cheng, Tao; Li, Changhao; Xu, Chao; Long, Ping; Chen, Chumming; Zhou, Shiliang

2014-03-01

The ideal DNA barcode for plants remains to be discovered, and the candidate barcode rbcL has been met with considerable skepticism since its proposal. In fact, the variability within this gene has never been fully explored across all plant groups from algae to flowering plants, and its performance as a barcode has not been adequately tested. By analysing all of the rbcL sequences currently available in GenBank, we attempted to determine how well a region of rbcL performs as a barcode in species discrimination. We found that the rbcLb region was more variable than the frequently used rbcLa region. Both universal and plant group-specific primers were designed to amplify rbcLb, and the performance of rbcLa and rbcLb was tested in several ways. Using blast, both regions successfully identified all families and nearly all genera; however, the successful species identification rates varied significantly among plant groups, ranging from 24.58% to 85.50% for rbcLa and from 36.67% to 90.89% for rbcLb. Successful species discrimination ranged from 5.19% to 96.33% for rbcLa and from 22.09% to 98.43% for rbcLb in species-rich families, and from 0 to 88.73% for rbcLa and from 2.04% to 100% for rbcLb in species-rich genera. Both regions performed better for lower plants than for higher plants, although rbcLb performed significantly better than rbcLa overall, particularly for angiosperms. Considering the applicability across plants, easy and unambiguous alignment, high primer universality, high sequence quality and high species discrimination power for lower plants, we suggest rbcLb as a universal plant barcode. © 2013 John Wiley & Sons Ltd.

DNA barcoding in the media: does coverage of cool science reflect its social context?

PubMed

Geary, Janis; Camicioli, Emma; Bubela, Tania

2016-09-01

Paul Hebert and colleagues first described DNA barcoding in 2003, which led to international efforts to promote and coordinate its use. Since its inception, DNA barcoding has generated considerable media coverage. We analysed whether this coverage reflected both the scientific and social mandates of international barcoding organizations. We searched newspaper databases to identify 900 English-language articles from 2003 to 2013. Coverage of the science of DNA barcoding was highly positive but lacked context for key topics. Coverage omissions pose challenges for public understanding of the science and applications of DNA barcoding; these included coverage of governance structures and issues related to the sharing of genetic resources across national borders. Our analysis provided insight into how barcoding communication efforts have translated into media coverage; more targeted communication efforts may focus media attention on previously omitted, but important topics. Our analysis is timely as the DNA barcoding community works to establish the International Society for the Barcode of Life.

A streamlined collecting and preparation protocol for DNA barcoding of Lepidoptera as part of large-scale rapid biodiversity assessment projects, exemplified by the Indonesian Biodiversity Discovery and Information System (IndoBioSys).

PubMed

Schmidt, Olga; Hausmann, Axel; Cancian de Araujo, Bruno; Sutrisno, Hari; Peggie, Djunijanti; Schmidt, Stefan

2017-01-01

Here we present a general collecting and preparation protocol for DNA barcoding of Lepidoptera as part of large-scale rapid biodiversity assessment projects, and a comparison with alternative preserving and vouchering methods. About 98% of the sequenced specimens processed using the present collecting and preparation protocol yielded sequences with more than 500 base pairs. The study is based on the first outcomes of the Indonesian Biodiversity Discovery and Information System (IndoBioSys). IndoBioSys is a German-Indonesian research project that is conducted by the Museum für Naturkunde in Berlin and the Zoologische Staatssammlung München, in close cooperation with the Research Center for Biology - Indonesian Institute of Sciences (RCB-LIPI, Bogor).

Combining and Comparing Coalescent, Distance and Character-Based Approaches for Barcoding Microalgaes: A Test with Chlorella-Like Species (Chlorophyta)

PubMed Central

Zou, Shanmei; Fei, Cong; Song, Jiameng; Bao, Yachao; He, Meilin; Wang, Changhai

2016-01-01

Several different barcoding methods of distinguishing species have been advanced, but which method is the best is still controversial. Chlorella is becoming particularly promising in the development of second-generation biofuels. However, the taxonomy of Chlorella–like organisms is easily confused. Here we report a comprehensive barcoding analysis of Chlorella-like species from Chlorella, Chloroidium, Dictyosphaerium and Actinastrum based on rbcL, ITS, tufA and 16S sequences to test the efficiency of traditional barcoding, GMYC, ABGD, PTP, P ID and character-based barcoding methods. First of all, the barcoding results gave new insights into the taxonomic assessment of Chlorella-like organisms studied, including the clear species discrimination and resolution of potentially cryptic species complexes in C. sorokiniana, D. ehrenbergianum and C. Vulgaris. The tufA proved to be the most efficient barcoding locus, which thus could be as potential “specific barcode” for Chlorella-like species. The 16S failed in discriminating most closely related species. The resolution of GMYC, PTP, P ID, ABGD and character-based barcoding methods were variable among rbcL, ITS and tufA genes. The best resolution for species differentiation appeared in tufA analysis where GMYC, PTP, ABGD and character-based approaches produced consistent groups while the PTP method over-split the taxa. The character analysis of rbcL, ITS and tufA sequences could clearly distinguish all taxonomic groups respectively, including the potentially cryptic lineages, with many character attributes. Thus, the character-based barcoding provides an attractive complement to coalescent and distance-based barcoding. Our study represents the test that proves the efficiency of multiple DNA barcoding in species discrimination of microalgaes. PMID:27092945

DNA barcoding reveals species level divergence between populations of the microhylid frog genus Arcovomer (Anura: Microhylidae) in the Atlantic Rainforest of southeastern Brazil.

PubMed

Jennings, W Bryan; Wogel, Henrique; Bilate, Marcos; Salles, Rodrigo de O L; Buckup, Paulo A

2016-09-01

The microhylid frogs belonging to the genus Arcovomer have been reported from lowland Atlantic Rainforest in the Brazilian states of Espírito Santo, Rio de Janeiro, and São Paulo. Here, we use DNA barcoding to assess levels of genetic divergence between apparently isolated populations in Espírito Santo and Rio de Janeiro. Our mtDNA data consisting of cytochrome oxidase subunit I (COI) nucleotide sequences reveals 13.2% uncorrected and 30.4% TIM2 + I + Γ corrected genetic divergences between these two populations. This level of divergence exceeds the suggested 10% uncorrected divergence threshold for elevating amphibian populations to candidate species using this marker, which implies that the Espírito Santo population is a species distinct from Arcovomer passarellii. Calibration of our model-corrected sequence divergence estimates suggests that the time of population divergence falls between 12 and 29 million years ago.

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.)

PubMed Central

Hartvig, Ida; Czako, Mihaly; Kjær, Erik Dahl; Nielsen, Lene Rostgaard; Theilade, Ida

2015-01-01

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions. PMID:26375850

Integrative taxonomy of Leptonetela spiders (Araneae, Leptonetidae), with descriptions of 46 new species

PubMed Central

Wang, Chun-Xia; Xu, Xin; Li, Shu-Qiang

2017-01-01

Extreme environments, such as subterranean habitats, are suspected to be responsible for morphologically inseparable cryptic or sibling species and can bias biodiversity assessment. A DNA barcode is a short, standardized DNA sequence used for taxonomic purposes and has the potential to lessen the challenges presented by a biotic inventory. Here, we investigate the diversity of the genus Leptonetela Kratochvíl, 1978 that is endemic to karst systems in Eurasia using DNA barcoding. We analyzed 624 specimens using one mitochondrial gene fragment (COI). The results show that DNA barcoding is an efficient and rapid species identification method in this genus. DNA barcoding gap and automatic barcode gap discovery (ABGD) analyses indicated the existence of 90 species, a result consistent with previous taxonomic hypotheses, and supported the existence of extreme male pedipalpal tibial spine and median apophysis polymorphism in Leptonetela species, with direct implications for the taxonomy of the group and its diversity. Based on the molecular and morphological evidence, we delimit and diagnose 90 Leptonetela species, including the type species Leptonetela kanellisi(Deeleman-Reinhold, 1971). Forty-six of them are previously undescribed. The female of Leptonetela zhai Wang & Li, 2011 is reported for the first time. Leptonetela tianxinensis (Tong & Li, 2008) comb. nov. is transferred from the genus Leptoneta Simon, 1872;the genus Guineta Lin & Li, 2010 syn. nov. is a junior synonym of Leptonetela; Leptonetela gigachela(Lin & Li, 2010) comb. nov. is transferred from Guineta. The genus Sinoneta Lin & Li, 2010 syn. nov. is a junior synonym of Leptonetela; Leptonetela notabilis(Lin & Li, 2010) comb. nov. and Leptonetela sexdigiti(Lin & Li, 2010) comb. nov. are transferred from Sinoneta; Leptonetela sanchahe Wang & Li nom. nov. is proposed as a replacement name for Sinoneta palmata(Chen et al, 2010) because Leptonetela palmata is preoccupied. PMID:29280363

Integrative taxonomy of Leptonetela spiders (Araneae, Leptonetidae), with descriptions of 46 new species.

PubMed

Wang, Chun-Xia; Xu, Xin; Li, Shu-Qiang

2017-11-18

Extreme environments, such as subterranean habitats, are suspected to be responsible for morphologically inseparable cryptic or sibling species and can bias biodiversity assessment. A DNA barcode is a short, standardized DNA sequence used for taxonomic purposes and has the potential to lessen the challenges presented by a biotic inventory. Here, we investigate the diversity of the genus Leptonetela Kratochvíl, 1978 that is endemic to karst systems in Eurasia using DNA barcoding. We analyzed 624 specimens using one mitochondrial gene fragment ( COI ). The results show that DNA barcoding is an efficient and rapid species identification method in this genus. DNA barcoding gap and automatic barcode gap discovery (ABGD) analyses indicated the existence of 90 species, a result consistent with previous taxonomic hypotheses, and supported the existence of extreme male pedipalpal tibial spine and median apophysis polymorphism in Leptonetela species, with direct implications for the taxonomy of the group and its diversity. Based on the molecular and morphological evidence, we delimit and diagnose 90 Leptonetela species, including the type species Leptonetela kanellisi (Deeleman-Reinhold, 1971). Forty-six of them are previously undescribed. The female of Leptonetela zhai Wang & Li, 2011 is reported for the first time. Leptonetela tianxinensis (Tong & Li, 2008) comb. nov. is transferred from the genus Leptoneta Simon, 1872; the genus Guineta Lin & Li, 2010 syn. nov. is a junior synonym of Leptonetela; Leptonetela gigachela (Lin & Li, 2010) comb. nov. is transferred from Guineta . The genus Sinoneta Lin & Li, 2010 syn. nov. is a junior synonym of Leptonetela ; Leptonetela notabilis (Lin & Li, 2010) comb. nov. and Leptonetela sexdigiti (Lin & Li, 2010) comb. nov. are transferred from Sinoneta ; Leptonetela sanchahe Wang & Li nom. nov. is proposed as a replacement name for Sinoneta palmata (Chen et al., 2010) because Leptonetela palmata is preoccupied.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Use of rbcL and trnL-F as a Two-Locus DNA Barcode for Identification of NW-European Ferns: An Ecological Perspective

PubMed Central

de Groot, G. Arjen; During, Heinjo J.; Maas, Jan W.; Schneider, Harald; Vogel, Johannes C.; Erkens, Roy H. J.

2011-01-01

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes—while playing an essential role in fern colonization and reproduction—generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology. PMID:21298108

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.