UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi

Identification of ectomycorrhizal (ECM) fungi is often achieved through comparisons of ribosomal DNA internal transcribed spacer (ITS) sequences with accessioned sequences deposited in public databases. A major problem encountered is that annotation of the sequences in these databases is not always complete or trustworthy. In order to overcome this deficiency, we report on UNITE, an open-access database. UNITE comprises well annotated fungal ITS sequences from well defined herbarium specimens that include full herbarium reference identification data, collector/source and ecological data. At present UNITE contains 758 ITS sequences from 455 species and 67 genera of ECM fungi. UNITE can be searched by taxon name, via sequence similarity using blastn, and via phylogenetic sequence identification using galaxie. Following implementation, galaxie performs a phylogenetic analysis of the query sequence after alignment either to pre-existing generic alignments, or to matches retrieved from a blast search on the UNITE data. It should be noted that the current version of UNITE is dedicated to the reliable identification of ECM fungi. The UNITE database is accessible through the URL http://unite.zbi.ee     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

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

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.     

A comprehensive and validated molecular taxonomy of beaked whales, family Ziphiidae

DNA sequences from orthologous loci can provide universal characters for taxonomic identification. Molecular taxonomy is of particular value for groups in which distinctive morphological features are difficult to observe or compare. To assist in species identification for the little known family Ziphiidae (beaked whales), we compiled a reference database of mitochondrial DNA (mtDNA) control region (437 bp) and cytochrome b (384 bp) sequences for all 21 described species in this group. This mtDNA database is complemented by a nuclear database of actin intron sequences (925 bp) for 17 of the 21 species. All reference sequences were derived from specimens validated by diagnostic skeletal material or other documentation, and included four holotypes. Phylogenetic analyses of mtDNA sequences confirmed the genetic distinctiveness of all beaked whale species currently recognized. Both mitochondrial loci were well suited for species identification, with reference sequences for all known ziphiids forming robust species-specific clades in phylogenetic reconstructions. The majority of species were also distinguished by nuclear alleles. Phylogenetic comparison of sequence data from "test" specimens to these reference databases resulted in three major taxonomic discoveries involving animals previously misclassified from morphology. Based on our experience with this family and the order Cetacea as a whole, we suggest that a molecular taxonomy should consider the following components: comprehensiveness, validation, locus sensitivity, genetic distinctiveness and exclusivity, concordance, and universal accessibility and curation.     

我国植物检疫性菌物数据库平台的建设及应用

近年来，随着我国对外贸易的不断增长，口岸检测样品量巨大，外来检疫性有害生物尤其是病原菌物传入我国的风险日趋增加，正成为我国国门生物安全的重要威胁。加强外来入侵菌物的防御能力建设，有效防范其造成的生物安全威胁迫在眉睫。由于外来入侵菌物各类群的基础研究薄弱，标准参比物质缺乏、基础数据和可检索数据库缺失，大部分物种缺乏准确、高效的鉴定手段，使得现有口岸菌物检疫存在准确性较低、速度较慢、误检漏检率较高等问题。针对上述问题，以我国进境检疫对象及主要进口农林作物上的高频检出但鉴定困难、误检率高的菌物类群为对象，建立了其标准参比物质库、形态特征信息库、多基因序列数据库，并通过整合多个信息库资源实现从检疫样品中初筛到物种精准鉴定的多模块服务平台www.casbrc.org/pqfungi，并开放共享。该数据库平台的应用有望促进我国口岸检疫部门的检测便利化水平大幅提升，在“智能海关”建设、维护国门生物安全及促进农林产品安全贸易中发挥重要作用。     

基于涉案兽类12S rRNA基因的NCBI数据库序列可靠性分析

本研究以常用于动物种属鉴定的12S rRNA基因位点为研究对象,利用所测得的17种常见涉案兽类12S rRNA基因部分片段序列及NCBI数据库中下载的该物种DNA序列及其近缘物种DNA序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从17种常见涉案兽类(共26份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的12S rRNA基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的12S rRNA基因序列共351条,利用MEGA7.0软件构建该物种及其近缘物种系统进化树。通过比对发现NCBI中登录号为KP202279等3个序列所对应物种拉丁名错误。登录号为AY184436等11个序列所对应物种拉丁名可能存在疑问。GenBank中某些物种拉丁名有同种异名现象。因此,NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。     

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens

AIMS: In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. METHODS AND RESULTS: For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. CONCLUSIONS: Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.     

YPL.db: the Yeast Protein Localization database

The Yeast Protein Localization database (YPL.db) contains information about the localization patterns of yeast proteins resulting from microscopic analyses. The data and parameters of the experiments to obtain the localization information, together with images from confocal or video microscopy, are stored in a relational database, building an archive of, and the documentation for, all experiments. The database can be queried based on gene name, protein localization, growth conditions and a number of additional parameters. All experiment parameters are selectable from predefined lists to ensure database integrity and conformity across different investigators. The database provides a structure reference resource to allow for better characterization of unknown or ambiguous localization patterns. Links to MIPS, YPD and SGD databases are provided to allow fast access to further information not contained in the localization database itself. YPL.db is available at http://ypl.tugraz.at.     

应用DNA条形码对植物标本的核查

DNA条形码主要目的是物种鉴定和新物种或隐存种的发现,而DNA条形码参考数据库是物种快速鉴定的重要基础。目前中国维管植物DNA条形码参考数据库正在建设之中,借助于公共数据库（NCBI）和初步建立的中国植物DNA条形码参考数据库,运用DNA条形码数据开展了植物标本鉴定的核查工作：（1）比较DNA序列信息与标本鉴定信息,从科、属、种级水平查找鉴定错误的标本;（2）基于有较好研究基础的DNA条形码参考数据库,开展未知标本的鉴定;（3）通过对标本核查的总结,提出DNA条形码参考数据库建设过程中的几点建议。     

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

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/     

Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals

Sequence-based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high-scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high-quality reference data can improve the reliability of sequence-based species identification.     

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

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.     

An assessment of the DNA barcodes of Indian freshwater fishes

Freshwater fishes in India are poorly known and plagued by many unresolved cryptic species complexes that masks some latent and endemic species. Limitations in traditional taxonomy have resulted in this crypticism. Hence, molecular approaches like DNA barcoding, are needed to diagnose these latent species. We have analyzed 1383 barcode sequences of 175 Indian freshwater fish species available in the databases, of which 172 sequences of 70 species were generated. The congeneric and conspecific genetic divergences were calculated using Kimura's 2 parameter distance model followed by the construction of a Neighbor Joining tree using the MEGA 5.1. DNA barcoding principle at its first hand approach, led to the straightforward identification of 82% of the studied species with 2.9% (S.E = 0.2) divergence between the nearest congeners. However, after validating some cases of synonymy and mislabeled sequences, 5% more species were found to be valid. Sequences submitted to the database under different names were found to represent single species. On the other hand, some sequences of the species like Barilius barna, Barilius bendelisis and Labeo bata were submitted to the database under a single name but were found to represent either some unexplored species or latent species. Overall, 87% of the available Indian freshwater fish barcodes were diagnosed as true species in parity with the existing checklist and can act as reference barcode for the particular taxa. For the remaining 13% (21 species) the correct species name was difficult to assign as they depicted some erroneous identification and cryptic species complex. Thus, these barcodes will need further assay and inclusion of barcodes of more specimens from same and sister species.     

Fish Karyome: A karyological information network database of Indian Fishes

'Fish Karyome', a database on karyological information of Indian fishes have been developed that serves as central source for karyotype data about Indian fishes compiled from the published literature. Fish Karyome has been intended to serve as a liaison tool for the researchers and contains karyological information about 171 out of 2438 finfish species reported in India and is publically available via World Wide Web. The database provides information on chromosome number, morphology, sex chromosomes, karyotype formula and cytogenetic markers etc. Additionally, it also provides the phenotypic information that includes species name, its classification, and locality of sample collection, common name, local name, sex, geographical distribution, and IUCN Red list status. Besides, fish and karyotype images, references for 171 finfish species have been included in the database. Fish Karyome has been developed using SQL Server 2008, a relational database management system, Microsoft's ASP.NET-2008 and Macromedia's FLASH Technology under Windows 7 operating environment. The system also enables users to input new information and images into the database, search and view the information and images of interest using various search options. Fish Karyome has wide range of applications in species characterization and identification, sex determination, chromosomal mapping, karyo-evolution and systematics of fishes.     

Protein identification in DNA databases by peptide mass fingerprinting.

Proteins can be identified using a set of peptide fragment weights produced by a specific digestion to search a protein database in which sequences have been replaced by fragment weights calculated for various cleavage methods. We present a method using multidimensional searches that greatly increases the confidence level for identification, allowing DNA sequence databases to be examined. This method provides a link between 2-dimensional gel electrophoresis protein databases and genome sequencing projects. Moreover, the increased confidence level allows unknown proteins to be matched to expressed sequence tags, potentially eliminating the need to obtain sequence information for cloning. Database searching from a mass profile is offered as a free service by an automatic server at the ETH, Zürich. For information, send an electronic message to the address cbrg/inf.ethz.ch with the line: help mass search, or help all.     

Sequence of specific mitochondrial 16S rRNA gene fragment from Egyptian buffalo is used as a pattern for discrimination between river buffaloes,cattle, sheep and goats

Characterization of molecular markers and the development of better assays for precise and rapid detection of domestic species are always in demand. This is particularly due to recent food scares and the crisis of biodiversity resulting from the huge ongoing illegal traffic of endangered species. The aim of this study was to develop a new and easy method for domestic species identification (river buffalo, cattle, sheep and goat) based on the analysis of a specific mitochondrial nucleotide sequence. For this reason, a specific fragment of Egyptian buffalo mitochondrial 16S rRNA gene (422 bp) was amplified by PCR using two universal primers. The sequence of this specific fragment is completely conserved between all tested Egyptian buffaloes and other river buffaloes in different places in the world. Also, the lengths of the homologous fragments were less by one nucleotide (421 bp) in case of goats and two nucleotides (420 bp) in case of both cattle and sheep. The detection of specific variable sites between investigated species within this fragment was sufficient to identify the biological origin of the samples. This was achieved by alignment between the unknown homologous sequence and the reference sequences deposited in GenBank database (accession numbers, FJ748599–FJ748607). Considering multiple alignment results between 16S rRNA homologous sequences obtained from GenBank database with the reference sequence, it was shown that definite nucleotides are specific for each of the four studied species of the family Bovidae. In addition, other nucleotides are detected which can allow discrimination between two groups of animals belonging to two subfamilies of family Bovidae, Group one (closely related species like cattle and buffalo, Subfamily Bovinae) and Group two (closely related species like sheep and goat, Subfamily Caprinae). This 16S DNA barcode character-based approach could be used to complement cytochrome c oxidase I (COI) in DNA barcoding. Also, it is a good tool for identification of unknown sample belonging to one of the four domestic animal species of family Bovidae quickly and easily.     

A fuzzy-set-theory-based approach to analyse species membership in DNA barcoding

Reliable assignment of an unknown query sequence to its correct species remains a methodological problem for the growing field of DNA barcoding. While great advances have been achieved recently, species identification from barcodes can still be unreliable if the relevant biodiversity has been insufficiently sampled. We here propose a new notion of species membership for DNA barcoding-fuzzy membership, based on fuzzy set theory-and illustrate its successful application to four real data sets (bats, fishes, butterflies and flies) with more than 5000 random simulations. Two of the data sets comprise especially dense species/population-level samples. In comparison with current DNA barcoding methods, the newly proposed minimum distance (MD) plus fuzzy set approach, and another computationally simple method, 'best close match', outperform two computationally sophisticated Bayesian and BootstrapNJ methods. The new method proposed here has great power in reducing false-positive species identification compared with other methods when conspecifics of the query are absent from the reference database.