Standard DNA Sequencing Research Articles

Molecular tools for investigating immunohaematology problems

Blood types are classically determined by haemagglutination methods. However, several backgrounds represent limitations for serological techniques, which may be overcome by molecular testing, for example blood typing in recently transfused patients or in case of a positive IgG direct antiglobulin test when indirect antiglobulin testing is required for antisera. A multitude of molecular tools are available and can be separated in five groups: low‐throughput tests, medium‐ to high‐throughput assays, genomic DNA sequencing, high‐throughput DNA sequencing devices and other specialized techniques. The low‐throughput tests especially include PCR‐restriction fragment length polymorphism (RFLP) and manual allele‐specific PCR techniques. The medium‐to high‐throughput systems, mainly real‐time PCR, microarray DNA chips and mass spectrometry, allow for the testing of multiple different SNPs. The so‐called ‘next‐generation sequencing’ technologies can analyse 10 Mb to 1000 Gb per run. Their implementation in transfusion medicine is currently developed by a few teams worldwide, and they may become a major investigating tool in the next future. Standard genomic DNA sequencing may be used when genotyping assays are unavailable or when screening for common mutations by genotyping devices is inconclusive. Specialized techniques, reserved for complex cases or research, are performed after messenger RNA extraction, followed by reverse transcription into complementary DNA, which may be directly sequenced or cloned with plasmid vectors for single‐allele sequencing. Finally, electronic databases are also very helpful tools in molecular immunohaematology, for example the resources from the National Center for Biotechnology Information (NCBI), Rhesus Base and the ISBT Website of the Working Party on Red Cell Immunogenetics and Blood Group Terminology.

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

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.

DNA barcoding for efficient identification of Ixiolirion species (Ixioliriaceae).

Ixiolirion is a genus of unresolved taxonomy. DNA barcoding is a technique that allows species identification using standardized DNA sequences. In this study, a total of 23 individuals, representing 2 Chinese Ixiolirion species, were sampled to test the effectiveness of 3 DNA barcodes [internal transcribed spacer (ITS), chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase] for species identification. Of the 3 DNA barcodes, ITS displayed the maximum level of polymerase chain reaction and sequencing success as well as the highest sequence variation. Intra-specific sequence distances of ITS, chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase were 0, 0, and 0-0.1%, respectively, with 8.3, 0.6, and 0.5% as mean inter-specific distances, respectively. All individuals of each species formed a monophyletic group (clade) in the neighbor-joining trees constructed using the 3 single-DNA barcodes. Our results demonstrated that ITS, chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase DNA markers could be used to identify Ixiolirion species. Our results indicate that DNA barcoding provides a reliable and effective means for discriminating Ixiolirion species, and is a robust tool for resolving taxonomic controversies of Ixiolirion in combination with morphology-based taxonomy.

Molecular identification of the genus Thryssa based on DNA barcoding.

DNA barcoding is an effective method for identifying species by analyzing one or a few short standardized DNA sequences. In this study, we examined the utility of mitochondrial cytochrome oxidase subunit I (COI) sequences as a DNA barcode for the identification of six species belonging to the genus Thryssa: T. dussumieri, T. hamiltonii, T. kammalensis, T. mystax, T. setirostris, and T. vitrirostris. We obtained an intraspecific distance of 0.000 for T. vitrirostris and T. hamiltonii, 0.006 for T. mystax, 0.002 for T. dussumieri, and 0.005 for T. kammalensis. The average intraspecific distance was 0.002, while the average interspecific distance was 0.137. Thus, the interspecific genetic distance was approximately 67-fold larger than the intraspecific genetic distance; the average genetic distance among species was greater than the minimum of 0.020 between species suggested elsewhere. The genetic distance between T. vitrirostris and T. mystax was 0.003. A maximum-likelihood phylogenetic tree constructed using best-fitting tree topology showed distinct clusters corresponding to the species (except for T. vitrirostris and T. mystax). The closest relationship was found between T. vitrirostris and T. mystax. These two species clustered together in the phylogenetic tree. This conclusion contradicts the evolutionary relationship based on morphological classification.

Novel association of FCGR2A polymorphism with age-related macular degeneration (AMD) and development of a novel CFH real-time genotyping method.

Age-related macular degeneration (AMD) is a degenerative ocular disease, which may lead to loss of central vision. In Caucasian populations, a strong correlation has been established with polymorphism Y402H (rs1061170) in the complement factor H gene (CFH). The H131R polymorphism (rs1801274) in the FCGR2A gene has been associated with many inflammatory diseases, but has not been investigated in relation to AMD. The goal of our study was the development of a novel method for Y402H (g.43097C>T) genotyping, the confirmation of its association with AMD in the Greek population and the investigation of the H131R polymorphism in AMD. DNAs were extracted from blood samples of 120 patients with the severe wet form of AMD and 103 age- and sex-matched controls, all of whom were clinically evaluated. A real-time PCR and melting curve analysis method for Y402H genotyping was developed in the LightCycler platform, after in silico design of appropriate primers and probes. Genotyping for H131R was performed using a real-time PCR method previously described by our group. The novel genotyping method for Y402H in the CFH gene is fast, reproducible (Efficiency=1.79, reproducibility CVCq=3.33%, Tm C allele 53.36 °C and T allele 61.91 °C, ΔTm=8.55) and accurate as results were confirmed with the gold standard DNA Sequencing method. The present study confirmed the association between CFH Y402H SNP and wet AMD in the Greek population (OR=1.77, p=0.002). FCGR2A H131R polymorphism was investigated for the first time in this present study for possible correlation with wet AMD and a statistically significant association was detected (OR=1.74, p=0.006), that awaits further confirmation in a larger set of samples.

Immunohistochemical NF1 analysis does not predict NF1 gene mutation status in pheochromocytoma.

Pheochromocytomas (PCCs) are tumors originating from the adrenal medulla displaying a diverse genetic background. While most PCCs are sporadic, about 40 % of the tumors have been associated with constitutional mutations in one of at least 14 known susceptibility genes. As 25 % of sporadic PCCs harbor somatic neurofibromin 1 gene (NF1) mutations, NF1 has been established as the most recurrently mutated gene in PCCs. To be able to pinpoint NF1-related pheochromocytoma (PCC) disease in clinical practice could facilitate the detection of familial cases, but the large size of the NF1 gene makes standard DNA sequencing methods cumbersome. The aim of this study was to examine whether mutations in the NF1 gene could be predicted by immunohistochemistry as a method to identify cases for further genetic characterization. Sixty-seven PCCs obtained from 67 unselected patients for which the somatic and constitutional mutational status of NF1 was known (49 NF1 wild type, 18 NF1 mutated) were investigated for NF1 protein immunoreactivity, and the results were correlated to clinical and genetic data. NF1 immunoreactivity was absent in the majority of the PCCs (44/67; 66 %), including 13 out of 18 cases (72 %) with a somatic or constitutional NF1 mutation. However, only a minority of the NF1 wild-type PCCs (18/49; 37 %) displayed retained NF1 immunoreactivity, thereby diminishing the specificity of the method. We conclude that NF1 immunohistochemistry alone is not a sufficient method to distinguish between NF1-mutated and non-mutated PCCs. In the clinical context, genetic screening therefore remains the most reliable tool to detect NF1-mutated PCCs.

Comparative molecular genetic study of the Asian population of Drosophila mercatorum (Diptera; Drosophilidae) at the COI gene fragment and the ITS1–ITS2 region of the rRNA genes

Drosophila mercatorum is a species of neotropical origin. It is represented by two subspecies, marcatorum and pararepleta. Synanthropic subspecies D. mercatorum mercatorum spread into Eurasia in the mid-20th century, and to date, it is widespread over the territory of Europe and the former Soviet Union. The experiments were performed using the specimens of D. m. mercatorum from natural Asian populations, laboratory stock 2328 of D. mercatorum (displaying some morphological differences from Asian wild-type flies), the specimens of D. busckii and D. virilis, and the COI gene sequence of D. mercatorum from NCBI database, accession number, DQ471607. The specimens were investigated for the variability of the standard DNA sequences used for species identification (the cytochrome oxidase subunit I (COI) gene fragment and the ITS1–ITS2 region of rRNA genes). Sequences of the COI gene fragment from Asian D. m. mercatorum were identical between the specimens, but differed from the sequences of stock 2328, as well as from the NCBI sequence. Asian populations of D. mercatorum and stock 2328 were identical in the ITS1–ITS2 sequences. Low sequence divergence between the COI gene fragments, along with the absence of differences at the ITS1–ITS2 region of the rRNA genes indicate that the difference between the Asian populations of D. m. mercatorum and morphologically different stock 2328 are within the frames of intraspecies divergence.

Abstract 1503: Rapid detection of somatic mutations in cancer genes

Abstract In the context of personalized cancer treatment, tumor mutational analysis by DNA sequencing has been described frequently as the “gold standard”, yet DNA sequencing is one of the least sensitive methods for characterizing mutation. At least 10-20% of a DNA population must be mutant for the mutation to be detected by standard DNA sequencing. Implicit acceptance that this level of sensitivity is sufficient to characterize tumor mutations only makes sense if one assumes that tumors are monoclonal and that all the critical driver mutations will be present in the bulk of the tumor. Yet these are incorrect assumptions. In fact, it has become clear that most if not all colon tumors are polyclonal and heterogeneous. Consequently, tumor driver mutations may not be detectable using standard DNA sequencing methods. In order to rapidly detect these low frequency mutations in tumor derived DNA we have developed a new technology that allows the detection of tumor driver and drug resistance somatic mutations at <0.1% sensitivity using quantitative PCR (qPCR) without the need for digital PCR or droplet digital PCR (ddPCR) methodology. We utilize xeno-nucleic acid (XNA) clamping probes that are specific for the target gene wild-type template and allow only the selective amplification of mismatched mutant templates. Direct detection of driver and drug resistance mutations within 2 hours of obtaining samples in KRAS, NRAS, BRAF, EGFR and JAK2 utilizing real-time qPCR with interchelating fluorescence dye detection. As well as a new end-point post-PCR microtiterplate based target gene mutant amplicon capture format that utilizes colorimetric or chemiluminescence detection will be described. Citation Format: Michael J. Powell, Larry Pastor, Rachel Diaz, Lily Chen, George Wu, Claudia Li, Aiguo Zhang. Rapid detection of somatic mutations in cancer genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1503. doi:10.1158/1538-7445.AM2014-1503

Historical perspective, development and applications of next-generation sequencing in plant virology.

Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology.

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH-psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity-based methods and tree-based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH-psbA. The ITS provided better results with 30.61-38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree-based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.

Discrimination between invasive Ponto-Caspian gobies using a PCR-RFLP method

Summary Accurate identification of invaders, and especially their juveniles and eggs, is a difficult task if several morphologically similar species co-occur. The aim of the study was to develop and test a rapid and cost-effective procedure for identification of five species of invasive gobies occurring in the middle Danube basin, namely round goby Neogobius melanostomus, bighead goby Ponticola kessleri, monkey goby Neogobius fluviatilis, racer goby Babka gymnotrachelus and tubenose goby Proterorhinus semilunaris. First, a 708 bp fragment of the cytochrome oxidase I gene was amplified and sequenced for representative samples of these five species. Appropriate sequences of the five species available in public databases were used for in silico analysis. A digestion of the amplified fragment with the BfaI enzyme was found to be suitable for the species identification, as it showed unique restriction patterns for each species. The technique was also successfully applied for fish remains from burbot Lota lota stomachs. Thus the technique could be a useful tool in monitoring biological invasions, especially by identifying specimens that could not be determined on the basis of morphological features. The results demonstrate that the PCR-RFLP method may in some cases be more reliable for species identification than a standard DNA sequencing.

Evaluation of seven DNA barcodes for differentiating closely related medicinal Gentiana species and their adulterants.

BackgroundSpecies identification of living organisms by standard DNA sequences has been well-accepted. Consortium for the Barcode of Life (CBOL) recommends chloroplast regions rbcL and matK as the DNA barcodes for the land plants. This study aims to evaluate the feasibility and limitations of rbcL, matK, and 5 other commonly used regions as the DNA barcodes for the medicinal Gentiana and their adulterants, Gentiana. rhodantha and Podophyllum hexandrum.MethodsThe species differentiation power of rbcL, matK, nuclear internal transcribed spacer (ITS) and 5S rRNA intergenic spacer, and chloroplast trnH-psbA, trnL-F and rpl36-rps8 intergenic spacers were tested in different medicinal Gentiana, including Gentiana scabra, Gentiana triflora, Gentiana manshurica and Gentiana rigescens, from common adulterants such as Gentiana rhodantha and Podophyllum hexandrum (a toxic herb producing podophyllotoxin).ResultsAll seven tested loci could be used to differentiate medicinal Gentiana species from their adulterants, and to distinguish Guanlongdan from Jianlongdan. In terms of general differentiation powers, rbcL and matK had no significant advantages over the other five loci. Only the 5S rRNA and trnL-F intergenic spacers were able to discriminate the closely related species G. triflora, G. scabra and G. manshurica.ConclusionThe DNA barcodes rbcL and matK are useful in differentiation of closely related medicinal species of Gentiana, but had no significant advantages over the other five tested loci.

Alveolar rhabdomyosarcoma after treatment of osteosarcoma

Secondary rhabdomyosarcoma (RMS) after treatment of osteosarcoma (OS) is rare. Reported here is the case of a metachronous RMS in the nasal cavity, developing 12 years after successful treatment of non-metastatic OS. The patient was diagnosed as having OS of the femur at 2 years of age. Chemotherapy for OS included doxorubicin (cumulative dose, 488 mg/m(2) ). No radiotherapy was given. There was no family history suggestive of cancer predisposition syndrome. At 14 years of age, alveolar RMS was diagnosed on histopathology. PAX3-FKHR fusion transcripts were detected on reverse transcription-polymerase chain reaction. Germline TP53 mutation was not seen on standard DNA sequencing. The occurrence of secondary sarcomas, in the Children's Cancer Survivor study conducted in North America, has been associated with high cumulative doses of anthracyclines, which may also have played a role in the development of RMS in the present case. In the future, novel molecular technologies might uncover genetic cancer predisposition in patients with metachronous cancers.

“Transcriptors,” Reliable Parts—Gateway to Programmable Cells?

Transcriptors, DNA-based versions of transistors, could make cellular computers commercially competitive, thanks in this case to Drew Endy and his fellow bioengineers at Stanford University in California. They and others in this field will benefit from having standardized DNA sequences or “parts” that can be reliably used and swapped from one experiment to the next, and one research group to another.

Rediscovery of Sagittalarva inornata n. gen., n. comb. (Gilbert, 1890) (Perciformes: Labridae), a long-lost deepwater fish from the eastern Pacific Ocean: a case study of a forensic approach to taxonomy using DNA barcoding.

Some of the more valuable contributions of a standardized DNA sequence database (the DNA barcode) are matching specimens of different life stages and confirming the species identity of individuals from distant locations. These applications can facilitate the detective work required to solve difficult taxonomic problems. In this case, a match was made between the COI mtDNA sequence of an adult male wrasse recently caught at the tip of Baja California in Mexico in deep water (30-100m) and sequences from a series of unusual larvae collected about 3500 km to the south, in the open ocean over the Galápagos Rift hydrothermal vents in 1985. The Baja adults fit the recent description of Halichoeres raisneri Baldwin & McCosker, 2001 from the Galápagos and Cocos Islands. However, another deepwater labrid is known from the same site and depth in Baja; it is the type locality for the century-old holotype and only specimen of the Cape Wrasse Pseudojulis inornatus Gilbert, 1890 (later as Pseudojuloides inornatus). Deepwater video images from the tip of Baja show wrasses identical to H. raisneri photographed in Galápagos but who also fit the description of Pseudojulis inornatus. This coincidence led to a closer investigation of the holotype with x-ray, which revealed unanticipated caniniform teeth (vs. incisiform in Pseudojuloides) and an error in the fin-ray count in the original description, both of which mistakenly separated Halichoeres raisneri. The two species now match in markings, meristics, and morphology as well as overlapping range and are therefore synonymized. Phenetic and phylogenetic trees using mtDNA and nuclear DNA sequences show the species is not close to any other lineage and does not group with the other julidine labrids of the New World or the Pseudojuloides or Halichoeres of the Indo-Pacific. The distinctive larval morphology, long, thin, and flattened with a sharply pointed black-tipped snout, resembles no other described labrid larvae and, without an available genus, the new genus Sagittalarva Victor, n. gen. and the new combination Sagittalarva inornata (Gilbert, 1890), n. gen., n. comb. are described.

Hidden Markov Model for Splicing Junction Sites Identification in DNA Sequences

Identification of coding sequence from genomic DNA sequence is the major step in pursuit of gene identification. In the eukaryotic organism, gene structure consists of promoter, intron, start codon, exons and stop codon, etc. and to identify it, accurate labeling of the mentioned segments is necessary. Splice site is the ‘separation’ between exons and introns, the predicted accuracy of which is lower than 90% (in general) though the sequences adjacent to the splice sites have a high conservation. As the accuracy of splice site recognition has not yet been satisfactory (adequate), therefore, much attention has been paid to improve the prediction accuracy and improvement in the algorithms used is very essential element. In this manuscript, Hidden Markov Model (HMM) based splice sites predictor is developed and trained using Modified Expectation Maximization (MEM) algorithm. A 12 fold cross validation technique is also applied to check the reproducibility of the results obtained and to further increase the prediction accuracy. The proposed system can able to achieve the accuracy of 98% of true donor site and 93% for true acceptor site in the standard DNA (nucleotide) sequence. Keywords: Algorithms, coding sequence, cross validation, gene finding, hidden markov model, modified expectation maximization (MEM), splice site.

Deep sequencing of phage display libraries to support antibody discovery

The use of Next Generation Sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

Concordance of KRAS mutation detection between Illumina next generation gene sequencing (NGS) and CLIA-approved, clinical assays in metastatic colorectal cancer (mCRC).

345 Background: It is well known that KRAS mutations limit the efficacy of anti-EGFR therapy in patients with mCRC. Therefore, accurate testing of KRAS is needed to ensure that appropriate patients receive anti-EFGR therapy. Most clinical institutions conduct KRAS testing in CLIA approved labs using standard DNA sequencing methods. The purpose of this study is to correlate KRAS mutation detection by Illumina KRAS gene sequencing to standard KRAS testing performed with CLIA. Methods: We analyzed tumor samples collected from 471 patients between 1998-2010. Patients were chosen randomly based on availability of sufficient tissue for DNA extraction. We performed targeted exome sequencing using an Illumina NGS platform with 50-100X coverage of KRAS. The BWA/GATK pipeline was used to identify variants and indels. Because matched normal samples were not available for comparison to identify somatic mutations, filtering of normal variants was performed using 1000 Genomes. Variants identified in 1000 Genomes with an MAF < 0.01 were filtered. Results: Out of 471 patients, 83 pts had KRAS testing done both by exome sequencing & CLIA approved labs. The concordance rate between the two testing methods was 89%. 39 pts (47%) were KRAS mutation negative (wild type) and 35 pts (46%) harbored KRAS mutation as determined by both methods. Of these, 31 pts had codon 12 mutations and 4 pts had codon 13 mutations. However, 6 pts (7%) were found to have no KRAS mutation using CLIA but were found to have KRAS mutations by NGS (two had A146V mutations, the others had Q61 mutations). In addition, 3 pts (4%) were KRAS mutants using CLIA but wild type by NGS. Thus, 10% of samples tested showed a discrepancy in outcome between the NGS & CLIA testing. Conclusions: Standard DNA sequencing methods (CLIA) was able to identify a majority of the KRAS mutants detected by NGS. However 10% of patients had either false positive or false negative results. The main explanation for this discrepancy is that CLIA approved labs generally only test for codons 12 & 13. The clinical role for NGS in tumor KRAS mutation detection should be further investigated to ensure that patients with mCRC receive appropriate therapy.

DNA barcoding for plant protection: applications and summary of available data for arthropod pests.

Abstract Over the last decade, DNA barcoding - the use of short, standardized DNA sequences for species identification - has emerged as an important tool to study biodiversity. However, given the challenge and importance of accurately identifying plant pests, we argue for the use of DNA barcoding as a tool to aid in phytosanitary regulation and integrated pest management. Additionally, the rigorous data quality standard of DNA barcoding complements both regulatory and management tasks. In this review, we describe the current and potential applications of DNA barcoding for plant pest management, with a particular focus on terrestrial arthropods, as well as introduce and discuss some analytical methodologies and tools used for DNA barcoding research. Furthermore, we summarize the currently available DNA barcode data for a composite global pest list of 1044 plant pests, compiled from six international pest lists.

A DNA Barcoding Method to Discriminate between the Model Plant Brachypodium distachyon and Its Close Relatives B. stacei and B. hybridum (Poaceae)

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 FindingsWe 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/SignificanceOur 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.