Deoxyribonucleic Acid Barcoding Research Articles

Identification of Colletotrichum spp. associated with fruit rot of Capsicum annuum in North Western Himalayan region of India using fungal DNA barcode markers

The DNA barcode approach was used to identify and establish association of Colletotrichum species complex with fruit rot disease of chili (Capsicum annuum L.) in North-Western Himalayan region of India. Twenty isolates of five morphologically identified Colletotrichum species collected from commercial chili growing areas were identified using deoxyribonucleic acid (DNA) barcode marker genes, 5.8S ribosomal ribonucleic acid flanking internal transcribed spacers 1 & 2 and β-tubulin gene. Morpho-cultural identification requires expertise to delineate C. gloeosporioides, C. boninense and C. acutatum complexes from each other, as these species possess minute variation in spore shape and size. Ribosomal DNA and β-tubulin sequence analysis along with species-specific marker amplification established the association of seven Colletorichum spp. viz., C. truncatum (syn. Colletotrichum capsici), C. coccodes, C. karstii, C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides complex with fruit rot of chili. Phylogenetic analysis of 35 Colletotrichum sequences including authentic type sequences validated the identified sequences with strong bootstrap support. This approach delineated morphologically identified species with great ease into more reliable genotype based speciation of various Colletorichum complexes. The DNA barcode markers have direct implications for plant pathologists in relation to diagnostics in fields and for the purpose of quarantine and disease management.

RibosomalRNA Gene Spacers as DNA Barcodes and Prelude of RibosomalRNA Gene Evolution in GenusPlantago

The importance of spacer regions in resolving the phylogenetic questions using a combination of molecular and cytological techniques illustrates the importance of spacer regions in determining the phylogeny of genus Plantago and in assigning them the name of gene relics in the pretext of evolution. 74Plantago and representative out-group tax a were chosen for sequence analysis of spacers of the ribosomal deoxyribonucleic acid (DNA) to evaluate their phylogenetic potential. The chromosome number of different species of the genus was matched in accordance to their positions in the phylogenetic tree with an aim to work out a reasonable resolution of sub-generic groups as recognised earlier. Internal transcribed spacer (ITS) sequences of 67 species from the GenBank showed a distinct 21base pair conserved motif within the ITS1 region, which was used for barcoding of different species of Plantago, which was further supported by developing a Polymerase Chain Reaction – Restriction fragment length polymorphism (PCR-RFLP) -based technique to help in the process of barcoding. The homology between the spacer region and the coding region of 5S ribosomal ribonucleic acid (rRNA), from various genera and species ascertained that these regions represent gene relics and constitute essential players in the regulation of rRNA genes contiguous to these sequences.

Microsatellite-Based DNA Fingerprinting of Guava (Psidium guajava) Genotypes

Molecular fingerprinting exploits the variation between individuals at the genome level. It is generally performed for clear identification of various accessions, verifying ambiguous nomenclature, i.e., if similar cultivars are known by different names and different cultivars are called by similar names; to address Intellectual Property Rights related issues in the case of developed hybrids. The objective of the present study was to genotype and deoxyribonucleic acid (DNA) fingerprint 79 guava (Psidium guajava) accessions. Statistical analysis was performed by CERVUS 3.0 software. DNA fingerprint of the guava genotypes was developed by using 13 microsatellite loci that showed high polymorphic information content (PIC), allelic number per loci, and heterozygosity values. A total of 86 different alleles were amplified; number of alleles per locus ranged from five to nine, with a mean of 6.5. Allelic sizes ranged from 86 to 256 bp and the observed heterozygosity values ranged from 0.00 to 0.364. The expected heterozygosity varied from 0.689 to 0.821. The PIC varied from 0.630 to 0.789. Probability of identity varied from 0.05 to 0.12. The DNA barcode developed is very discriminative. So, it can be used for efficient and unambiguous identification of guava accessions, thereby increasing the accuracy in maintenance of genetic resources. Apart from the DNA fingerprint, interpretation of the results clearly indicated that each accession had a unique allelic combination at the 13 nuclear microsatellite loci, indirectly reflecting the fact that none of accessions were similar.

Identification of a Herbal Powder by Deoxyribonucleic Acid Barcoding and Structural Analyses.

Background: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.Objective:To identify a herbal powder obtained from a herbalist in the local vicinity of Rajkot, Gujarat, using deoxyribonucleic acid (DNA) barcoding and molecular tools.Materials and Methods: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.Results: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.Conclusion: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.SUMMARY 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.

Discovery of new populations and DNA barcoding of the Arapahoe snowfly Arsapnia arapahoe (Plecoptera: Capniidae).

The Arapahoe Snowfly, Arsapnia arapahoe (Nelson & Kondratieff)was recently discovered in six different first-order streams outside of the Cache la Poudre River Basin where it was previously considered endemic. Specimens of A. arapahoe were always collected in much lower relative abundance, 1.09% (±2.3SD), than other sympatric adult capniids. The first mitochondrial deoxyribonucleic acid (DNA) barcodes for A. arapahoe and A. coyote (Nelson & Baumann) are presented and compared with those of A. decepta. DNA barcoding was not able to differentiate between A. arapahoe and A. decepta Banks but it was able to indicate that A. coyote is specifically distinct.

Morphological taxonomy, DNA barcoding, and species diversity in southern Rocky Mountain headwater streams

Elevation gradients allow scientists to observe changes in fauna over a range of abiotic conditions. A variety of trends in aquatic insect diversity patterns across elevation have been reported. However, many of these studies are confounded because they include streams at lower elevations, which are often larger in size and more polluted than their higher-elevation counterparts. Moreover, such studies always relied solely on morphological delineation of taxa, thereby potentially overlooking cryptic diversity. We reduced these limitations by sampling only minimally impacted wadeable streams across an elevation gradient and by combining morphological taxonomy with deoxyribonucleic acid (DNA) barcoding to identify taxa. We collected numerically abundant Ephemeroptera, Plecoptera, and Trichoptera (EPT) from single streams at ∼200-m elevation intervals across >1000-m transects in 3 watersheds draining the eastern slope of the Colorado Rocky Mountains. Based on morphology alone, we identified 49 numerically abundant EPT morphospecies across 26 sites. Using DNA barcoding, we found 69 distinct lineages that probably represent distinct species. EPT species richness was highest at mid-elevations, and rates of turnover along elevation transects showed no consistent elevation trend or trend among ecological zones defined by vegetation. β-diversity across sites at comparable elevations in different watersheds showed a negative trend with increasing elevation that was marginally significant for DNA barcode taxa (p = 0.051) but not for morphospecies. Furthermore, significant (p < 0.05) differences in taxon richness, turnover, and lateral β-diversity values generated by DNA barcoding underscore the ability of molecular tools to quantify patterns in aquatic insect diversity across elevations.

Comparison of four species-delimitation methods applied to a DNA barcode data set of insect larvae for use in routine bioassessment

Abstract Species delimitation (grouping individuals into distinct taxonomic groups) is an essential part of evolutionary, conservation, and molecular ecology. Deoxyribonucleic acid (DNA) barcodes, short fragments of the cytochrome c oxidase subunit I (COI) gene, are being used in environmental bioassessments to assign specimens to putative species, but no method for delimiting DNA barcodes into species-level entities is universally accepted. We investigated the effect of delimitation methods on outcomes of bioassessments based on DNA barcodes. We used 2 tree-construction methods (neighbor joining [NJ], maximum likelihood [ML]) and 4 classes of species-delimitation criteria (distance-based, bootstrap support, reciprocal monophyly, and coalescent-based) with a DNA barcode data set consisting of 3 genera and 2202 COI sequences. We compared species delimitations for Baetis (Ephemeroptera:Baetidae), Eukiefferiella (Diptera:Chironomidae), and Simulium (Diptera:Simuliidae) from different streams. We assessed con...

Does DNA barcoding improve performance of traditional stream bioassessment metrics?

Benthic macroinvertebrate community composition is used to assess wetland and stream condition and to help differentiate the effects of stressors among sites. Deoxyribonucleic acid (DNA) barcoding has been promoted as a way to increase taxonomic resolution and, thereby, to increase the sensitivity of bioassessment metrics. We compared the ability of several commonly used bioassessment metrics calculated with data derived from morphology and from DNA barcoding to detect differences in stream condition of 6 paired sites in southern California with relatively subtle impacts to habitat. At each site, we sampled an upstream (reference) reach and a downstream (impact) reach with armored stream banks. We counted and identified ∼600 organisms/sample based on morphology (generally to species, but to genus for midges). We then extracted mitochondrial (mt)DNA from each individual and sequenced the ∼658-base pair (bp) barcoding region of the cytochrome c oxidase subunit I (COI) gene. Most (91%) organisms yielded sequences >350 bp in length, but high failure rates for all taxa collected from 1 stream required that we exclude it from analysis. Sixteen metrics calculated with morphological data showed subtle but not significant differences in community composition between armored and unarmored reaches. The statistical power of 10 of the 16 metrics was substantially higher when calculated with DNA than with morphological data, and we were able to discern differences between armored and unarmored reaches with the DNA data. These differences were associated with increased taxonomic richness detected for midges, mayflies, noninsects, caddisflies, and black flies when DNA data were used. Our results suggest that identifications based on DNA barcoding have the potential to improve power to detect small changes in stream condition.

Cryptic biodiversity in streams: a comparison of macroinvertebrate communities based on morphological and DNA barcode identifications

AbstractSpecies-level identifications are difficult or impossible for many larval aquatic macroinvertebrates. We described the taxonomic composition of macroinvertebrate communities from 5 coastal streams in 3 neighboring catchments in southern California. We compared taxonomic identifications based on deoxyribonucleic acid (DNA) barcoding (cytochrome c oxidase subunit I [COI]) with morphological identifications of the same specimens. We examined 5870 individuals, and barcodes with sequence lengths >350 base pairs (bp) for 91% of those specimens. We used the naturally occurring gaps in divergence frequencies for each order (usually 2% level of genetic divergence) to delimit putative species for all taxonomic groups except Simulium (3%) and Baetis (1%). We identified 200 species across these 5 streams. We identified 104 more species via barcodes than via morphology (200 vs 96, a 108% increase). Richness increases were greatest for Chironomidae (60 more species), Ephemeroptera (10 species), Acari (10 specie...

An efficient protocol for total DNA extraction from the members of order Zingiberales- suitable for diverse PCR based downstream applications

Different protocols are usually used for extracting total deoxyribonucleic acid (DNA) from different plant species of same order and DNA of the associated viruses. Here, we describe a rapid, efficient and universal protocol for isolating total DNA from the members of Zingiberales which harbor a high amount of polysaccharides and secondary metabolites. DNA isolated with this protocol was successfully used for PCR based downstream applications viz. random amplified polymorphic DNA (RAPD), Inter-simple sequence repeats (ISSR), DNA barcoding gene (Internal transcribed spacer and trnl-f) amplification and detection of the viruses.

DNA Barcode of Thief Ant Complex (Hymenoptera: Formicidae)

The thief ant, Solenopsis molesta (Say), a common nuisance species found throughout the United States is genetically related to red imported fire ants, S. invicta Buren. Therefore, its identification at the molecular level is very important. The deoxyribonucleic acid (DNA) barcoding, a recent technique was used to identify thief ant complex at species and subspecies levels using a short DNA sequence from the cytochrome oxidase subunit 1 (COI) mitochondrial region. The DNA from thief ants collected from 9 states was extracted using Qiagen's Gentra PUREGENE® DNA Isolation Kit. The polymerase chain reactions (PCR) were run on the extracted DNA to amplify partial sequence of COI using primers Lep-F1 (forward) and Lep-R1 (reverse). The resulting DNA products were concentrated, purified and sequenced. The 600 bp sequences of the COI generated were submitted to GenBank that issued accessions numbers from HM179641 to HM179653. The sequences associated with these accession numbers were used as DNA barcodes for distinguishing species and subspecies. Based on this molecular analysis, thief ants collected from New York, Indiana and 1 location in Nebraska were separated in 1 group as S. molesta validiuscula (Emery) and another with ants from Louisiana identified as S. carolinensis (Forel). The third group was comprised of ants from South Dakota, Washington, New Jersey, Tennessee, Kansas and 2 other locations in Nebraska was identified as S. molesta molesta (Say).

Species identification of Rhododendron (Ericaceae) using the chloroplast deoxyribonucleic acid PsbA-trnH genetic marker.

Background:Rhododendron is a group of famous landscape plants with high medicinal value. However, there is no simple or universal manner to discriminate the various species of this group. Deoxyribonucleic acid (DNA) barcoding technique is a new biological tool that can accurately and objectively identify species by using short and standard DNA regions.Objective:To choose a suitable DNA marker to authenticate the Rhododendron species.Materials and Methods:Four candidate DNA barcodes (rbcL, matK, psbAtrnH, and ITS2 intergenic spacer) were tested on 68 samples of 38 species.Results:The psbAtrnH candidate barcode yielded 86.8% sequencing efficiency. The highest interspecific divergence was provided by the psbA-trnH intergenic spacer, based on six parameters, and the Wilcoxon signed rank tests. Although there was not a clear barcoding gap, the Wilcoxon Two sample tests indicated that the interspecific divergence of the psbA-trnH intergenic spacer was significantly higher than the relevant intraspecific variation. The psbA-trnH DNA barcode possessed the highest species identification efficiency at 100% by the BLAST1 method. The present results showed that the psbA-trnH intergenic spacer was the most promising one of the four markers for barcoding the Rhododendron species. To further evaluate the ability of the psbA-trnH marker, to discriminate the closely related species, the samples were expanded to 94 samples of 53 species in the genus, and the rate of successful identification was 93.6%. The psbA-trnH region would be useful even for unidentified samples, as it could significantly narrow their possible taxa to a small area.Conclusion:The psbA-trnH intergenic region is a valuable DNA marker for identifying the Rhododendron species.

Application of deoxyribonucleic acid barcoding in Lauraceae plants

Background:This study aims to determine the candidate markers that can be used as DNA barcode in the Lauraceae family.Material and Methods:Polymerase chain reaction amplification, sequencing efficiency, differential intra- and interspecific divergences, DNA barcoding gap, and identification efficiency were used to evaluate the four different DNA sequences of psbA-trnH, matK, rbcL, and ITS2. We tested the discrimination ability of psbA-trnH in 68 plant samples belonging to 42 species from 11 distinct genera and found that the rate of successful identification with the psbA-trnH was 82.4% at the species level. However, the correct identification of matK and rbcL were only 30.9% and 25.0%, respectively, using BLAST1. The PCR amplification efficiency of the ITS2 region was poor; thus, ITS2 was not included in subsequent experiments. To verify the capacity of the identification of psbA-trnH in more samples, 175 samples belonging to 117 species from the experimental data and from the GenBank database of the Lauraceae family were tested.Results:Using the BLAST1 method, the identification efficiency were 84.0% and 92.3% at the species and genus level, respectively.Conclusion:Therefore, psbA-trnH is confirmed as a useful marker for differentiating closely related species within Lauraceae.

Identification of Meconopsis species by a DNA barcode sequence: The nuclear internal transcribed spacer (ITS) region of ribosomal deoxyribonucleic acid (DNA)

Deoxyribonucleic acid (DNA) barcoding is a novel technology that uses a standard DNA sequence to facilitate species identification. Species identification is necessary for the authentication of traditional plant based medicines. Although a consensus has not been agreed regarding which DNA sequences can be used as the best plant barcode, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA has been tested and applied extensively in recent years. In this study, we used the ITS region to test its possibility to serve as barcodes for Meconopsis species which are widely used in traditional Tibetan medicines that are used throughout the Himalayas. We sequenced 14 accessions representing four Meconopsis species. The result shows ITS sequences were found to be unique for each species, with the interspecies percentages of nucleotide differences averaging 5.1% and ranging from 0.2 to 9.6%. In contrast, the intraspecies percentages of nucleotide differences among the Meconopsis species studied ranged from 0 to 1.9%. Our results indicate that the ITS region can be used as a barcode to distinguish Meconopsis species.

DNA barcoding facilitates description of unknown faunas: a case study on Trichoptera in the headwaters of the Tigris River, Iraq

Monitoring water quality with aquatic insects as sentinels requires taxonomic knowledge of adult and immature life stages that is not available in many parts of the world. We used deoxyribonucleic acid (DNA) barcoding to expedite identification of larval caddisflies from 20 sites in the headwaters of the Tigris River in northern Iraq by comparing their mitochondrial cytochrome c oxidase subunit I (COI) sequences to a global reference library (the Trichoptera Barcode of Life). We obtained full-length DNA barcodes for 16 COI haplogroups from 11 genera in 9 Trichoptera families. The most haplogroups and genera were recorded from Sulaimani Province. Two distinct COI haplogroups were found for the genus Psychomyia, and 5 haplogroups were found for Hydropsyche. The Hydropsyche COI haplogroups do not form a monophyletic clade with reference to the world fauna, but 4 out of 5 haplogroups are related to other Palearctic species. Three larval Rhyacophila specimens in a single COI haplogroup are closely related to specimens of Rhyacophila nubila Zetterstedt and Rhyacophila dorsalis (Curtis) from Europe, but adults from Iraq are needed to confirm their species identity.

Applying DNA barcoding in benthology: the state of the science

AbstractThe application of deoxyribonucleic acid (DNA) barcoding in benthic ecology is proceeding rapidly, and a special session held at the 2009 North American Benthological Society Annual Meeting in Grand Rapids, Michigan, USA, brought together benthic scientists working in areas varying from taxonomy to biomonitoring to present the state of the science. The papers arising from this meeting are grouped in this special series of papers. Here, we present a brief introduction to this emerging area of benthic science, highlight the contributions of previous researchers, and provide an overview of these latest contributions to this highly active field. Paper topics include the development of DNA libraries for identification of material from field studies, application of DNA-based taxonomy in the discovery of new faunas, and the use of DNA-based identification in field biomonitoring studies, present and future. In addition, a critique is presented of the use of DNA barcoding in benthology and the potential pi...

Incorporation of DNA barcoding into a large-scale biomonitoring program: opportunities and pitfalls

Taxonomic identification of benthic macroinvertebrates is critical to protocols used to assess the biological integrity of aquatic ecosystems. The time, expense, and inherent error rate of species-level morphological identifications has necessitated use of genus- or family-level identifications in most large, statewide bioassessment programs. Use of coarse-scale taxonomy can obscure signal about biological condition, particularly if the range of species tolerances is large within genera or families. We hypothesized that integration of deoxyribonucleic acid (DNA) barcodes (partial cytochrome c oxidase subunit I sequences) into bioassessment protocols would provide greater discriminatory ability than genus-level identifications and that this increased specificity could lead to more sensitive assessments of water quality and habitat. Analysis of DNA barcodes from larval specimens of Ephemeroptera, Plecoptera, and Trichoptera (EPT) taxa collected as part of Maryland’s Biological Stream Survey (MBSS) revealed ∼2 to 3× as many DNA-barcode groups or molecular operational taxonomic units (mOTUs) as morphologically identified genera. As expected, geographic distributions for several mOTUs were tighter than for the parent genus, but few mOTUs showed closer associations with water-quality variables or physical-habitat features than did the genus in which they belonged. The need for improved protocols for the consistent generation of DNA barcodes is discussed.

DNA barcoding: a taxonomic point of view

AbstractA taxonomic perspective of deoxyribonucleic acid (DNA) barcoding is presented here with respect to its use within the freshwater science community. The goals and methods of DNA barcoding and the criticisms leveled at the procedure by the taxonomic community are explained. The major goal of DNA-barcoding efforts is to aid identification of specimens by matching sequences to a sequence library. This goal is achievable, but barcoding efforts must be coupled with a much wider sampling regime and should be done with the full cooperation and collaboration of the taxonomic community. Through collaborative efforts, taxonomists and barcode advocates can build capacity for taxonomy and sequence the vast number of specimens needed to build a truly comprehensive barcode library for use by benthic scientists. Other barcoding goals, such as species diagnosis and biodiversity surveys, are more problematic, but solutions are possible.

A DNA-barcoding approach to identifying juvenile freshwater mussels (Bivalvia:Unionidae) recovered from naturally infested fishes

AbstractWe developed a multilocus deoxyribonucleic acid (DNA)-barcoding approach to identify newly transformed juvenile mussels collected from naturally infested fishes in a federally protected waterway that is home to a diverse mussel community, the St Croix River (Minnesota/Wisconsin, USA). We used new and publicly available data downloaded from GenBank to build reference databases for identified adult mussels. We assessed the efficacy of the mitochondrial loci cytochrome oxidase c subunit I (COI) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) for DNA barcoding. We concluded that the barcoding gap between average intra- and interspecific genetic distances is wider for ND1 than for COI, but both loci perform well for species identification in character-based phylogenetic analyses. Almost every species formed a monospecific clade with high bootstrap and posterior-probability support. We obtained newly transformed juvenile mussels by collecting individuals of 3 different fish species t...