LSU rDNA D5 region: the DNA barcode for molecular classification and identification of Demodex

Whether ribosomal genes can be used as DNA barcodes for molecular identification of Demodex (Acariformes: Demodicidae) is unclear. To examine this, Demodex folliculorum, D. brevis, D. canis, and D. caprae were collected for DNA extraction, rDNA fragments amplification, sequencing, and analysis. The V2 and V4 regions of SSU rDNA; D5, D6, and D8 regions of LSU rDNA; and ITS region were obtained from the four morphospecies. BLAST analysis showed that the obtained sequences matched those of Demodex or Aplonobia (Acariformes: Tetranychidae) in Raphignathae. Phylogenetic trees derived from V2, V4, D5, D6, and D8 regions, but not from ITS region, showed that the four species of Demodex clustered independently. Sequence divergence analysis further demonstrated that D5, D6, and D8 regions had obvious barcoding gap between intraspecific and interspecific divergences, with the gap of D5 (16.91%) larger than that of D6 (11.82%) and D8 (4.66%). The V2 and V4 regions did not have a barcoding gap, as the intraspecific and interspecific divergences partially overlapped. For the ITS region, intraspecific and interspecific divergences completely overlapped. These results suggest that the D5, D6, and D8 regions of LSU rDNA, especially D5, are suitable DNA barcodes for Demodex.

Download Full-text

Comparative efficacy of four candidate DNA barcode regions for identification of Vicia species

Plant Genetic Resources ◽

10.1017/s1479262115000623 ◽

2015 ◽

Vol 15 (4) ◽

pp. 286-295 ◽

Cited By ~ 5

Author(s):

Sebastin Raveendar ◽

Jung-Ro Lee ◽

Donghwan Shim ◽

Gi-An Lee ◽

Young-Ah Jeon ◽

...

Keyword(s):

Phylogenetic Trees ◽

Dna Barcode ◽

Resolving Power ◽

Conservation Strategies ◽

Species Discrimination ◽

Dna Barcodes ◽

Additional Tool ◽

Germplasm Collections ◽

Classification Tool ◽

Individual Locus

AbstractThe genus Vicia L., one of the earliest domesticated plant genera, is a member of the legume tribe Fabeae of the subfamily Papilionoideae (Fabaceae). The taxonomic history of this genus is extensive and controversial, which has hindered the development of taxonomic procedures and made it difficult to identify and share these economically important crop resources. Species identification through DNA barcoding is a valuable taxonomic classification tool. In this study, four DNA barcodes (ITS2, matK, rbcL and psbA-trnH) were evaluated on 110 samples that represented 34 taxonomically best-known species in the Vicia genus. Topologies of the phylogenetic trees based on an individual locus were similar. Individual locus-based analyses could not discriminate closely related Vicia species. We proposed a concatenated data approach to increase the resolving power of ITS2. The DNA barcodes matK, psbA-trnH and rbcL were used as an additional tool for phylogenetic analysis. Among the four barcodes, three-barcode combinations that included psbA-trnH with any two of the other barcodes (ITS2, matK or rbcL) provided the best discrimination among Vicia species. Species discrimination was assessed with bootstrap values and considered successful only when all the conspecific individuals formed a single clade. Through sequencing of these barcodes from additional Vicia accessions, 17 of the 34 known Vicia species could be identified with varying levels of confidence. From our analyses, the combined barcoding markers are useful in the early diagnosis of targeted Vicia species and can provide essential baseline data for conservation strategies, as well as guidance in assembling germplasm collections.

Download Full-text

Efficiency of ITS1-5.8S-ITS2 region in identifying Cordyceps species

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/16/4/13220 ◽

2020 ◽

Vol 16 (4) ◽

pp. 705-712

Author(s):

Le Thi Thu Hien ◽

Ha Hong Hanh

Keyword(s):

Dna Sequences ◽

Phylogenetic Trees ◽

Nearest Neighbor ◽

Dna Barcode ◽

Morphological Characteristics ◽

Pcr Amplification ◽

Cordyceps Sinensis ◽

Its2 Region ◽

Dna Barcodes ◽

Plastid Genomes

Cordyceps genus is a well-known traditional medicine worldwide. It contains abundant physiological active compounds that were demonstrated to perform benefit in reducing progression of cancer as well as protecting human health. Accurately classifying species in this genus is essential in order to prevent commercial counterfeit medicines. Nowadays, a taxonomic classification of species based on DNA sequences can overcome the existed limitation in identifying by using only morphological characteristics of this genus. DNA barcodes are standard short genomic regions that are universally present in target lineages and has sufficient sequence variation to discriminate species in the genus. A variety of loci has been suggested as DNA barcodes for plants, including genes and non-coding regions in the nuclear and plastid genomes such as psbA-trnH, matK, rbcL, and ITS. Thus, the objective of this study was to identify selected species of Cordyceps genus using DNA barcodes. Seven strains of Cordyceps were collected. Total DNA extraction and purification, PCR amplification and DNA sequencing were performed with standard chemicals and kits. The candidate ITS1-5.8S-ITS2 region was amplified and sequenced. Data were analyzed using Bioedit 7.2.6 and MEGA 7 softwares. Analysis of seven obtained DNA barcode sequences of collected samples revealed that the ITS1-5.8S-ITS2 region provided high species discriminating power for Cordyceps genus. Accordingly, phylogenetic trees based on this DNA barcode exhibited six samples had closed relationship to Cordyceps militaris, while another specimen was the nearest neighbor to Cordyceps sinensis with average similarities at 99.82% and 99.81%, respectively. Our results support the identification of valuable medicinal plant species within Cordyceps genus.

Download Full-text

DNA barcoding of Zygaenidae (Lepidoptera): results and perspectives

Nota Lepidopterologica ◽

10.3897/nl.42.33190 ◽

2019 ◽

Vol 42 (2) ◽

pp. 137-150

Author(s):

Konstantin A. Efetov ◽

Anna V. Kirsanova ◽

Zoya S. Lazareva ◽

Ekaterina V. Parshkova ◽

Gerhard M. Tarmann ◽

...

Keyword(s):

Dna Barcoding ◽

Sequence Data ◽

Dna Barcode ◽

Sequence Divergence ◽

Coi Gene ◽

Dna Barcodes ◽

The Family ◽

The World ◽

Biological Characters

The present study provides a DNA barcode library for the world Zygaenidae (Lepidoptera). This study reports 1031 sequence data of the COI gene DNA barcodes for more than 240 species in four of the five subfamilies of the family Zygaenidae. This is about 20% of the world Zygaenidae species. Our results demonstrate the specificity of the COI gene sequences at the species level in most of the studied Zygaenidae and agree with already established taxonomic opinions. The study confirms the effectiveness of DNA barcoding as a tool for determination of most Zygaenidae species. However, some of the results are contradictory. Some cases of shared barcodes have been found, as well as cases of deep intraspecific sequence divergence in species that are well separated by morphological and biological characters. These cases are discussed in detail. Overall, when combined with morphological and biochemical data, as well as biological and ecological observations, DNA barcoding results can be a useful support for taxonomic decisions.

Download Full-text

Feasibility of nuclear ribosomal region ITS1 over ITS2 in barcoding taxonomically challenging genera of subtribe Cassiinae (Fabaceae)

PeerJ ◽

10.7717/peerj.2638 ◽

2016 ◽

Vol 4 ◽

pp. e2638 ◽

Cited By ~ 6

Author(s):

Priyanka Mishra ◽

Amit Kumar ◽

Vereena Rodrigues ◽

Ashutosh K. Shukla ◽

Velusamy Sundaresan

Keyword(s):

Dna Barcoding ◽

Phylogenetic Trees ◽

Sequence Similarity ◽

Its Region ◽

Genetic Distances ◽

Discrimination Power ◽

Distance Method ◽

Tree Reconciliation ◽

Its1 Region ◽

Barcoding Gap

Premise of the StudyThe internal transcribed spacer (ITS) region is situated between 18S and 26S in a polycistronic rRNA precursor transcript. It had been proved to be the most commonly sequenced region across plant species to resolve phylogenetic relationships ranging from shallow to deep taxonomic levels. Despite several taxonomical revisions in Cassiinae, a stable phylogeny remains elusive at the molecular level, particularly concerning the delineation of species in the generaCassia, SennaandChamaecrista. This study addresses the comparative potential of ITS datasets (ITS1, ITS2 and concatenated) in resolving the underlying morphological disparity in the highly complex genera, to assess their discriminatory power as potential barcode candidates in Cassiinae.MethodologyA combination of experimental data and an in-silico approach based on threshold genetic distances, sequence similarity based and hierarchical tree-based methods was performed to decipher the discriminating power of ITS datasets on 18 different species of Cassiinae complex. Lab-generatedsequences were compared against those available in the GenBank using BLAST and were aligned through MUSCLE 3.8.31 and analysed in PAUP 4.0 and BEAST1.8 using parsimony ratchet, maximum likelihood and Bayesian inference (BI) methods of gene and species tree reconciliation with bootstrapping. DNA barcoding gap was realized based on the Kimura two-parameter distance model (K2P) in TaxonDNA and MEGA.Principal FindingsBased on the K2P distance, significant divergences between the inter- and intra-specific genetic distances were observed, while the presence of a DNA barcoding gap was obvious. The ITS1 region efficiently identified 81.63% and 90% of species using TaxonDNA and BI methods, respectively. The PWG-distance method based on simple pairwise matching indicated the significance of ITS1 whereby highest number of variable (210) and informative sites (206) were obtained. The BI tree-based methods outperformed the similarity-based methods producing well-resolved phylogenetic trees with many nodes well supported by bootstrap analyses.ConclusionThe reticulated phylogenetic hypothesis using the ITS1 region mainly supported the relationship between the species of Cassiinae established by traditional morphological methods. The ITS1 region showed a higher discrimination power and desirable characteristics as compared to ITS2 and ITS1 + 2, thereby concluding to be the locus of choice. Considering the complexity of the group and the underlying biological ambiguities, the results presented here are encouraging for developing DNA barcoding as a useful tool for resolving taxonomical challenges in corroboration with morphological framework.

Download Full-text

Assessing Temporal Patterns and Species Composition of Glass Eel (Anguilla spp.) Cohorts in Sumatra and Java Using DNA Barcodes

Diversity ◽

10.3390/d13050193 ◽

2021 ◽

Vol 13 (5) ◽

pp. 193

Author(s):

Arif Wibowo ◽

Nicolas Hubert ◽

Hadi Dahruddin ◽

Dirk Steinke ◽

Rezki Antoni Suhaimi ◽

...

Keyword(s):

Species Delimitation ◽

Economic Value ◽

Species Conservation ◽

Dna Barcode ◽

Temporal Patterns ◽

Molecular Classification ◽

Dna Barcodes ◽

Glass Eel ◽

Silver Eels ◽

Glass Eels

Anguillid eels are widely acknowledged for their ecological and socio-economic value in many countries. Yet, knowledge regarding their biodiversity, distribution and abundance remains superficial—particularly in tropical countries such as Indonesia, where demand for anguillid eels is steadily increasing along with the threat imposed by river infrastructure developments. We investigated the diversity of anguillid eels on the western Indonesian islands of Sumatra and Java using automated molecular classification and genetic species delimitation methods to explore temporal patterns of glass eel cohorts entering inland waters. A total of 278 glass eels were collected from monthly samplings along the west coast of Sumatra and the south coast of Java between March 2017 and February 2018. An automated, DNA-based glass eel identification was performed using a DNA barcode reference library consisting of 64 newly generated DNA barcodes and 117 DNA barcodes retrieved from BOLD for all nine Anguilla species known to occur in Indonesia. Species delimitation methods converged in delineating eight Molecular Operational Taxonomic Units (MOTUs), with A. nebolusa and A. bengalensis being undistinguishable by DNA barcodes. A total of four MOTUs were detected within the glass eel samples, corresponding to Anguilla bicolor, A. interioris, A. marmorata, and A. nebulosa/A. bengalensis. Monthly captures indicated that glass eel recruitment peaks in June, during the onset of the dry season, and that A. bicolor is the most prevalent species. Comparing indices of mitochondrial genetic diversity between yellow/silver eels, originating from several sites across the species range distribution, and glass eels, collected in West Sumatra and Java, indicated a marked difference. Glass eels displayed a much lower diversity than yellow/silver eels. Implications for the management of glass eel fisheries and species conservation are discussed.

Download Full-text

Validity of Pampus liuorum Liu & Li, 2013, Revealed by the DNA Barcoding of Pampus Fishes (Perciformes, Stromateidae)

Diversity ◽

10.3390/d13120618 ◽

2021 ◽

Vol 13 (12) ◽

pp. 618

Author(s):

Jiehong Wei ◽

Renxie Wu ◽

Yongshuang Xiao ◽

Haoran Zhang ◽

Laith A. Jawad ◽

...

Keyword(s):

Dna Barcode ◽

Valid Species ◽

Dna Barcodes ◽

Operational Taxonomic Units ◽

Barcoding Gap ◽

Coi Barcoding ◽

Commercially Important ◽

Coi Sequences ◽

Morphological Species ◽

Fishery Species

The genus Pampus contains seven valid species, which are commercially important fishery species in the Indo-Pacific area. Due to their highly similar external morphologies, Pampus liuorum has been proposed as a synonym of Pampus cinereus. In this study, partial sequences of COI (582 bp) and Cytb (1077 bp) were presented as potential DNA barcodes of six valid Pampus species and the controversial species P. liuorum. A species delimitation of the seven Pampus species was performed to verify their validities. Explicit COI barcoding gaps were found in all assessed species, except for P. liuorum and P. cinereus, which resulted from their smaller interspecific K2P distance (0.0034–0.0069). A Cytb barcoding gap (0.0200) of the two species was revealed, with a K2P distance ranging from 0.0237 to 0.0277. The longer Cytb fragment is thus a more suitable DNA barcode for the genus Pampus. In the genetic tree, using concatenated Cytb and COI sequences, the seven species reciprocally formed well-supported clades. Species delimitations with ABGD, GMYC, and bPTP models identified seven operational taxonomic units, which were congruent with the seven morphological species. Therefore, all of the seven analyzed species, including P. liuorum, should be kept as valid species.

Download Full-text

Feasibility of nuclear ribosomal region ITS1 over ITS2 in barcoding taxonomically challenging genera of subtribe Cassiinae (Fabaceae)

10.7287/peerj.preprints.2434v1 ◽

2016 ◽

Author(s):

Priyanka Mishra ◽

Amit Kumar ◽

Vereena Rodrigues ◽

Ashutosh K Shukla ◽

Velusamy Sundaesan

Keyword(s):

Dna Barcoding ◽

Phylogenetic Trees ◽

Sequence Similarity ◽

Its Region ◽

Genetic Distances ◽

Discrimination Power ◽

Distance Method ◽

Tree Reconciliation ◽

Its1 Region ◽

Barcoding Gap

Premise of the Study. The internal transcribed spacer (ITS) region is situated between 18S and 26S in a polycistronic rRNA precursor transcript. It had been proved to be the most commonly sequenced region across plant species to resolve phylogenetic relationships ranging from shallow to deep taxonomic levels. Despite several taxonomical revisions in Cassiinae, a stable phylogeny remains elusive at the molecular level, particularly concerning the delineation of species in the genera Cassia, Senna and Chamaecrista. This study addresses the comparative potential of ITS datasets (ITS1, ITS2 and concatenated) in resolving the underlying morphological disparity in the highly complex genera, to assess their discriminatory power as potential barcode candidates in Cassiinae. Methodology. A combination of experimental data and an in-silico approach based on threshold genetic distances, sequence similarity based and hierarchical tree-based methods was performed to decipher the discriminating power of ITS datasets on 18 different species of Cassiinae complex. Lab-generated sequences were compared against those available in the GenBank using BLAST and were aligned through MUSCLE 3.8.31 and analysed in PAUP 4.0 and BEAST1.8 using parsimony ratchet, maximum likelihood and Bayesian inference (BI) methods of gene and species tree reconciliation with bootstrapping. DNA barcoding gap was realized based on the Kimura two-parameter distance model (K2P) in TaxonDNA and MEGA. Principal Findings. Based on the K2P distance, significant divergences between the inter- and intra-specific genetic distances were observed, while the presence of a DNA barcoding gap was obvious. The ITS1 region efficiently identified 81.63% and 90% of species using TaxonDNA and BI methods, respectively. The PWG-distance method based on simple pairwise matching indicated the significance of ITS1 whereby highest number of variable (210) and informative sites (206) were obtained. The BI tree-based methods outperformed the similarity-based methods producing well-resolved phylogenetic trees with many nodes well supported by bootstrap analyses. Conclusion. The reticulated phylogenetic hypothesis using the ITS1 region mainly supported the relationship between the species of Cassiinae established by traditional morphological methods. The ITS1 region showed a higher discrimination power and desirable characteristics as compared to ITS2 and ITS1+2 there by concluding to be the locus of choice. Considering the complexity of the group and the underlying biological ambiguities, the results presented here are encouraging for developing DNA barcoding as a useful tool for resolving taxonomical challenges in corroboration with morphological framework.

Download Full-text

Evaluation of five regions as DNA barcodes for identification of Lepista species (Tricholomataceae, Basidiomycota) from China

PeerJ ◽

10.7717/peerj.7307 ◽

2019 ◽

Vol 7 ◽

pp. e7307

Author(s):

Siyu Wang ◽

Hongbo Guo ◽

JiaJia Li ◽

Wei Li ◽

Qin Wang ◽

...

Keyword(s):

Ribosomal Rna ◽

Internal Transcribed Spacer ◽

Dna Barcode ◽

Pcr Amplification ◽

Intergenic Spacer ◽

Its Region ◽

Small Subunit ◽

Dna Barcodes ◽

Success Rates ◽

Small Subunit Rdna

Background Distinguishing among species in the genus Lepista is difficult because of their similar morphologies. Methods To identify a suitable DNA barcode for identification of Lepista species, we assessed the following five regions: internal transcribed spacer (ITS), the intergenic spacer (IGS), nuclear ribosomal RNA subunit, mitochondrial small subunit rDNA, and tef1. A total of 134 sequences from 34 samples belong to eight Lepista species were analyzed. The utility of each region as a DNA barcode was assessed based on the success rates of its PCR amplification and sequencing, and on its intra- and inter-specific variations. Results The results indicated that the ITS region could distinguish all species tested. We therefore propose that the ITS region can be used as a DNA barcode for the genus Lepista. In addition, a phylogenetic tree based on the ITS region showed that the tested eight Lepista species, including two unrecognized species, formed eight separate and well-supported clades.

Download Full-text

Cryptococcus rajasthanensis sp. nov., an anamorphic yeast species related to Cryptococcus laurentii, isolated from Rajasthan, India

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.64543-0 ◽

2007 ◽

Vol 57 (2) ◽

pp. 414-418 ◽

Cited By ~ 11

Author(s):

Puja Saluja ◽

G. S. Prasad

Keyword(s):

Large Subunit ◽

Its Region ◽

Molecular Data ◽

Lsu Rdna ◽

Cryptococcus Laurentii ◽

The Novel ◽

D2 Domain ◽

Its Regions ◽

Anamorphic Yeast ◽

Physiological Tests

Two novel anamorphic yeast strains (S-15LT and 3-C1) were isolated from the inflorescences of plants collected in two different towns in Rajasthan State, India. Sequencing of the D1/D2 domains of the large-subunit (LSU) rDNA and the internal transcribed spacer (ITS) regions suggested they are strains of the same species. Phenotypic characteristics such as the absence of fermentation, the absence of sexual structures and ballistoconidia, the assimilation of myo-inositol and d-glucuronate, and positive Diazonium blue B and urease reactions indicated that these strains belong to the genus Cryptococcus. The novel strains differed from Cryptococcus laurentii in six physiological tests and differed from other related species in more than six tests. A phylogenetic analysis of the sequences of the D1/D2 domains of the LSU rDNA and the ITS regions placed these strains in the Bulleromyces clade within the order Tremellales, with C. laurentii as their closest described relative. The novel strains showed 1.6 and 7.5 % divergence in the D1/D2 domain of the LSU rDNA and ITS regions, respectively, with respect to C. laurentii. The divergence from other species was more than 3 % for the D1/D2 domain and more than 9 % for the ITS region. On the basis of the phenotypic and molecular data, strains S-15LT and 3-C1 represent a novel species within the genus Cryptococcus, for which the name Cryptococcus rajasthanensis sp. nov. is proposed. The type strain is S-15LT (=MTCC 7075T=CBS 10406T).

Download Full-text

Biosystematic Study on Some Egyptian Species of Astragalus L. (Fabaceae)

Agriculture ◽

10.3390/agriculture11020125 ◽

2021 ◽

Vol 11 (2) ◽

pp. 125

Author(s):

Monier M. Abd El-Ghani ◽

Ashraf S. A. El-Sayed ◽

Ahmed Moubarak ◽

Rabab Rashad ◽

Hala Nosier ◽

...

Keyword(s):

Genetic Relationships ◽

Dna Barcode ◽

Plant Morphology ◽

Its Region ◽

Its2 Region ◽

Whole Plant ◽

Three Samples ◽

Two Samples ◽

Region 10 ◽

Unique Banding Pattern

Astragalus L. is one of the largest angiosperm complex genera that belongs to the family Fabaceae, subfamily Papilionoideae or Faboideae under the subtribe Astragalinae of the tribe Galegeae. The current study includes the whole plant morphology, DNA barcode (ITS2), and molecular marker (SCoT). Ten taxa representing four species of Astragalus were collected from different localities in Egypt during the period from February 2018 to May 2019. Morphologically, identification and classification of collected Astragalus plants occurred by utilizing the light microscope, regarding the taxonomic revisions of the reference collected Astragalus specimens in other Egyptian Herbaria. For molecular validation, ten SCoT primers were used in this study, producing a unique banding pattern to differentiate between ten samples of Astragalus taxa which generated 212 DNA fragments with an average of 12.2 bands per 10 Astragalus samples, with 8 to 37 fragments per primer. The 212 fragments amplified were distributed as 2 monomorphic bands, 27 polymorphic without unique bands, 183 unique bands (210 Polymorphic with unique bands), and ITS2 gene sequence was showed as the optimal barcode for identifying Astragalus L. using BLAST searched on NCBI database, and afterward, analyzing the chromatogram for ITS region, 10 samples have been identified as two samples representing A. hauarensis, four samples representing A. sieberi, three samples representing A. spinosus and one sample representing A. vogelii. Based on the ITS barcode, A. hauarensis RMG1, A. hauarensis RMG2, A. sieberi RMG1, A. sieberi RMG2, A. sieberi RMG3, A. sieberi RMG4, A. spinosus RMG1, A. spinosus RMG2, A. spinosus RMG3, A. vogelii RMG were deposited into GenBank with accession # MT367587.1, MT367591.1, MT367593.1, MT367585.1, MT367586.1, MT367588.1, MT160347.1, MT367590.1, MT367589.1, MT367592.1, respectively. These results indicated the efficiency of SCoT markers and ITS2 region in identifying and determining genetic relationships between Astragalus species.

Download Full-text