The Information Systems for DNA Barcode Data

Objective To make up the limitations of traditional morphological classification methods, we identified vector fleas by DNA barcoding in Qinghai Province. Methods The mt DNA cytochrome c oxidase subunit Ⅰ(COⅠ) gene was amplified by PCR from 36 muscle tissues of fleas in 3 states, 2 cities and 5 counties of Qinghai Province, and the obtained COⅠ gene fragments were sequenced and aligned. The intra- and inter-species genetic distances were calculated with Mega 6 software using K2-P model and a phylogenetic tree was constructed with neighbor-joining (NJ) method. Results Totally 36 COⅠ gene sequences of 2 superfamilies, 4 genera and 6 kinds of vector fleas were measured, the average genetic distance was 0.119, and the intraspecific distance was 0.002 - 0.027, the interspecific distance was 0.039 - 0.207, and the interspecific genetic distance was significantly greater than the intraspecific genetic distance. NJ tree showed the same species had formed a single line with high support rate and interspecific branch was clear. Conclusion DNA barcoding is suitable for identification of vector fleas in Qinghai Province, may make up the limitations of traditional morphological classification methods. Key words: Siphonaptera; Cytochrome c oxidase subunit Ⅰ; DNA barcoding

<p class="Els-Abstract-text">Even though not yet considered as endangered, White-bellied Sea Eagle’s global population is decreasing due to illegal hunting, bird trading, and deforestation. So far, there hasn’t been any report regarding the phylogenetic study of the White-bellied Sea Eagle inhabiting the coastal regions of Java. Moreover, there hasn’t been any report on the genetic data, especially COI gene, of the White-bellied Sea Eagle living in coastal area of Java. Thus, in this research, two individuals of <em>Heliaeetus leucogaster</em><em> </em>(<a title="Johann Friedrich Gmelin" href="https://en.wikipedia.org/wiki/Johann_Friedrich_Gmelin">Gmelin</a>, 1788); <em> </em>are compared based on its COI gene sequence to the member of genus <em>Haliaeetu</em>s to determine their position in the phylogenetic tree of genus Haliaeetus. COI gene amplification is performed using <em>Forward</em> primer BirdF1 5’- TTC TCC AAC CAC AAA GAC ATT GGC AC-3’ and <em>Reverse </em>primer BirdR2 5’ ACT ACA TGT GAG ATG ATT CCG AAT-3’. The phylogenetic analysis using MEGA6 with <em>Maximum Likelihood </em>method shows that <em>Haliaeetus leucogaster</em> in this study is related to <em>Haliaeetus leucocephalus</em> (Linnaeus, 1766), <em>Haliaeetus pelagicus</em> (Pallas, 1811), and <em>Haliaeetus albicilla</em> (Linnaeus, 1758).</p><p> </p><div><p class="Els-keywords"><strong>Keywords:</strong> phylogenetic study; <em>Heliaeetus leucogaster</em><em> </em>(<a title="Johann Friedrich Gmelin" href="https://en.wikipedia.org/wiki/Johann_Friedrich_Gmelin">Gmelin</a>, 1788); DNA barcoding, <em>C</em><em>ytochrome-c </em><em>O</em><em>xidase </em><em>S</em><em>ubunit</em> I (COI).</p></div>

Identification and DNA barcoding of Tabanid flies in the pasture area of sheep at Mannavanur, Palani Hills, Tamil Nadu, India.

Sequence analysis based on the mitochondrial cytochrome c oxidase subunit Ⅰ(COI) gene fragment was conducted for 21 clam species belonging to the family Veneridae to determine the utility of this sequence in identifying phylogeographic and phylogenetic structure.The DNAs of 21 clam species were extracted and subjected to PCR using primers COI-F and COI-R.The resultant COI fragment was 707 bp in all species,with A+T contents ranging from 62.4%—67.8%.The COI gene sequences of all 21 species were analyzed using MEGA 5.0 software.Interspecific COI sequence divergences was very high,ranging from 7.2%—38.2%,mainly as a result of transitions and transversions.The minimum value of 7.2% was observed between Meretrix meretrix and Meretrix lusoria,and the maximum value of 38.2% was between Tapes literata and Cyclina sinensis.M.lusoria exhibited 0.4% intraspecific sequence divergence among three sequenced individuals.However,for other species,the COI sequences were identical among the three sequenced individuals for each species.An analysis of COI gene sequence variation among 21 species revealed 379 variable sites,of which 334 were parsimony-informative sites,with 72 transitions(si) and 93 transversions(sv)(si/sv=0.8).Meanwhile,no indels(insertions/deletions) were detected in the COI sequences from any samples.The percentage of interspecific sequence divergence within the same genus,intergeneric sequence divergence within the same subfamily,and those between subfamilies were analyzed.The mean values were 12.5%—27.3%,19.7%—29.0% and 29.2%,respectively.The COI fragment encodes 235 amino acids,of which 100 sites were variable.A phylogenetic tree of 35 Veneridae clams was constructed based on the COI nucleotide sequences,with Mactra chinensis as an outgroup species.Tree topologies showed that all species of Veneridae are in the same group,forming a clade distinct from the outgroup species.Within Veneridae,six species of the subfamily Dosininae formed a clade with a bootstrap value of 75%.Two species of the subfamily Venerinae formed another clade with a bootstrap value of 93%.All species of the subfamily Meretricinae formed a monophyletic group with a bootstrap value of 70%,in which M.meretrix,M.lusoria and Meretrix petechialis were first clustered with a high bootstrap value of 99%.This result indicated that M.meretrix,M.lusoria,and M.petechialis have a closer relationship than other species.An analysis of tree topology and sequence alignment indicated that M.lusoria,M.petechialis,and M.meretrix are closely related species.The data support the viewpoint that M.petechialis and M.lusoria should be treated as synonymous with M.meretrix,and may be described as two geographical subspecies.The data also support the perspective that Dosinia corrugata and Dosinia angulosa are two distinct species.The validity of Paphia undulata and Paphia textile as different species is also supported.This result reveals that the COI gene is an appropriate marker for studies in phylogeny and phylogeography of the Veneridae clams.

Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5' fragment of the cytochrome c oxidase I (COI) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, the COI gene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful.

Bivalve molluscs are a diverse group of animals with particular economic and ecological importance. Their morphological characteristics frequently confuse their identification leading to mislabelling of edible species. Genetic diversity is critical to the resilience of marine bivalve populations in the face of environmental stressors such as ocean acidification and warming. In this study, we characterized the phylogeny and defined the first DNA barcodes of six marine bivalves [Ostrea edulis (Linnaeus, 1758) Arca noae (Linnaeus, 1758), Pinctada radiata (Leach, 1814), Venus verrucosa (Linnaeus, 1758), Calllista chione (Linnaeus, 1758) and Ruditapes decussatus (Linnaeus, 1758)] sampled from different coastal areas of Aegean and Ionian Seas using the molecular markers cytochrome c oxidase subunit I (COI) and 18S ribosomal RNA (18S rRNA). Further, COI gene was employed to investigate the population genetic diversity since 18S rRNA exhibited no conspecific differences. The sequence of 18S rRNA successfully discriminated the bivalves at family or superfamily level but occasionally proved insufficient for species identification. Contrariwise, COI was highly informative and could reliably distinguish all species. Population haplotype diversity was moderate to high and was always accompanied by generally low nucleotide diversity, indicating genetically closely related haplotypes. The invasive Pinctada radiata was found to be panmictic even among distant sampling areas, while Ostrea edulis was the only species that exhibited moderate levels of population subdivision. Finally, here we report for the first time the presence of Ostrea stentina in Thermaikos Gulf sampled among Ostrea edulis specimens, demonstrating a new invasive bivalve species in Eastern Mediterranean.

Objective To apply the DNA barcoding technology for identification of rodent animals and to establish a rodent animal DNA barcode database in Gansu Province. Methods A total of 54 rodent animals were detected. DNA barcoding technology was used to analyze the DNA mitochondrial cytochrome C oxidase subunit I (CO I) gene sequence in Gansu Province. Results The intra-specific genetic distance was 0-2% while the inter-specific distance ranged from 18% to 30%. Eight major clusters were apparently showed on a Neighbor joining tree. Conclusion DNA barcoding technology could overcome the shortcomings of the morphological identification, so it could be used to identify the rodent animals and has important implications for disease control and prevention in the natural focus of Gansu Province. Key words: Rodentia; DNA barcoding; Mitochondrial cytochrome C oxidase subunit I; Species identification

Genes for URF2, tRNAtrp, tRNAcys, tRNAtyr and cytochrome c oxidase subunit I (COI) have been identified within a sequenced segment of the Drosophila yakuba mtDNA molecule. The five genes are arranged in the order given. Transcription of the tRNAcys and tRNAtyr genes is in the same direction as replication, while transcription of the URF2, tRNAtrp and COI genes is in the opposite direction. A similar arrangement of these genes is found in mammalian mtDNA except that in the latter, the tRNAala and tRNAasn genes are located between the tRNAtrp and tRNAcys genes. Also, a sequence found between the tRNAasn and tRNAcys genes in mammalian mtDNA, which is associated with the initiation of second strand DNA synthesis, is not found in this region of the D. yakuba mtDNA molecule. As the D. yakuba COI gene lacks a standard translation initiation codon, we consider the possibility that the quadruplet ATAA may serve this function. As in other D. yakuba mitochondrial polypeptide genes, AGA codons in the URF2 and COI genes do not correspond in position to arginine-specifying codons in the equivalent genes of mouse and yeast mtDNAs, but do most frequently correspond to serine-specifying codons.

Mitochondrial genes for cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) of the sea anemone Metridium senile (phylum Cnidaria) each contain a group I intron. This is in contrast to the reported absence of introns in all other metazoan mtDNAs so far examined. The ND5 intron is unusual in that it ends with A and contains two genes (ND1 and ND3) encoding additional subunits of NADH dehydrogenase. Correctly excised ND5 introns are not circularized but are precisely cleaved near their 3' ends and polyadenylylated to provide bicistronic transcripts of ND1 and ND3. COI introns, which encode a putative homing endonuclease, circularize, but in a way that retains the entire genome-encoded intron sequence (other group I introns are circularized with loss of a short segment of the intron 5' end). Introns were detected in the COI and ND5 genes of other sea anemones, but not in the COI and ND5 genes of other cnidarians. This suggests that the sea anemone mitochondrial introns may have been acquired relatively recently.

BackgroundPlasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources.ResultsA population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable.ConclusionsThis two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

We evaluated the extent of intraspecific and interspecific genetic distances and the effectiveness of predefined threshold values using the main genes for estimates of biodiversity and specimens' identification in anurans. Partial sequences of the mitochondrial genes for small (12S) and large (16S) ribosomal subunits, cytochrome c oxidase subunit I (COI) and Cytochrome b (Cytb) of the family Hylidae were downloaded from GenBank and curated for length, coverage, and potential contaminations. We performed analyses for all sequences of each gene and the same species present in these datasets by distance and tree (monophyly)-based evaluations. We also evaluated the ability to identify specimens using these datasets applying "nearest neighbor" (NN), "best close match" (BCM) and "BOLD ID" tests. Genetic distance thresholds were generated by the function 'threshVal' and "localMinima" from SPIDER package and traditional threshold values (1%, 3%, 6% and 10%) were also evaluated. Coding genes, especially COI, had a better identification capacity than non-coding genes on barcoding gap and monophyly analysis and NN, BCM, BOLD ID tests. Considering the multiple factors involved in global DNA barcoding evaluations, we present a critical assessment of the use of these genes for biodiversity estimation and specimens' identification in anurans (e.g. hylids).

Tree frog (Polypedates celebensis) is an endemic amphibi found in Southeast Sulawesi. Database ofthis species, in particular genetic databases is still very limited. The purpose of this research was tocharacterize the partial gene cytochrome C oxidase subunit I (COI) of tree frog (P. celebensis). Frog sampleswere collected from Tanjung Peropa Wildlife Reserve, Moramo, Southeast Sulawesi, and extracted theirDNA genome, and amplified using the polymerase chain reaction (PCR) method, with specific primers,and then sequenced. Characterization was done by comparing the COI gene with another COI gene fromother frog (data available on GenBank). The results showed that the COI gene fragment P. celebensisconsisted of 665 base pairs. These fragment gene had 99% conserved nucleotides, and only 1% nucleotidesthat varied. Nine sites of nucleotide varied namely: 65th, 98th, 101th, 358th, 396th, 403th, 405th, 424th, and426th. These sites of nucleotide varied can be used as genetic markers for P. celebensis. Composition ofnitrogenous bases P. celebensis COI gene fragment were 29.9% timine, 28.9% cytocine, 22.0% adenine, and19.2% guanine. Molecular phylogeny of P. celebensis based on 665 bp of their COI gene suggests that thesespecies was monophyletic. It is concluded that the gene fragment of cytochrome C oxidase of tree frog (P.celebensis) the origin of Wildlife Moramo, Southeast Sulawesi, has conserved nucleotides more thannucleotides that varied. These varied nucleotides can be used as genetic markers especially for the treefrog origin of Wildlife Moramo, Southeast Sulawesi. The most nitrogenous bases is thymine, while thefewest is guanine. Molecular phylogeny based on cytochrome C oxidase gene is monophyletic

Beetles (Coleoptera) represent one of the most diverse insect groups and play vital ecological roles, yet their accurate identification is often challenging due to morphological similarities among taxa. DNA barcoding has emerged as a powerful and reliable tool for species-level identification and biodiversity monitoring. In this study, we established a local DNA barcode reference database for beetles in the Liancheng Nature Reserve, Gansu Province, China. From May to August 2024, beetle specimens were collected and identified using both morphological traits and DNA barcoding. Three species delimitation methods—Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Bayesian Poisson Tree Processes (bPTP)—were employed as complementary analytical tools, and phylogenetic relationships were inferred from cytochrome c oxidase subunit I (COI) sequences. A total of 164 COI sequences (650 bp) were obtained, representing 126 beetle species from 95 genera and 20 families. DNA barcoding successfully resolved morphologically ambiguous taxa, with many sequences reported here for the first time. Phylogenetic analysis revealed that species within the same genus formed cohesive clades before clustering at the family level, confirming the species-level discriminative power of the COI gene. Collectively, these findings demonstrate that COI-based DNA barcoding is a powerful complement to traditional taxonomy. The establishment of this preliminary reference database provides a valuable molecular resource for beetle identification and a practical tool to support biodiversity conservation, resource management, and long-term monitoring in the Liancheng Nature Reserve.

In wildlife forensics, precise species identification is essential, particularly in cases involving poaching, illegal wildlifetrade, and biodiversity conservation. Molecular technologies are necessary for accurate forensic analysis since traditionalmorphological methods frequently fail when biological specimens are processed, incomplete, or degraded. MitochondrialDNA (mtDNA) has proven particularly useful in these situations because of its large copy number, maternal mode ofinheritance, and increased stability in damaged tissues. The cytochrome c oxidase subunit I (COI) gene is one of the mostcommonly used mtDNA markers for DNA barcoding due to its high interspecies variability, evolutionary conservation,and inclusion in extensive international databases.This study examined the COI gene sequences of ten often encountered domestic and wild Indian species: Elephas maximus(elephant), Bos gaurus (gaur), Bos taurus (domestic cow), Axis axis (chital), Axis porcinus (hog deer), Macaca mulatta (rhesusmacaque), Pavo cristatus (Indian peacock), Sus scrofa (wild boar), and Panthera tigris (tiger) and Panthera pardus (leopard).COI sequences that are publicly accessible were obtained from NCBI GenBank database. BLAST was used for sequencevalidation, and Clustal Omega was used for multiple sequence alignment. Using MEGA12, a neighbor-joining tree wasconstructed and pairwise distance analysis was performed to evaluate genetic distance and phylogenetic relationships.Significant genetic divergence was found among the species under study, and the phylogenetic tree showed groupingpatterns that aligned with established taxonomic relationships. These results demonstrate that COI-based DNA barcodingis a reliable method for differentiating between closely related species.

Amphibians, a diverse and ecologically important group, are facing global declines due to various factors, including habitat loss and climate change. Accurate species identification is crucial for effective conservation efforts, and DNA barcoding has emerged as a powerful tool in this regard. This study compares the efficacy of two DNA barcoding primer sets, targeting the 16S ribosomal RNA gene and the Cytochrome Oxidase I (COI) gene, for identifying 20 amphibian species. While both primer sets successfully amplified sequences, the 16S rRNA gene region identified all 20 samples, whereas the COI region identified 14. The amplified sequences, approximately 550 base pairs for 16S rRNA and 658 base pairs for COI facilitated precise taxonomic placement within amphibian families using Neighbor-Joining phylogenetic trees. These findings enhance DNA barcoding methodology and aid in understanding amphibian diversity, crucial for effective conservation strategies amidst global declines drives by habitat loss, diseases, and climate change.