DNA Barcoding Technique Research Articles

Fish product mislabeling identified in the Russian far east using DNA barcoding

DNA barcoding techniques combined with the BOLD (www.boldsystems.org) identification engine and the locally assembled barcode library were applied to investigate mislabeling of 22 specimens of frozen fish and fillets from food stores. Processed fish (fillets) and whole fish exemplars, sold under the labels Halibut, Flounder, Cod, Walleye Pollock, and Tilapia, from two food/fish stores of Vladivostok, Russia were analyzed. Based on the BOLD identification system, as well as using the local sequence reference library, it was found that four different species representing three genera of the family Pleuronectidae were labeled at two stores by the common name “flounder”. In addition, in one case walleye pollock was mislabeled as cod fillet. Tilapia fillets were not identified accurately due to the lack of a comprehensive comparative data, i.e. reference library. Some cases of labeling were also non-compliant with the rules of Sea-Fisheries Protection Authority's (SFPA), Russian Federation (Information notice on Labeling Changes) that raises questions regarding risk assessment and risk management. The necessity of additional studies to trace the major sources and stages of seafood trade, where mislabeling is most likely to occur, has become evident.

Molecular identification of two gobi fishes of Bangladesh using cytochrome oxidase subunit I (CoI) gene sequences

DNA barcoding has been proposed as a means of quick species identification using a short standardized segment of DNA. Two species (Eleotris fusca and Glossogobius giuris) from the family Gobiidae and Eleotridae were selected for DNA barcoding using samples collected from different regions of Bangladesh. Cytochrome Oxidase Subunit I (COI) gene was sequenced from two different gobi fishes and compared with two previously published similar sequences from the genera Eleotris and Glossogobius. Multiple sequence alignment and the molecular systematic study were performed. The DNA barcode technique identified the two species. The study provides a good example of how DNA barcoding can build upon its primary mission of species identification and use available data to integrate genetic variation investigated at the local scale into a global framework.Bangladesh J. Zool. 44(2): 175-184, 2016

A DNA Barcoding Based Study to Identify Main Mosquito Species in Taiwan and its Difference from Those in Mainland China.

Mosquitoes can transmit many types of viruses such as West Nile virus and Zika virus and are responsible for a number of virus-causing diseases including malaria, dengue fever, yellow fever, lymphatic filariasis, and Japanese B encephalitis. On January 19, 2016, the first case of Zika virus infection was identified in Taiwan, which presents the need for studying the mosquito species in the Taiwan Strait and evaluating the risk of the outbreak of this infection. In this study, we have collected 144 mosquito specimens from 42 species belonging to nine genera from both sides of the Taiwan Strait during 2013 and 2014. We then applied the COI DNA Barcoding technique to classify the specimens and performed a phylogenetic analysis to infer the evolutionary history of these mosquitoes. Based on the analyses, we found that though the mosquitoes from different sides of the Taiwan Strait share a lot of commonality, they have a few regional specificities. Our results also suggested a very small divergences (1%~9%) between specimens from the same mosquito species and relatively large divergences (8%~25%) between specimens from different mosquito species. Within the same species, the divergence of specimens from the same region is significantly smaller than that between two regions. A few highly divergent species between Fujian and Taiwan (e.g., An.maculatus and Ae.elsiae) might be formed due to the so-called "cryptic evolutionary events", in which the species has differentiation into cryptic species due to geographical differences without changing morphological characteristics. In conclusion, the phylogenetic analyses showed a very similar taxonomy to the historical one based on morphological characteristics, validating again the application of COI DNA Barcoding technique in classifying mosquito species. However, there are also some inconsistencies between COI DNA Barcoding and historical taxonomy, which points out the differences between mosquito DNA and morphological characteristics and suggests the possibility to improve mosquito taxonomy based on DNA techniques.

Morphological and genetic analyses of the first record of longrakered trevally, Ulua mentalis (Perciformes: Carangidae) and of the pinjalo snapper, Pinjalo pinjalo (Perciformes: Lutjanidae) in the Odisha coast, Bay of Bengal

Identification of fish species have so far been carried out mostly by classical morpho-taxonomy. In the present study, however, an attempt has been taken to identify two species of fishes Ulua mentalis and Pinjalo pinjalo of order Perciformes which happens to be the first record in Odisha coast Bay of Bengal, India during the year 2015, using DNA barcoding technique for reconfirmation over conventional morpho-taxonomy. During recent past, study of molecular-taxonomical profile of mitochondrial DNA in general and Cytochrome Oxidase subunit I (COI) gene in particular has gained enormous importance for accurate identification of species. In the present study, the partial COI sequence of Ulua mentalis and Pinjalo pinjalo were generated. Analysis using the COI gene produced phylogenetic trees in concurrence with other multi gene studies and we came across the identical phylogenetic relationship considering Neighbor-Joining and Maximum Likelihood tree. Moreover, these molecular data set further testified in Bayesian framework to reevaluate the exact taxonomic groupings within the family. Surprisingly, Ulua mentalis and Pinjalo pinjalo seems to be closely related to their sister taxa.

DNA Barcoding of Cheimerius nufar (Valenciennes, 1830) (Perciformes: Sparidae) From the Sultanate of Oman

Santer sea bream Cheimerius nufaris one of the most important commercial fish of the Oman.Presented work aims to use DNA barcoding techniques to identify Cheimerius nufer. Five representative samples were taken from three locations in the Sultanate of Oman (Ashkarah, Lakbi and Salalah). Species identification is done by extracted DNA which used for PCR amplification of COI gene and sequencing the mitochondrial gene cytochrome oxidase COI gene. Then the species identification compared the generated sequence with the databases at NCBI and BOLD. Neighbour Joint (NJ) clustering analysis showed that all samples formed single monophyletic clade further suggesting that they belonged to Cheimerius nuferwith percent identity more than 98%.

Identification of the Holothurian species of the Red Sea and Gulf of Aqaba using DNA barcoding technique.

Identification of the Holothurian species of the Red Sea and Gulf of Aqaba using DNA barcoding technique.

Characterization of Three Species of Thrips on Weeping Fig, Nutmeg, and Marine Seruni Plants Based on Mtcoi DNA Sequences

<p>Thrips are widely reported as pests in vegetable crops. However, the existence of Phlaeothripidae members has a less concern in Indonesia. Phlaeothripidae is the only family of Tubulifera Suborder and some reports suggested that they had potential to be pests in several crops due to their ability to roll up and to make galls on leaves. The first step in pest management attempt is to identify the pest accurately and quickly, so the pest management can be on target and more efficient. One of the identification methods is the molecular identification using DNA barcoding techniques. This study aimed to characterize and to compare species thrips in banyan, nutmeg, and marine seruni based on their molecular characteristics. This research was conducted in Bogor and Kuningan. The process of molecular characterization consisteds of four steps DNA total extraction, amplification by using PCR, COI gene sequence, and data analysis. PCR programme was succesfully to amplified mt<em>COI</em> gene fragment at 710 bp. The length of mt<em>COI </em>gene of <em>Gynaikothrips uzeli, Haplothrips ganglbaueri</em>, and <em>Pseudophilothrips ichini</em> were 704, 686, and 702 bp dominated by A and T bases with nucleotide variation value of 27.8%. This results confirmed that molecular characterization using mt<em>COI </em>gene mitochondrial had successfully supported the morphological data. </p><p><strong>How to Cite</strong></p><p>Kurniawaty, N., Hidayat, P. & Rauf, A. (2016). Characterization of Three Species of Thrips on Banyan, Nutmeg, and Marine Seruni Plants Based on Coi Gene. <em>Biosaintifika: Journal of Biology & Biology Education</em>, 8(2), 185-192.</p>

Forensic species identification of elasmobranch products sold in Costa Rican markets

One barrier to establishing catch limits to help protect shark populations is a lack of accurate species-specific extraction rates. This is due to many species looking similar, distinguishing characteristics (fins and head) of sharks commonly being removed, or sharks being grouped together in fisheries data. For this study, we collected elasmobranch (shark and ray) tissue samples from the central markets in San Jose (10 fish vendors or pescadarias) and Heredia (5 pescadarias) from June 2013 to September 2014. We used DNA barcoding techniques to amplify approximately 1050bp of the NADH dehydrogenase subunit 2 (NADH2) gene (n=833). We found that at least nine species of shark (Alopias pelagicus, Carcharhinus falciformis, C. limbatus, C. obscurus, Mustelus lunulatus, Nasolamia velox, Rhizoprionodon longurio, Sphyrna lewini, S. zygaena) and one ray (Dasyatis longa) are being sold in local markets, with C. falciformis representing 87.3% of shark samples tested (n=637) and D. longa representing 100% of ray samples tested (n=85). Our results suggest that C. falciformis continues to be under intense fishing pressure in the waters around Costa Rica despite recent concern over continued population declines. Although the number of Endangered S. lewini (4%) being sold in the markets is much less than for C. falciformis (87.3%), the numbers are still concerning given their current conservation status.

DNA Barcoding for the Identification of Botanicals in Herbal Medicine and Dietary Supplements: Strengths and Limitations.

In the past decades, the use of traditional medicine has increased globally, leading to a booming herbal medicine and dietary supplement industry. The increased popularity of herbal products has led to a rise in demand for botanical raw materials. Accurate identification of medicinal herbs is a legal requirement in most countries and prerequisite for delivering a quality product that meets consumer expectations. Traditional identification methods include botanical taxonomy, macroscopic and microscopic examination, and chemical methods. Advances in the identification of biological species using DNA-based techniques have led to the development of a DNA marker-based platform for authentication of plant materials. DNA barcoding, in particular, has been proposed as a means to identify herbal ingredients and to detect adulteration. However, general barcoding techniques using universal primers have been shown to provide mixed results with regard to data accuracy. Further technological advances such as mini-barcodes, digital polymerase chain reaction, and next generation sequencing provide additional tools for the authentication of herbs, and may be successful in identifying processed ingredients used in finished herbal products. This review gives an overview on the strengths and limitations of DNA barcoding techniques for botanical ingredient identification. Based on the available information, we do not recommend the use of universal primers for DNA barcoding of processed plant material as a sole means of species identification, but suggest an approach combining DNA-based methods using genus- or species-specific primers, chemical analysis, and microscopic and macroscopic methods for the successful authentication of botanical ingredients used in the herbal dietary supplement industry.

DNA barcode for the identification of the sand fly Lutzomyia longipalpis plant feeding preferences in a tropical urban environment.

Little is known about the feeding behavior of hematophagous insects that require plant sugar to complete their life cycles. We studied plant feeding of Lutzomyia longipalpis sand flies, known vectors of Leishmania infantum/chagasi parasites, in a Brazilian city endemic with visceral leishmaniasis. The DNA barcode technique was applied to identify plant food source of wild-caught L. longipalpis using specific primers for a locus from the chloroplast genome, ribulose diphosphate carboxylase. DNA from all trees or shrubs within a 100-meter radius from the trap were collected to build a barcode reference library. While plants from the Anacardiaceae and Meliaceae families were the most abundant at the sampling site (25.4% and 12.7% of the local plant population, respectively), DNA from these plant families was found in few flies; in contrast, despite its low abundance (2.9%), DNA from the Fabaceae family was detected in 94.7% of the sand flies. The proportion of sand flies testing positive for DNA from a given plant family was not significantly associated with abundance, distance from the trap, or average crown expansion of plants from that family. The data suggest that there may indeed be a feeding preference of L. longipalpis for plants in the Fabaceae family.

DNA-Based Identification and Chemical Characteristics of Hypnea musciformis from Coastal Sites in Ghana

This work reveals new, important insights about the influence of broad spatial variations on the phylogenetic relationship and chemical characteristics of Ghanaian Hypnea musciformis—a carrageenan-containing red seaweed. DNA barcoding techniques alleviate the difficulty for accurate morphological identification. COI barcode sequences of the Ghanaian H. musciformis showed <0.7% intraspecies divergence, indicating no distinct phylogenetic variation, suggesting that they actually belong to the same species. Thus, the spatial distribution of the sampling sites along the coast of Ghana did not influence the phylogenetic characteristics of H. musciformis in the region. The data also showed that the Ghanaian Hypnea sp. examined in this work should be regarded as the same species as the H. musciformis collected in Brazilian Sao Paulo (KP725276) with only 0.8%–1.3% intraspecies divergence. However, the comparison of COI sequences of Ghanaian H. musciformis with the available COI sequence of H. musciformis from other countries showed intraspecies divergences of 0%–6.9% indicating that the COI sequences for H. musciformis in the GenBank may include different subspecies. Although samples did not differ phylogenetically, the chemical characteristics of the H. musciformis differed significantly between different sampling locations in Ghana. The levels of the monosaccharides, notably galactose (20%–30% dw) and glucose (10%–18% dw), as well as the seawater inorganic salt concentration (21–32 mg/L) and ash content (19%–33% dw), varied between H. musciformis collected at different coastal locations in Ghana. The current work demonstrated that DNA-based identification allowed a detailed understanding of H. musciformis phylogenetic characteristics and revealed that chemical compositional differences of H. musciformis occur along the Ghanaian coast which are not coupled with genetic variations among those samples.

DNA Barcoding of Birds at a Migratory Hotspot in Eastern Turkey Highlights Continental Phylogeographic Relationships.

The combination of habitat loss, climate change, direct persecution, introduced species and other components of the global environmental crisis has resulted in a rapid loss of biodiversity, including species, population and genetic diversity. Birds, which inhabit a wide spectrum of different habitat types, are particularly sensitive to and indicative of environmental changes. The Caucasus endemic bird area, part of which covers northeastern Turkey, is one of the world’s key regions harboring a unique bird community threatened with habitat loss. More than 75% of all bird species native to Turkey have been recorded in this region, in particular along the Kars-Iğdır migratory corridor, stopover, wintering and breeding sites along the Aras River, whose wetlands harbor at least 264 bird species. In this study, DNA barcoding technique was used for evaluating the genetic diversity of land bird species of Aras River Bird Paradise at the confluence of Aras River and Iğdır Plains key biodiversity areas. Seventy three COI sequences from 33 common species and 26 different genera were newly generated and used along with 301 sequences that were retrieved from the Barcoding of Life Database (BOLD). Using the sequences obtained in this study, we made global phylogeographic comparisons to define four categories of species, based on barcoding suitability, intraspecific divergence and taxonomy. Our findings indicate that the landbird community of northeastern Turkey has a genetical signature mostly typical of northern Palearctic bird communities while harboring some unique variations. The study also provides a good example of how DNA barcoding can build upon its primary mission of species identification and use available data to integrate genetic variation investigated at the local scale into a global framework. However, the rich bird community of the Aras River wetlands is highly threatened with the imminent construction of the Tuzluca Dam by the government.

Duplex DNA barcoding allows accurate species determination of morphologically similar limpets (Patellaspp.) from non-destructive sampling

Accurate discrimination of two morphologically similar species ofPatellalimpets has been facilitated by using qPCR amplification of species-specific mitochondrial genomic regions. Cost-effective and non-destructive sampling is achieved using a mucus swab and simple sample lysis and dilution to create a PCR template. Results show 100% concurrence with dissection and microscopic analysis, and the technique has been employed successfully in field studies. The use of highly sensitive DNA barcoding techniques such as this hold great potential for improving previously challenging field assessments of species abundance.

DNA barcoding, species-specific PCR and real-time PCR techniques for the identification of six Tribolium pests of stored products.

Flour beetles of the genus Tribolium Macleay (Coleoptera: Tenebrionidae) are important stored product pests in China and worldwide. They are often found or are intercepted in grain depots, flour mills, and entry-exit ports, etc. Traditionally, Tribolium species are identified according to the morphological characteristics of the adult. However, it is almost impossible to rapidly identify adult fragments and non-adult stages based on external morphological characteristics. Molecular techniques for the rapid and accurate identification of Tribolium species are required, particularly for pest monitoring and the quarantine of stored products pests. Here, we establish DNA barcoding, species-specific PCR, and real-time PCR techniques for the identification of six stored-product pest Tribolium species including T. castaneum, T. confusum, T. destructor, T. madens, T. freemani and T. brevicornis. We detected the mitochondrial DNA cytochrome oxidase subunit I (COI) barcodes for Tribolium from 18 geographic populations and 101 individuals, built a Tribolium DNA barcode library, and designed species-specific primers and TaqMan probes for the above six Tribolium species. The three techniques were applied to identify Tribolium collected from stored samples and samples captured from quarantine ports. The results demonstrated that three techniques were all able to identify the six species of Tribolium both rapidly and accurately.

Isolation of a sp. nov. Ljungan virus from wild birds in Japan.

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

Application of DNA Barcode Technique in Identifying and Tracing Invasive Pest Insects in Agriculture and Forestry

Application of DNA Barcode Technique in Identifying and Tracing Invasive Pest Insects in Agriculture and Forestry

DNA Barcoding of the Endangered Aquilaria (Thymelaeaceae) and Its Application in Species Authentication of Agarwood Products Traded in the Market.

The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication.

Genetic diversity and phylogenetic analysis of two Tunisian bivalves (Mactridae) Mactra corallina (Linnaeus, 1758) and Eastonia rugosa (Helbling, 1799) based on COI gene sequences

A partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) was used as a genetic marker for a genetic diversity and phylogenetic analysis (DNA barcoding) of two Mactridae species, Mactra corallina and Eastonia rugosa, collected from the Tunisian coast. These Mactridae species could be distinguished by DNA barcoding techniques and they will be considered as monophyletic clades with the Neighbor-Joining (NJ) tree. The genetic structure detected that E. rugosa presents three haplotypes with a high frequency of HER1 (0.89). However, M. corralina shared 14 haplotypes. The haplotypic diversity (H) was equal to 0.205 and 0.954, respectively, for E. rugosa and M. corallina. While the nucleotide diversity (π) was higher for M. corallina (π=0.0818), the mismatch distribution showed a unimodal curve for E. rugosa (a recent sudden demographic expansion) and a multimodal distribution for M. corallina (size stability).

Molecular phylogeny and DNA barcoding confirm cryptic species in the African freshwater oysterEtheria ellipticaLamarck, 1807 (Bivalvia: Etheriidae)

Recent molecular approaches to taxonomy have led to a steady increase in the identification of cryptic species. Within the Etheriidae, the species Etheria elliptica (freshwater oyster) is widespread and common and exists in most of the major African drainages. Within the African freshwater ecosystems, there are major threats to biodiversity and cryptic species complicate conservation strategies; unknown species exist and no conservation status has been assigned. Our objective here was to determine if E. elliptica from several locations in the Congo drainage are correctly classified as representing a single species. We analysed the genetic diversity at two mitochondrial loci (COI and 16S) and two nuclear loci (H3 and 28S), and estimated evolutionary relationships using phylogenetic and DNA barcoding techniques. Bayesian inference yielded three cryptic species of Etheria, and mismatch analysis revealed discrete differences between the cryptic species. We identified three cryptic species within these collections, and evidence indicates that the third species may resolve further with more sampling. In conclusion, the taxonomic history of E. elliptica makes finding cryptic species unsurprising. However, molecular studies such as this may finally help to resolve the number of species within this genus.

Identification of Species in Tripterygium (Celastraceae) Based on DNA Barcoding.

Species of genus Tripterygium (Celastraceae) have attracted much attention owing to their excellent effect on treating autoimmune and inflammatory diseases. However, due to high market demand causing overexploitation, natural populations of genus Tripterygium have rapidly declined. Tripterygium medicinal materials are mainly collected from the wild, making the quality of medicinal materials unstable. Additionally, identification of herbal materials from Tripterygium species and their adulterants is difficult based on morphological characters. Therefore, an accurate, convenient, and stability method is urgently needed. In this wok, we developed a DNA barcoding technique to distinguish T. wilfordii HOOK. f., T. hypoglaucum (LÉVL.) HUTCH, and T. regelii SPRAGUE et TAKEDA and their adulterants based on four uniform and standard DNA regions (internal transcribed spacer 2 (ITS2), matK, rbcL, and psbA-trnH). DNA was extracted from 26 locations of fresh leaves. Phylogenetic tree was constructed with Neighbor-Joining (NJ) method, while barcoding gap was analyzed to assess identification efficiency. Compared with the other DNA barcodes applied individually or in combination, ITS2+psbA-trnH was demonstrated as the optimal barcode. T. hypoglaucum and T. wilfordii can be considered as conspecific, while T. regelii was recognized as a separate species. Furthermore, identification of commercial Tripterygium samples was conducted using BLAST against GenBank and Species Identification System for Traditional Chinese Medicine. Our results indicated that DNA barcoding is a convenient, effective, and stability method to identify and distinguish Tripterygium and its adulterants, and could be applied as the quality control for Tripterygium medicinal preparations and monitoring of the medicinal herb trade in markets.