DNA Barcoding Technique Research Articles

Comparison of morphological and molecular methods to identify the diet of a generalist omnivore

Abstract ContextEcologists need robust and effective methods to quantify the diet of animals. However, assessing dietary composition can be challenging because most animals are seldom observed eating, especially when studying rare or cryptic species. AimsMorphological analysis of scats has been extensively used previously, and recent advances in the accessibility of DNA barcoding techniques have also made molecular approaches a viable alternative for diet analysis from scats. We compared the results from two methods of scat analysis, to trial the use of contemporary approaches in scat analysis. MethodsIn the present study, we used morphological analysis and DNA barcoding of matter in scats to catalogue the diet of a generalist omnivore, the greater bilby (Macrotis lagotis Thylacomyidae), in the West Kimberley. The composition and diversity of diet items, as well as the taxonomic identification level, were compared between methods. Key resultsEach method provided complimentary results; morphological analysis uncovered the type of matter consumed (e.g. root, seed) and relative proportion of the total undigested content, whereas DNA barcoding could assign such matter to a taxon. Even though dietary DNA could be extracted from only 38% of scats, DNA barcoding identified a greater diversity of taxa in scats than did morphological analyses. Barcoding could detect the presence of highly-digestible items such as cossid moths (Cossidae) and spiders (Araneae). ConclusionsMorphological analysis was useful for quantifying relative abundance of diet categories; however, DNA barcoding detected a greater diversity of dietary items within scats. Despite the expense of DNA barcoding, the method can more accurately identify the taxa consumed, whereas morphology can greatly underestimate dietary species diversity. However, the technical requirements for performing DNA analysis make it expensive, while resource-limited field ecologists can generally perform morphological analysis with appropriate training. ImplicationsResearchers and land managers will benefit from using both approaches in concert to gain a robust understanding of the local bilby diet. However, the cost and limitations of DNA barcoding (particularly when dealing with degraded DNA) mean that this approach should only be employed when the quality of the genetic material within samples is suitable. We recommend conducting exploratory analysis using morphological analysis (potentially in the field), with follow-up DNA barcoding to detect highly digestible items in fresh scats.

Genetic Diversity of Pediculus humanus capitis (Phthiraptera: Pediculidae) in Peninsular Malaysia and Molecular Detection of Its Potential Associated Pathogens.

Pediculosis capitis caused by Pediculus humanus capitis (De Geer) is endemic all over the world, and children are mostly affected, particularly those living in overcrowded institutions. Several studies have shown that P. h. capitis carried human pathogenic bacteria, suggesting the potential role of head lice in the transmission of pathogens to humans. In this study, we determined the genetic diversity of head lice collected from welfare homes sheltering underprivileged children by using DNA barcoding and demonstrated the presence of Acinetobacter spp., Serratia marcescens, and Staphylococcus aureus in head lice, which have never been investigated before in Malaysia. Cox1 DNA barcoding identified the head lice, P. h. capitis collected from welfare homes across two geographical areas of Peninsular Malaysia as belonging to clades A, B, and D. Acinetobacter bacteria: Acinetobacter guillouiae, Acinetobacter junii, Acinetobacter baumannii, and Acinetobacter nosocomialis were detected in head lice belonging to clades A and also D. In addition, DNA from S. marcescens and S. aureus were also detected in both clades A and D. To our knowledge, this is the first report on the genetic diversity of head lice in Malaysia through DNA barcoding, as well as the first to provide molecular evidence on the type of bacteria occurring in head lice in Malaysia. It is anticipated that the DNA barcoding technique used in this study will be able to provide rapid and accurate identification of arthropods, in particular, medically important ectoparasites.

Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing

BackgroundMore than 80% of all animal species remain unknown to science. Most of these species live in the tropics and belong to animal taxa that combine small body size with high specimen abundance and large species richness. For such clades, using morphology for species discovery is slow because large numbers of specimens must be sorted based on detailed microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA sequences could be used for sorting specimens to species. Morphological verification of such “molecular operational taxonomic units” (mOTUs) could then be based on dissection of a small subset of specimens. However, this approach requires cost-effective and low-tech DNA barcoding techniques because well-equipped, well-funded molecular laboratories are not readily available in many biodiverse countries.ResultsWe here document how MinION sequencing can be used for large-scale species discovery in a specimen- and species-rich taxon like the hyperdiverse fly family Phoridae (Diptera). We sequenced 7059 specimens collected in a single Malaise trap in Kibale National Park, Uganda, over the short period of 8 weeks. We discovered > 650 species which exceeds the number of phorid species currently described for the entire Afrotropical region. The barcodes were obtained using an improved low-cost MinION pipeline that increased the barcoding capacity sevenfold from 500 to 3500 barcodes per flowcell. This was achieved by adopting 1D sequencing, resequencing weak amplicons on a used flowcell, and improving demultiplexing. Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% accuracy, 0.46% Ns) and thus yielded very similar species units (match ratio 0.991). Morphological examination of 100 mOTUs also confirmed good congruence with morphology (93% of mOTUs; > 99% of specimens) and revealed that 90% of the putative species belong to the neglected, megadiverse genus Megaselia. We demonstrate for one Megaselia species how the molecular data can guide the description of a new species (Megaselia sepsioides sp. nov.).ConclusionsWe document that one field site in Africa can be home to an estimated 1000 species of phorids and speculate that the Afrotropical diversity could exceed 200,000 species. We furthermore conclude that low-cost MinION sequencers are very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. MinION sequencing could quickly reveal the extent of the unknown diversity and is especially suitable for biodiverse countries with limited access to capital-intensive sequencing facilities.

Standard reference material (SRM) DNA barcode library approach for authenticating Antidesma bunius (L.) Spreng. (bignay) derived herbal medicinal products

ABSTRACTIn the Philippines, the herbal medicinal product (HMP) market is flourishing due to the abundance of pharmacologically important species, and the high level of ethnomedicinal knowledge still widely accepted by the public. As such, herbal products from Antidesma bunius (L.) Spreng., locally known as bignay, are popular as medicine for various ailments of the circulatory and digestive systems. Though efficacy is guaranteed, the authenticity of the marketed products is still in question as several other herbal plants can provide the said benefits. Similar morphology between wild species also hinders species identification and contributes confusion especially to the general consumer. The authenticity of the marketed HMPs was established by means of DNA barcoding techniques which offers quick and reliable species identification by means of (1) the Basic Local Alignment Search Tool (BLASTn) and (2) the establishment of the first Standard Reference Material (SRM) Herbal barcode library for Antidesma spp. A total of 56 gene accessions from matK-psbA-trnH-rbcL sequences of 9 wild Antidesma spp. comprised the SRM which was then used to confirm the identity of 11 randomly sampled bignay-derived HMPs. Following the BLASTn and the SRM (maximum likelihood tree reconstruction) criterion, the subjected sequences revealed that only three of the 11 HMPs were authentic A. bunius-derived products. The other eight HMPs contained substitutes that were either fillers or different herbal medicinal plant not indicated in the product labels. These results indicate that product safety should be reinforced with complete HMP authentication using traditional methods supported by molecular data.

The combination of ITS2 and psbA-trnH region is powerful DNA barcode markers for authentication of medicinal Terminalia plants from Thailand.

The dried fruits of Terminalia plant (Combretaceae) called "Samo" have been used as herbal medicine in Thai traditional medicine. Four "Samo" crude drugs, namely, Samo thai, Samo thed, Samo dee-ngu, and Samo phiphek, are used as the main ingredients in Triphala and Trisamo recipes. Their commercial products are available in processed and powdered form, but are difficult to authenticate by conventional methods. In this study, we aimed to discriminate species of genus Terminalia for the identification of their crude drugs by a DNA barcoding technique. A total of 208 closely related nucleotide sequences were obtained from nine Terminalia species collected from Thailand and the DDBJ/EMBL/GenBank database. An effective DNA barcode marker was selected from six DNA loci (matK, rbcL, psbA-trnH, ITS, ITS1, and ITS2) and their two-locus combination. All sequences were analyzed by three major methods: (1) BLAST search; (2) the genetic divergence method using Kimura 2-parameter (K2P) distance matrices; and (3) tree topology analysis based on the neighbor-joining method. Comparison of the six candidate DNA loci indicated that ITS identified Terminalia with 100% accuracy at the species and genus levels in the BLAST1 method. ITS2 showed the highest K2P variability. The data from the single markers and the two-locus combinations revealed that only the two-locus combinations, namely, the combinations of rbcL, ITS, ITS1, and ITS2 with psbA-trnH, clearly discriminated all the species. From the results of DNA sequence analysis and the three methods, ITS2 is recommended for the identification of Terminalia species to supplement psbA-trnH.

Using DNA Barcoding for Fingerprinting of Two Important Forage Crops Varieties (Alfalfa And Egyptian Clover)

Study was executed to differentiate and discriminate (Medicago sativa andTrifolium alexandrinum) using DNA barcoding genes [rbcl]and Cox1 genes. Identification of (Medicago sativa (Rammah 1) was completed via rbcl and Cox1 genesand was identified as Medicago sativa voucher G00199095 ribulose1,5 bisphosphate carboxylase / oxygenase large subunit gene, partial cds; chloroplast Sequence ID: KJ204375.1 or Medicago sativa voucher Ahrendsen 23 for rbcl and Cox1 genes. Identity estimation were listed with 90% as alfalfa, Rammah 1 Genotype ribulose1 /5 bisphosphate carboxylase / oxygenase large subunit gene sequences ID: KJ206375.1] also, identity values of 91.24% were recorded with for alfalfa Rammah 1 Genotype, cytochrome c oxidase subunit I gene cox 1 sequences ID: KJ 204375.1). Trifolium alexandrinum Helaly genotype was identified as Trifolium alexandrinum (Sequence ID: HM850407.1) and Trifolium alexandrinum voucher K-016Hv (Sequence ID: KU234213.1) by rbcl and Cox1 genes respectively. Affiliation of genetic source was revealed for Trifolium alexandrinum with 100 % match with origin which indicate rising possibility for applying discrimination through comparing with Medicago sativa which reflect the lowest genetic likeness with the source. Moreover, we might detect from the available data that we can use DNA Bar-coding Technique in Discriminating the local Egyptian Clover Genotypes and Protect Them internationally. Also, DNA Bar-coding can be used to determine the genetic polymorphism in identifying superior genotypes as source of parental genotypes in Egyptian clover breeding program in future.

Assessment of seasonal variation of diet composition in rodents using DNA barcoding and Real-Time PCR

The study of animal diet and feeding behaviour is a fundamental tool for the illustration of the ecological role of species in the ecosystem. However, size and quality of food intake samples make it hard for researchers to describe the diet composition of many small species. In our study, we exploited genomic tools for the analysis of the diet composition of the Savi’s pine vole (Microtus savii) using DNA barcoding and qPCR techniques for the identification of ingested plant species retrieved from stomach contents. In contrast with previous studies, we found that, despite being a fossorial species, the Savi’s pine vole is a selective feeder that undergoes intense superficial activity in search for food. In addition, our study shows that with a a priori knowledge of the candidate plant species included in animal diet, qPCR is a powerful tool to assess presence/absence, frequency of occurrence and electivity of ingested species. We conclude that this approach offers new opportunities to implement the analysis of food selection in small animals, thereby revealing a detailed picture of plant-animal interactions.

DNA barcoding application of garfish (Hemirhampus sp.) in North Maluku Sea

The purpose of this study was to identify the species of garfish in the waters of north Maluku by applying DNA bar-coding techniques. The method used in the study was the primary design of DNA extraction and isolation, and DNA amplification through polymerase chain reaction. The results showed that Gen Cyt b analysis amplified ± 350 bp amplicon and for the alignment analysis showed that the amplification sample sequence had similarity with the Cyt b gene sequence of Hemiramphus balao with value of 97%. Sequence analysis showed that the six specimens had very low genetic distances. It can be concluded that the isolate samples of garfish have a very close kinship.

DNA Barcoding of Cyprinid Fish Chagunius chagunio Hamilton,1822 from Phewa Lake, Nepal

The present study is the first of its type that uses a technique of DNA barcoding to determine identification and relationship of a species of fish from Phewa lake, Nepal. The mitochondrial DNA from two ethanol-preserved samples of fish, randomly collected from Phewa lake, was extracted using Gene AllExgene TMtissue extraction kit. 650 base pair of mitochondrial cytochrome c oxidase subunit 1 (CO1) was amplified using a cocktail of four primers and was sequenced bidirectionaly using Sanger sequence method. The DNA sequences were edited using AliView software. The sequences confirmed Chagunius chagunio as their alignment with 16 reference sequences belonging to Chagunius chagunio in the NCBI GenBank, scored highest percentage of Query Cover (75% to 100%) and Percentage Identity (97.29% to 100%). The MEGA software analysed the DNA sequences to obtain their corresponding protein sequences. The DNA sequences were submitted to the GenBank and accession numbers (MN087472 and MN087473) were obtained. Clustal Omega software analysed multiple sequence alignment among 19 homologous DNA sequences of Chagunius chagunio from India, Bangladesh and Phewa lake, Nepal. The percentage of similarity among the aligned sequences was calculated as 39.3%. Based on the neighbour joining tree, the Chagunius chagunio of Phewa lake is found closely related with Chagunius chagunio of Bangladesh.

A taxonomic review of the genus Hypnea (Gigartinales, Rhodophyta) in Brazil based on DNA barcode and morphology

The red algal genus Hypnea has a wide geographical distribution along the coast of Brazil, where it has economic and ecological importance. The relatively simple and plastic morphology, often influenced by the conditions of its habitat, complicates the identification of Hypnea species. In the past years, several studies dealing with the taxonomy of Hypnea on the coast of Brazil have changed considerably the known diversity of species in the region. Studies using molecular markers led to the description of new species, while other names were placed in synonymy. In this review, revaluation of the morphology and the addition of new sequences for COI-5P, UPA and rbcL-3P markers were used in order to better understand the diversity of Hypnea species and their range of distribution. An identification key is presented for eleven species of the genus Hypnea occurring on the coast of Brazil: H. brasiliensis P.B. Jesus, Nauer & J.M.C. Nunes, H. cervicornis J. Agardh, H. cornuta (Kutzing) J. Agardh, H. cryptica P.B. Jesus & J.M.C. Nunes, H. edeniana Nauer, Cassano & M.C. Oliveira, H. flava Nauer, Cassano & M.C. Oliveira, H. pseudomusciformis Nauer, Cassano & M.C. Oliveira, H. platyclada P.B. Jesus & J.M.C. Nunes, H. spinella (C. Agardh) Kutzing, H. yokoyana Nauer, Cassano & M.C. Oliveira and H. wynnei Nauer, Cassano & M.C. Oliveira. Of all taxonomic characteristics proposed by earlier studies, the only ones that have taxonomic value considering the species analyzed in this work were: (1) habit of the thallus, (2) main axis, (3) apex shape, (4) form of the branchlets and (5) tetrasporangial sori position in the branches. However, the identification of the species based only on morphological characteristics should be made with caution, once phenotypic plasticity and convergent morphologies are present in the group. The DNA barcode technique, especially the COI-5P marker, proved to be very effective in the identification and delimitation of the species, revealing scenarios that would go unnoticed by morphology alone.

A case study in forensic soil examination from China

Abstract Soil examination can provide useful forensic information about the spatial location and human activities of a suspect. Soil is widely used in criminal investigations. In a case that occurred in the countryside of Jilin Province, in the NE of China, soil was found adhering to clothing on a body. Examination of plant debris in the soil using plant DNA barcoding technique found it to be ginseng root, which indicated that the soil might have come from a ginseng plantation. In the first instance, it helped in finding the place where the body was initially buried. Then a comparison was made between the soil recovered from the body and from the ginseng plantation. Composite analysis of minerals, pollen types and elements provided additional information to assist in making comparisons. Trace amounts of soil located on the body played an important role in locating the burial site and was regarded as the most valuable evidence in convicting the suspect of murder even without the suspect's DNA being available.

The taxonomic position of Asian Holopedium (Crustacea: Cladocera) confirmed by morphological and genetic analyses

The cladoceran Holopedium gibberum Zaddach, 1855 (Ctenopoda: Holopediidae) was once thought to occur broadly in the northern hemisphere, but its cryptic sister species was recently separated from H. gibberum sensu stricto (s.s.) as a new species, Holopedium glacialis. In East Asia, although “H. gibberum” occurrence has been recorded in many water bodies, the identity of the surveyed populations has rarely been confirmed via molecular analyses. Thus, it is unclear whether it is actually H. gibberum s.s. or H. glacialis that is distributed in East Asia. We used DNA-barcoding techniques to check the taxonomic status of Holopedium samples collected in Japan. We sequenced mitochondrial cytochrome c oxidase subunit 1 (mtCOI) and nuclear 18S ribosomal DNA (nr18S) of Japanese Holopedium and compared the results with those of H. gibberum s.s. collected in Norway and H. glacialis collected in Canada. The mtCOI sequence divergences between Norwegian H. gibberum s.s. and Japanese Holopedium were at most 2.4%, which was within the degree of intraspecific differentiation in cladocerans. Norwegian H. gibberum s.s. and Japanese Holopedium shared identical nr18S haplotypes. Individuals of Canadian H. glacialis were genetically different from those of Japanese Holopedium. We therefore concluded that Japanese Holopedium can be identified as H. gibberum s.s.

Identification of Mislabelling in Frozen Fish Fillets Based on DNA Barcoding Analysis

A number of studies have shown that mislabelling and species substitutions in fish products are very common worldwide. This fraud has two major aspects: economics and health. Moreover, poor trading, and neglecting the species conservation status are growing threats for fish stocks. First the type and extend of this fraud in fish must be detected in order to take proper actions. As some markers (e.g. protein analysis and morphological features) can fail, DNA markers, especially sequencing of cytochrome oxidase I gene (or DNA barcoding), is becoming a more widely preferred methodology for species identification. In this study, DNA barcoding technique was employed to confirm the species names written on the product packages of fish fillets purchased from the market. The fillets were labeled as Nile tilapia (Oreochromis niloticus). Among the 15 fillet samples analyzed, only 4 of them were labeled correctly. Seven (47%) of them were found to originate from pangasius (Pangasianodon hypophthalmus) and three of them were found to originate from a different tilapia species (Oreochromis mossambicus). This paper revealed a significant mislabelling of frozen fish fillets in Turkey. Customers are making informed decisions based on many reasons (like health issues or palate) and they have the right to eat what they think they are paying for. The results indicate the necessity for taking immediate actions and regulations against fraud in food items to sustain food quality and safety.

Dates and rates in grape's plastomes: evolution in slow motion.

The family Vitaceae includes the domesticated grapevine (Vitis vinifera), one of the most economically important crops in the world. Despite the importance of Vitaceae, there is still considerable controversy surrounding their phylogenetic relationships and evolutionary timescales. Moreover, variation in rates of molecular evolution among Vitaceae remains mostly unexplored. The present research aims to fill these knowledge gaps through the analysis of plastome sequences. Thirteen newly sequenced grape plastomes are presented and their phylogenetic relationships examined. Divergence times and absolute substitution rates are inferred under different molecular clocks by the analysis of 95 non-coding plastid regions and 43 representative accessions of the major lineages of Vitaceae. Furthermore, the phylogenetic informativeness of non-coding plastid regions is investigated. We find strong evidence in favor of the random local clock model and rate heterogeneity within Vitaceae. Substitution rates decelerate in Ampelocissus, Ampelopsis, Nekemias, Parthenocissus, Rhoicissus, and Vitis, with genus Vitis showing the lowest values up to a minimum of ~ 4.65 × 10-11s/s/y. We suggest that liana-like species of Vitaceae evolve slower than erect growth habit plants and we invoke the "rate of mitosis hypothesis" to explain the observed pattern of the substitution rates. We identify a reduced set of 20 non-coding regions able to accurately reconstruct the phylogeny of Vitaceae and we provide a detailed description of all 152 non-coding regions identified in the plastomes of subg. Vitis. These polymorphic regions will find their applications in phylogenetics, phylogeography, and population genetics as well in grapes identification through DNA barcoding techniques.

First DNA Barcoding-Based Record of Tetranychus and their Predators in Egypt

Tetranychus sp. is a globally spread economic pest that affects multiple plant species affecting the quality and the quantity of the crop. Because of its economic importance, the ecological and molecular perspective was investigated deeply for this pest. From the ecological perspective, the numbers and the relative infestation of Tetranychus were investigated for different cultivars of maize, and we had found that there wer three cultivars that were relatively resistant in Egypt; namely, Pioneer 3057, 321 and 352 under the prevailing conditions of temperature and humidity. From the molecular perspective, the DNA barcoding technique was applied for the first time in Egypt for Tetranychus species and its predators, and we had found two species of Tetranychus; namely, Tetranychus cinnabarinus and Tetranychus ludeni Zacher which would have been mistakenly identified as Tetranychus urticae Koch., in most of the cases due to the high resemblance in morphology,and also we had found a predator ;namely, Neoseiulus barkeri, as one of the predators of Tetranychus species. This research proved that combining both ecological and molecular analysis of the pest is very important for understanding the best protection and prevention options. Moreover, the molecular DNA barcoding is a very reliable tool in tetranychus species identification and its predators.

A rapid protocol for generating arthropod DNA barcodes suitable for use with undergraduate students

ABSTRACT We provide a protocol for rapid DNA extraction from spiders suitable for undergraduate practical sessions. Students who were previously naïve to the theory and laboratory technique of DNA barcoding were successfully able to extract and recover 29 DNA sequences from 16 species of small spiders in the family Linyphiidae. We anticipate that with careful selection of specimens, undergraduate students could participate in sessions which both benefit their professional development and further taxonomic understanding across a variety of organisms.

Cytotoxic Withanolides from the Whole Herb of Physalis angulata L.

Physalis angulata L. is a medicinal plant of the Solanaceae family, which is used to produce a variety of steroids. The present study reports on the cytotoxic withanolides of this plant. The species of Physalis angulata L. was identified by DNA barcoding techniques. Two new withanolides (1–2), together with six known analogues (3–8), were isolated from the whole plant of Physalis angulata L. The structures of these new compounds were determined on the basis of extensive spectroscopic data analyses and electronic circular dichroism (ECD) calculations. The withanolides exhibited strong cytotoxic activities against A549, Hela and p388 cell lines. Furthermore, compounds 1 and 2 induced typical apoptotic cell death in A549 cell line according to the evaluation of the apoptosis-inducing activity by flow cytometric analysis.

The First Attempt at Studying the Species Diversity of Fish in Lake Khanka Using DNA Barcoding Techniques

Lake Khanka is the largest lake in the northeastern part of Asia, being a valuable part of the resources of freshwater fisheries. Lake Khanka has a low value of endemism owing to the recent origin and connection to large river systems of the Amur and Ussuri. The list of fish species present in the lake has been constantly updated owing to invasions that occur both unintentionally and because of the introduction of hydrobionts to maximize the potential of natural productivity. In this regard, the Lake Khanka is a suitable testing ground for the development and implementation of complex methods for monitoring species diversity. The cases of invasion have become more frequent in the lake recently. Moreover, the introduction of some fish species for stabilization of relationships among communities and exhaustive utilization of the natural productivity is now being carried out, which makes challenging the development and implementation of powerful techniques for monitoring of the local fauna species diversity. For the first time, we present a complex approach to the solution of that problem. We collected 64 fish specimens from 16 species representing four families of ray-finned fishes and analyzed their taxonomy and species diversity using both classic methods and DNA barcoding techniques. Specimens have been genotyped based on the mitochondrial Co-1 gene marker. The mean values of K2P-corrected intraspecific genetic distances were 0.15 ± 0.01%; the distances between different species within the same genera were 4.08 ± 0.01%. Phylogenetic analysis revealed monophyletic origin of all species clusters with high support (98–100% of bootstrap values). Thus, reciprocal complement of morphological and genetic techniques for the purpose of species delimitation and high effectiveness of DNA barcoding in application for documentation of species diversity in Lake Khanka has been demonstrated. New molecular genetic data generally are in agreement with taxonomic relationships previously shown for these fish taxa, but an additional investigation is required to verify the composition of genus Acheilognathus in Lake Khanka.

DNA barcoding detects floral origin of Indian honey samples.

The unique medicinal and nutritional properties of honey are determined by its chemical composition. To evaluate the quality of honey, it is essential to study the surrounding vegetation where honeybees forage. In this study we used conventional melissopalynological and DNA barcoding techniques to determine the floral source of honey samples collected from different districts of the state of Mizoram, India. Pollen grains were isolated and genomic DNA was extracted from the honey samples. PCR amplification was carried out using universal barcode candidates ITS2 and rbcL to identify the plant species. Furthermore, TA cloning was carried out to screen the PCR amplicon libraries to identify the presence of multiple plant species. Results from both the melissopalynological and DNA barcoding analyses identified almost exactly the same 22 species, suggesting that both methods are suitable for analysis. However, DNA barcoding is easier and widely practiced. Hence, it can be concluded that DNA barcoding is a useful tool in determining the medicinal and commercial value of honey.

DNA BARCODING APPLICATION OF MITOCHONDRIAL COI GENE TO IDENTIFY SOME FISH SPECIES OF FAMILY GOBIIDAE IN VIETNAM

The family Gobiidae is a relatively high abundance family of coastal fish with about 2000 species in 210 genera described. In Vietnam, the study on Gobiidae is very complex due to the large number of species, small size and high morphological variation which makes difficulties in identification and classification. In this study, the DNA barcoding technique has been used through mitochondrial cytochrome oxidase I (COI) gene to classify 30 specimens of Gobiidae from three coastal areas (North, Central and South) in Vietnam to evaluate the effectiveness compared to the morphological classification method. Results showed that 26 species belonging to 21 genera were determined by the COI barcode while 19 species (17 genera) were determined when using morphological method. Mahidolia mystacina was new recorded in Vietnam. The DNA barcodes of COI gene developed in this study could be useful for estimating phylogenetic diversity as well as other studies of gobiids in terms of conservation, management and utilization of fisheries resources in Vietnam. In addition, the results showed the high potentiality in using COI barcode to identify marine fish.