DNA barcoding as a molecular tool to track down mislabeling and food piracy

DNA barcoding is a molecular technology that allows the identification of any biological species by amplifying, sequencing and querying the information from genic and/or intergenic standardized target regions belonging to the extranuclear genomes. Although these sequences represent a small fraction of the total DNA of a cell, both chloroplast and mitochondrial barcodes chosen for identifying plant and animal species, respectively, have shown sufficient nucleotide diversity to assess the taxonomic identity of the vast majority of organisms used in agriculture. Consequently, cpDNA and mtDNA barcoding protocols are being used more and more in the food industry and food supply chains for food labeling, not only to support food safety but also to uncover food piracy in freshly commercialized and technologically processed products. Since the extranuclear genomes are present in many copies within each cell, this technology is being more easily exploited to recover information even in degraded samples or transformed materials deriving from crop varieties and livestock species. The strong standardization that characterizes protocols used worldwide for DNA barcoding makes this technology particularly suitable for routine analyses required by agencies to safeguard food safety and quality. Here we conduct a critical review of the potentials of DNA barcoding for food labeling along with the main findings in the area of food piracy, with particular reference to agrifood and livestock foodstuffs

Sampling Local Fungal Diversity in an Undergraduate Laboratory using DNA Barcoding

Traditional methods for fungal species identification require diagnostic morphological characters and are often limited by the availability of fresh fruiting bodies and local identification resources. DNA barcoding offers an additional method of species identification and is rapidly developing as a critical tool in fungal taxonomy. As an exercise in an undergraduate biology course, we identified 9 specimens collected from the Hendrix College campus in Conway, Arkansas, USA to the genus or species level using morphology. We report that DNA barcoding targeting the internal transcribed spacer (ITS) region supported several of our taxonomic determinations and we were able to contribute 5 ITS sequences to GenBank that were supported by vouchered collection information. We suggest that small-scale barcoding projects are possible and that they have value for documenting fungal diversity

Marine nematode taxonomy in the DNA age: the present and future of molecular tools to access their biodiversity

Molecular taxonomy is one of the most promising yet challenging fields of biology. Molecular markers such as nuclear and mitochondrial genes are being used in a variety of studies surveying marine nematode taxa. Sequences from more than 600 species have been deposited to date in online databases. These barcode sequences are assigned to 150 nominal species from 104 genera. There are 41 species assigned to Enoplea and 109 species to Chromadorea. Morphology-based surveys are greatly limited by processing speed, while barcoding approaches for nematodes are hampered by difficulties in matching sequence data with morphology-based taxonomy. DNA barcoding is a promising approach because some genes contain variable regions that are useful to discriminate species boundaries, discover cryptic species, quantify biodiversity and analyse phylogeny. We advocate a combination of several approaches in studies of molecular taxonomy, DNA barcoding and conventional taxonomy as a necessary step to enhance the knowledge of biodiversity of marine nematodes

Combining DNA Barcoding and Macroinvertebrate Sampling to Assess Water Quality

DNA barcoding (using a standardized sequence of the mitochondrial CO1 gene) was used to determine the aquatic insect species richness of two sites along White Clay Creek in Pennsylvania. Water quality assessment at the sites did not change from good (14.6, previous MAIS score 13.2) and fair (9.4, earlier MAIS 7.3), but barcoding increased the species richness and provided a much more detailed analysis by detecting cryptic species. Aquatic insect identifications by an amateur biologist and by expert taxonomists using traditional methods based on morphology were compared to DNA barcoding. The amateur biologist’s identifications were limited to order and family while expert taxonomists were able to identify 44 different species and DNA barcoding indicated 128 different species. 84% of the 1786 specimens that were submitted for barcoding generated a successful DNA sequence. DNA barcoding revealed the presence of more species than expert taxonomists identified as shown in the following listing of insect orders with comparison of numbers of species identified by expert taxonomists and DNA barcoding: Diptera (23 expert spp. and 128 barcoding spp.), Ephemeroptera (6 expert spp. and 16 barcoding spp.), Plecoptera (0 expert spp. and 6 barcoding spp), Trichoptera (9 expert spp. and 14 barcoding spp), and Coleoptera (6 expert spp. and 6 barcoding spp). Station 12 had an overall higher species richness and abundance of Chironomidae; Chironomids accounted for 63% of the specimens with 64 species. Chironomids made up only 30% of the specimens at Station 11 and EPT richness was higher. The increase in the abundance and species richness of Chironomidae at Station 12 supported the previous findings of a lower water quality than that occurring at Station 11. Barcoding, when combined with traditional aquatic macroinvertebrate sampling, provides the most accurate and cost effective method to determine the water quality of fresh water ecosystems

Wolbachia and DNA barcoding insects: patterns, potential and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein – wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor – for which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region

Identification of Meat and Poultry Species in Food Products Using DNA Barcoding

DNA barcoding is a promising method for the sequencing-based identification of meat and poultry species in food products. However, DNA degradation during processing may limit recovery of the full-length DNA barcode from these foods. The objective of this study was to investigate the ability of DNA barcoding to identify species in meat and poultry products and to compare the results of full-length barcoding (658 bp) and mini-barcoding (127 bp). Sixty meat and poultry products were collected for this study, including deli meats, ground meats, dried meats, and canned meats. Each sample underwent full and mini-barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were queried against the Barcode of Life Database (BOLD) and GenBank for species identification. Overall, full-barcoding showed a higher sequencing success rate (68.3%) as compared to mini-barcoding (38.3%). Mini-barcoding out-performed full barcoding for the identification of canned products (23.8% vs. 19.0% success), as well as for turkey and duck products; however, the primer set performed poorly when tested against chicken, beef, and bison/buffalo. Overall, full barcoding was found to be a robust method for the detection of species in meat and poultry products, with the exception of canned products. Mini-barcoding shows high potential to be used for species identification in processed products; however, an improved primer set is needed for this application

Using molecular tools to differentiate closely related blackfly species of the genus Simulium

Biodiversity data are the foundation for conservation and managemet and taxonomy provides the reference system, skills and tools used to identify organisms. Species level data such as species richness, composition and diversity are common metrics. However, species level identification of organisms tends to be neglected within ecological work, especially within monitoring programmes, but also in conservation biology (Giangrande, 2003). This is because collection of species level data is time consuming, with identification of species-specific characteristics traditionally involving lengthy examination of samples using microscopy. In addition it is costly and species level data is almost impossible to collect if the taxa involved are species rich and difficult to identify (Báldi 1999). Other reasons why species level identification is neglected include the fact that sample collection can damage organisms, so diagnostic morphological features are lost, or that individuals may be in a life history stage or of a sex that does not have diagnostic morphological characteristics. Furthermore, the numbers of available expert taxonomists needed for species identification are in decline and have been for several decades. Species identification using molecular taxonomy where DNA is used as a marker is championed as a tool for resolving a range of morphological problems, such as the association of all life history stages, correlating male and female specimens to the same species and identifying partial specimens. Traditional taxonomy is built around morphological variations between species, with systematic inferences based upon shared physical characters. In molecular taxonomy on the other hand, proteins and genes are used to determine evolutionary relationships. ’DNA barcoding’ aims to provide an efficient method for species-level identification and it is thought that it will provide a powerful tool for taxonomic and biodiversity research (Hajibabaei et al. 2007). Cited strengths of a molecular based approach to species identification include the potential universality and objective nature of DNA data as taxonomic information, the usefulness of molecular data in animal groups characterized by morphological cryptic characters and the use of DNA sequence information to determine otherwise ‘unidentifiable’ biological material (such as incomplete specimens or immature specimens). Its aim is to increase the speed, precision and efficiency of field studies involving diverse and difficult to identify taxa and it has the potential to be automated to provide a rapid and consistently accurate supplementary identification system to traditional taxonomy. This project was a proof-of-concept study that investigated the feasibility of using DNA barcodes to differentiate closely related blackfly species of the genus Simulium. The longer term objective would be to apply such molecular approaches to organisms used in water quality monitoring and to biodiversity studies to provide a quick, robust but practical and cost effective tool for species identification. Great Britain is currently home to 33 morphospecies of blackfly many of which are morphologically close to other species and have been the cause of much systematic revision. In addition to evaluating the use of DNA barcodes in species identification, a non-destructive DNA extraction method was developed to preserve voucher pecimens that will allow a complete morphological classification to be carried after DNA extraction. Using molecular tools to differentiate closely related blackfly species of the genus Simulium v Finding an effective DNA barcode for an individual species involves accurate taxonomic identification and the retention of voucher specimens for future morphological studies. A rapid non-destructive method for DNA extraction from small insects was developed where no clean-up step was required prior to amplification and it was possible to extract DNA of sufficient quality in minutes retaining diagnostic morphological characteristics. For any molecular tool used for species discrimination, an important consideration is defining the specific genetic loci (e.g. the position of genes on a chromosome) to be monitored. All blackfly species in this study were successfully amplified with the standard barcoding coxI gene primer pair LCO1490 5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3' and HCO2198 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3' (Folmer et al. 1994) and we did not need to optimise or redesign the primer sequence

DNA BARCODING: FOUNDATIONS AND APPLICATIONS FOR SOUTHEAST ASIAN FRESHWATER FISHES

Identifying and delineating species are the primary tasks of taxonomy. Owing to the decreasing interest of the nations for taxonomy and the inventory of living beings, funds have been drastically decreasing during the last two decades for taxonomic studies. As a consequence, the worldwide pool of taxonomists has dramatically decreased. DNA barcoding, as an automated tool for species delineation and identification, proved to rejuvenate the field of taxonomy and open new perspectives in ecology and conservation. In the present review, we will discuss how DNA barcoding established as a new paradigm in taxonomy and how DNA barcoding has been recently integrated in taxonomic studies. We will further detail the potential applications for species identifications and discuss how DNA barcoding may positively impact the inventory and conservation of living beings, particularly in biodiversity hotspots. We emphasise the benefit of DNA barcoding for the conservation of Southeast Asian freshwater fishes

The Utilization of Polymerase Chain Reaction, DNA Barcoding and Bioinformatics in Identifying Plant Species

Bioinformatics and DNA barcoding is a process used to identify plants, animals, and fungi. DNA barcoding in plants utilizes a key variable region in the genome, the RuBisCo large subunit (RbcL) on Chloroplast DNA. Once the DNA is extracted, Polymerase Chain Reaction (PCR) amplifies that region and that sample is sent off for sequencing. Bioinformatics and DNA barcoding helps taxonomists determine the sequence of the RbcL gene as well as obtain a unique barcode that can be used to identify plants. Several plant species from our local campus were sequenced and identified using the previously described methods