DNA Barcoding Methods Research Articles

Assembly-free reads accurate identification (AFRAID) approach outperforms other methods of DNA barcoding in the walnut family (Juglandaceae)

DNA barcoding has been extensively used for species identification. However, species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods. In this study, we use plant species in Juglandaceae to evaluate an assembly-free accurate reads identification (AFRAID) method of species identification, a novel approach for precise species identification in plants. Specifically, we determined (1) the accuracy of DNA barcoding approaches in delimiting species in Juglandaceae, (2) the minimum size of chloroplast dataset for species discrimination, and (3) minimum amount of next generation sequencing (NGS) data required for species identification. We found that species identification rates were highest when whole chloroplast genomes were used, followed by taxon-specific DNA barcodes, and then universal DNA barcodes. Species identification of 100% was achieved when chloroplast genome sequence coverage reached 20% and the original sequencing data reached 500,000 reads. AFRAID accurately identified species for all samples tested after 500,000 clean reads, with far less computing time than common approaches. These results provide a new approach to accurately identify species, overcoming limitations of traditional DNA barcodes. Our method, which uses next generation sequencing to generate partial chloroplast genomes, reveals that DNA barcode regions are not necessarily fixed, accelerating the process of species identification.

Molecular identification of Nemacheilus sp. from Bangka Island, Indonesia Based on the Cytochrome C oxidase Subunit I (COI) Gene

Abstract Nemacheilus is a genus of stone loaches native to Asia. There are 13 species of the genus Nemacheilus that can be found in western Indonesia, from 44 species that exist in Asia. During our expedition to Bangka Island, which took place between the 20th and the 25th of January 2023, we discovered 15 live specimens of Nemacheilus sp. These specimens were collected using a fish trap in the Bencah River, Bangka Island, Indonesia. We identified Nemacheilus sp based molecular analysis by using the DNA Barcoding method based on the Cytochrome C oxidase Subunit I (COI) gene. DNA barcoding is a solution to provide accurate, fast, and automatable identification of species and species discovery. Based aon the COI gene, Nemacheilus sp. Nemacheilus from Bangka Island is genetically closest to Nemacheilus pfeifferae, which is found in Sumatra. Sumatra and Bangka Island have a history of river connection before the maximum glacial phase II. However, based on the similarity of DNA base pairs, these two species are only 90% similar. This indicates that the genetic difference between these two species is 10%, which is far below the threshold for what is considered to be an identical species, which is under 3%. Therefore, we hypothesize that the Nemacheilus that was found on the island of Bangka is not the species known as Nemacheilus pfeifferae, but rather another species. Since the data for this other species are not yet available in Genbank, we are referring to it as Nemacheilus sp. Bangka for the time being.

Complementary studies of the Perlidae (Insecta: Plecoptera) fauna from the Paranapiacaba Mountains using DNA barcode data

The Paranapiacaba Mountains are a region of Brazil where the stonefly fauna is relatively well known. Despite this, there are still gaps in knowledge that need to be filled. In this study, through field sampling and molecular analyses, we have updated the taxonomic knowledge of Perlidae species in this region. Our study employed DNA barcoding methods to complement traditional morphological approaches, facilitating the association and description of the nymphs of Anacroneuria itajaimirim Bispo & Froehlich 2004. Additionally, we generated DNA barcode sequences for Anacroneuria iporanga Bispo & Froehlich 2004. Our results contributed to knowledge about Perlidae from the Paranapiacaba Mountains, highlighting the importance of DNA barcode data in the association of immature stages and species delimitations. Although the stoneflies of Paranapiacaba Mountains have been relatively well-studied, our results reveal that we still have deficits in knowledge of the fauna of this region.

DNA Barcoding Unveils Novel Discoveries in Authenticating High-Value Snow Lotus Seed Food Products.

Snow Lotus Seed (SLS), esteemed for its nutritional and market value, faces challenges of authentication due to the absence of appropriate testing standards and methods. This results in frequent adulteration of SLS sourced from Gleditsia sinensis (G. sinensis) with other plant seeds endosperm. Traditional chloroplast DNA barcoding methods are inadequate for species identification due to the absence of chloroplasts in G. sinensis seeds endosperm. In this study, the homology of 11 ITS genes among 6 common Gleditsia species was analyzed. Universal primers suitable for these species were designed and screened. A DNA barcoding method for distinguishing SLS species was developed using Sanger sequencing technology, leveraging existing GenBank and Barcode of Life Data System (BOLD) databases. Optimized sample pretreatment facilitated effective DNA extraction from phytopolysaccharide-rich SLS. Through testing of commercial SLS products, the species origin has been successfully identified. Additionally, a novel instance of food fraud was uncovered, where the Caesalpinia spinosa endosperm was used to counterfeit SLS for the first time. The study established that the developed DNA barcoding method is effective for authenticating SLS species. It is of great significance for combating food fraud related to SLS, ensuring food safety, and promoting the healthy development of the SLS industry.

Cost-efficient PCR based DNA barcoding of marine invertebrate specimens with NovaSeq amplicon sequencing.

The marine environment harbors high biodiversity; however, it is poorly understood. Nucleotide sequence data of all marine organisms should be accumulated before natural and/or anthropogenic environmental changes jeopardize the marine environment. In this study, we report a cost-effective and easy DNA barcoding method. This method can be readily adopted without using library preparation kits. It includes multiplex PCR of short targets, indexing PCR, and outsourcing to a sequencing service using the NovaSeq system. We targeted four mitochondrial genes [cytochrome c oxidase subunit I (COI), COIII, 16S rRNA (16S), and 12S rRNA (12S)] and three nuclear genes [18S rRNA (18S), 28S rRNA (28S), internal transcribed spacer 2 (ITS2)] in 95 marine invertebrate specimens, which were primarily annelids. The primers, including adapters and indices for NovaSeq sequencing, were newly designed. Two PCR runs were conducted. The 1st PCR amplified specific loci with universal primers and the 2nd added sequencing adapters and indices to the 1st PCR products. The gene sequences obtained from the FASTQ files were subjected to BLAST search and phylogenetic analyses. One run using 95 specimens yielded sequences averaging 2816bp per specimen for a total length of six loci. Nuclear genes were more successfully assembled compared with mitochondrial genes. A weak but significantly negative correlation was observed between the average length of each locus and success rate of the assembly. Some of the sequences were almost identical to the sequences obtained from specimens collected far from Japan, indicating the presence of potentially invasive species identified for the first time. We obtained gene sequences efficiently using next-generation sequencing rather than Sanger sequencing. Although this method requires further optimization to increase the success rate for some loci, it is used as a first step to select specimens for further analyses by determining the specific loci of the targets.

Can DNA Barcode Study be Done from a Museum Specimen Fixed in a Formaldehyde Solution? A Case of Emys orbicularis (Linnaeus, 1758)

DNA barcoding, a molecular taxonomy technique, has been increasingly used by herptile taxonomists in recent years. In DNA barcoding studies with museum specimens, there are difficulties in achieving success in specimens that have been exposed to formaldehyde, which is usually used as a fixative, for a long time and intensively. Here we studied the effect of formaldehyde on the application of the DNA barcode method in Emys orbicularis specimens stored in 4% formaldehyde and 70% ethanol solution since 2008 and 2014. Sanger sequence analysis of tissues taken from samples stored in both ethanol and formaldehyde solution successfully yielded sequences of 623 bp. In conclusion, the use of ethanol solutions should be preferred for mid or long-term sample storage, especially in the context of molecular studies. In cases where the use of formaldehyde is unavoidable, it may be advisable to use extremely low concentrations to increase success in molecular research.

Bidirectional linkage of DNA barcodes for the multiplexed mapping of higher-order protein interactions in cells.

Capturing the full complexity of the diverse hierarchical interactions in the protein interactome is challenging. Here we report a DNA-barcoding method for the multiplexed mapping of pairwise and higher-order protein interactions and their dynamics within cells. The method leverages antibodies conjugated with barcoded DNA strands that can bidirectionally hybridize and covalently link to linearize closely spaced interactions within individual 3D protein complexes, encoding and decoding the protein constituents and the interactions among them. By mapping protein interactions in cancer cells and normal cells, we found that tumour cells exhibit a larger diversity and abundance of protein complexes with higher-order interactions. In biopsies of human breast-cancer tissue, the method accurately identified the cancer subtype and revealed that higher-order protein interactions are associated with cancer aggressiveness.

One Step beyond Species Description: Unveiling a Fine-Scale Diversity within the Genus Dzhanokmenia Kostjukov (Hymenoptera: Eulophidae).

Although Chalcidoidea is one of the megadiverse superfamilies in Hymenoptera, numerous species are still being discovered and described. However, the difficulties in delimiting intra- and interspecific variation hinder this process. In this study, DNA barcoding methods using the COI gene were employed to investigate the morphological variation within Dzhanokmenia Kostjukov, 1977. The nuclear locus, 28S D2, was used to infer a phylogeny to gain an understanding of the relationship of Dzhanokmenia with other potentially close genera. Through a preliminary DNA barcode library established here, including eight species, we calibrated the intraspecific variation in certain diagnostic characters for the new species described here, D. brevifunis Ganbaatar & Cao sp. nov. Maximum likelihood results show that Dzhanokmenia is clustered with the genera associated with Tetrastichus, such as Chaenotetrastichus Graham, 1987, Baryscapus Förster, 1856, Tetrastichus Haliday, 1844, and Oomyzus Rondani, 1870 involved in this study. Our results indicate that the species diversity of Dzhanokmenia is understudied and tentatively confirm that Dzhanokmenia has a potential close relationship with Baryscapus. Along with the DNA barcode library, the referenced phylogeny datasets improve the understanding of the systematic position of Dzhanokmenia within the subfamily Tetrastichinae and the definition of this genus in terms of morphology, thereby facilitating species delimitation, discovery, and description within Dzhanokmenia.

Phylogenetic Analysis of Rare and Endangered Tulipa Species (Liliaceae) of Kazakhstan Based on Universal Barcoding Markers.

In Kazakhstan, the genus Tulipa is represented by 35 species, 18 of which are listed in the Red Data Book of Kazakhstan and protected by the state. Recent studies of tulip specimens from regions bordering Kazakhstan emphasize the significance of species inventory and report the discovery of several hybrids. In this study, eight tulip species were identified based on morphological characteristics and using DNA barcoding methods. Molecular genetic markers, including nrDNA (ITS) and cpDNA markers (rbcL, matK), of the studied species were sequenced and analyzed using the Bayesian inference and maximum likelihood phylogenetic analysis methods. Our work demonstrates that DNA barcodes based on the ITS, rbcL, and matK marker regions have successful practical applicability, with ITS being the most informative at the intragenic level. However, for distinguishing closely related taxa, the most effective approach would be to use a combined dataset of sequences from multiple DNA markers. The results showed discrepancies in the placement of several taxa (T. kaufmanniana, T. patens), likely due to introgression and natural spontaneous hybridization. The molecular phylogenetic analysis suggests the existence of a previously undescribed hybrid between T. patens and T. alberti. Further detailed population studies are needed to validate this hypothesis.

Sequence variation of commercially available kratom products at universal DNA barcode regions.

Mitragyna speciosa, commonly known as kratom, is a narcotic plant that is used for its unique mood-enhancing and pain-relieving effects. It is marketed throughout the United States as a 'legal high' and has gained popularity as an alternative to opioids. However, kratom's increasing involvement in accidental overdoses, especially among polydrug users, has prompted warnings from the Drug Enforcement Agency (DEA) and the Food and Drug Administration (FDA). Despite these warnings, kratom remains legal federally, although it is banned in six states. This legal disparity complicates monitoring and enforcement efforts in states where kratom is illegal. Common forensic techniques using morphology or chemical analysis are beneficial in some instances but are not useful in source attribution because most seized kratom is powdered and the alkaloid content of samples can vary within products, making sourcing unreliable. This study focused on developing a DNA barcoding method to access sequence variation in commercial kratom products. It evaluated the utility of one nuclear barcode region (ITS) and three chloroplast barcode regions (matK, rbcL, and trnH-psbA) in assessing sequence variation across commercially available kratom products. Novel polymorphisms were discovered, and the ITS region showed the greatest variation between samples. Among the 15 kratom products tested, only two haplotypes were identified across the four barcoding regions. The findings highlight the potential of DNA barcoding as a forensic tool in the traceability and enforcement against illegal kratom distribution. Nonetheless, the limited haplotypic diversity points to a need for further development and expansion of the M. speciosa DNA sequence database.

Advancing One Health in Urban Seafood Markets: A Genetic and Social Analysis of Dried Sea Cucumber in Three New York City Chinatowns

This study employs a multidisciplinary methodology across natural and social sciences to examine relationships between biodiversity loss at sea and urban consumption with a focus on sea cucumber and dried seafood markets in New York City (NYC). The study identified 34 dried seafood retailers across three NYC Chinatown boroughs: Manhattan, Brooklyn, and Queens. Samples of sea cucumber were collected with Chinese-language labels indicating the commodity was from South America, a region of conservation concern. Comparison samples were taken from sea cucumbers labeled from Mexico and Japan. A mitochondrial DNA barcoding method was used to examine the taxonomic origin of 103 samples. Sequence data were successfully obtained from 74 of the samples, 8 of which were classified as brown sea cucumber (Isostichopus fuscus), an endangered species for which harvest is banned in several locations. Semi-structured interviews with dried seafood retailers and consumers (n = 64), moreover, revealed associations between consuming sea cucumber and enhancing human health and limited knowledge of product origins. Collectively, the findings reveal socio-ecological dynamics wherein endangered species on the market coupled with geographic market labeling practices and varying degrees of retailer and consumer knowledge negatively bear on marine biodiversity. Furthermore, given that brown sea cucumbers are abundant on the market, there is a need for developing genetic markers that can trace geographic origin to determine if species were legally harvested. These results indicate that more robust market labeling, training, genetic research, and public outreach are required to advance One Health in urban seafood markets.

Creating a novel genetic diversity of Trichoderma afroharzianum by γ-radiation for xylanase-cellulase production

Creating novel sources of a microbial strain using induced mutation can increase enzyme production for industrial use. According to this, we have developed a mutant strain of Trichoderma afroharzianum by Co60 gamma irradiation. Trichoderma mutants were isolated from an optimum dose of 250 Gy. The qualitative and quantitative screening were used for evaluating their enzyme production and the DNA barcoding method was used to identify the best Trichoderma mutant isolates. The highest cellulase (exo-glucanase, endoglucanase, β-glucosidase, and total cellulase) and xylanase activities were observed in superior mutant isolates of Trichoderma afroharzianum NAS107-M44 and Trichoderma afroharzianum NAS107-M82, which is approximately 1.6–2.5 times higher than its parent strain, respectively. The electrophoretic pattern of proteins showed that the exo-glucanase I, endo-glucanase III, and the xylanase I enzymes hydrolyzed the corn bran, synergistically. Overall, gamma irradiation-induced mutation could be an expedient technique to access such superior mutants for the bioconversion of corn bran wastes.

GENETIC DISTANCE REVEALS SYNONYMY AND NEW FISH SPECIES IN BALIKESİR STREAMS, TÜRKİYE

The Mediterranean-Aegean region play a significant role in the context of conserving biodiversity. The protection of endemic and local species becomes attainable only with a thorough understanding of their biology. Specifically, aquatic species dwelling in inland waters may be more vulnerable due to the irregular composition of their habitats, characterized by natural barriers. It becomes imperative to initiate the conservation process by identifying the fauna and flora of ecosystems, thereby facilitating the development of comprehensive conservation plans. In this context, the primary objective of the present study is to identify freshwater fish species inhabiting Madra and Havran Streams in Balıkesir, Türkiye using the DNA barcoding method. The procedure involves DNA isolation through the Chelex protocol, followed by the amplification of the mitochondrial CO1 region using various primer combinations. The results obtained from the gene sequences of 29 individuals in total provide valuable information on species diversity, genetic relationships, and variations. This research emphasizes the importance of DNA barcoding as a valuable tool for species identification, genetic exploration, and conservation plans. The acquired outcomes establish a foundation for the effective management of aquatic biodiversity, particularly within these vulnerable ecosystems.

Dominant species of squid in the waters of Western Kalimantan and Northern Java identified by DNA barcoding method

Squid resources in Indonesia have high economic value, and their exploitation tended to increase due to declining fish resources in many waters. Previous research mentioned that 21 squid species were identified in Indonesian waters. However, many researchers argued that the identification of cephalopod morphology, especially squids, was challenging, and that is why supporting methods were needed, one of which was DNA analysis. The study aimed to determine the type and distribution of squid in the western waters of Kalimantan and northern Java using the DNA barcoding method on the COI gene. Squid samples were collected from six locations, consisting of Tanjung Pinang, Bangka Belitung (Babel), Kalimantan Barat (Kalbar), Muara Angke, Pekalongan, and Lamongan. The analysis results showed that Uroteuthis chinensis was present in all sampling locations except Tanjung Pinang, where Uroteuthis edulis and U. sibogae were found. The results of this study are very important in the management of squid resources because they can contribute to the sustainable management of marine resources and the maintenance of the sustainability of marine ecosystems.

Integrated DNA barcoding methods to identify species in the processed fish products from Chinese market

DNA-based methods are reliable for a precise identification of species in processed products. In this study, we assessed five typical DNA extraction methods from multiple aspects. Full-length and mini-length DNA barcoding were performed to detect the species substitution and mislabeling of 305 processed fish products from the Chinese market covering six processed fish products. The salt extraction method that exhibited the best overall performance was applied. All samples were successfully extracted; however, only 19.3 % of samples could be amplified using the full-DNA barcode primer set, and 90.2 % of samples could be amplified using the newly designed mini-DNA barcode primer sets (401 and 320 bp). Overall, the molecular identification results revealed that 36.4 % (111/305) of the samples were inconsistent with the labels, with commercial fraud observed in all six types of processed fish products. The survey findings provide technical references for effective fish authentication monitoring, offering insights into the seafood safety in markets.

DNA Barcoding for an Undergraduate Class.

DNA technique is a topic mandatorily covered in a biology and biochemistry undergraduate curriculum. Inquiry-based pedagogy is proven to be the most effective way of learning, and DNA barcoding method allows to merge necessary-to-study experimental techniques such as DNA isolation and purification, PCR, and basic BLAST search into a two- or three-week inquiry-based student project. It also provides a research-based experience to the students, who, when organized in groups, can design their own DNA-barcoding project if they wish. Here, we describe how DNA barcoding can be offered in an undergraduate college or advanced high school settings. This chapter is intended to help college and high school instructors to include DNA barcoding in their classes.

Morphological, molecular identification and evaluation of antioxidant activity of seahorses from the Moroccan coasts

Seahorses, part of the small marine teleost fish family Syngnathidae, are increasingly under threat due to habitat degradation and overfishing. Notably used in traditional Chinese medicine, these fish have demonstrated significant pharmacological and cosmetic properties. In Morocco, however, seahorses are minimally exploited. This study aims to explore the biodiversity of Moroccan seahorses, focusing on identifying species from the Atlantic and Mediterranean coasts both morphologically and molecularly, and evaluating their antioxidant activity.The research involved collecting 62 dried seahorses from local fishermen. These specimens were subjected to detailed morphological and molecular identification through the DNA barcoding method, concentrating on the mitochondrial marker Cytochrome Oxidase I (COI) gene. Following DNA extraction and amplification, the sequences were analyzed for species identification and phylogenetic relationships. Additionally, the antioxidant activities of the seahorses were quantified using assays such as ABTS, reducing power, phosphomolybdenum, and β-carotene-linoleic acid.The combined morphological and molecular analyses consistently identified all specimens as Hippocampus hippocampus, and phylogenetic trees suggested a close relation with European and Turkish counterparts. Furthermore, the antioxidant assays revealed significant activity, with the ABTS assay showing an IC50 of 14.571 mg/mL ± 0.334, and the β-carotene-linoleic acid assay showing an IC50 of 1.273 mg/mL ± 0.166. The reducing power and phosphomolybdenum assays recorded EC50 values of 1.868 mg/mL ± 0.033 and 1.156 mg/mL ± 0.112, respectively. These results confirm the high antioxidant potential of Moroccan seahorses, suggesting their therapeutic value and necessitating measures for their biodiversity preservation at a national level.

First record and phylogenetic relationship of the endangered species Betta foerschi (Vierke, 1979) (Anabantiformes : Osphronemidae) from Belitung Island, Indonesia

Betta foerschi (Vierke, 1979) is a betta fish group from the Osphronemidae family which is only known from Central and West Borneo Island, Indonesia. This species is classified as an Endangered (EN) by the IUCN Red List, where the population has recently been moderately decreased. The new distribution of B. foerschi fish has been found on Belitung Island based on morphological similarities. In this research, we report the first record and phylogenetic relationship of B. foerschi based on the DNA Barcoding method using the Cytochrome C Oxidase Subunit I (COI) gene from Belitung Island Indonesia. Additionally, the DNA barcoding for B. foerschi not only represents the first DNA record for Belitung Island, but also the first sequence reported in the Genbank database. The result showed that B. foerschi forms the same clade within the Osphronemidae family and Betta genus solidifying its position in this taxonomic group with the closest genetic distance that occurs between B. foerschi and B. persephone of 0.15 using the Maximum Composite Likelihood model analysis.

DNA barcoding identification of grafted Semen Ziziphi Spinosae and transcriptome study of wild Semen Ziziphi Spinosae.

Semen Ziziphi Spinosae (SZS) is the dried and ripe seeds of Ziziphus jujuba var. spinosa. Currently, the yield of naturally grown SZS is unstable owing to environmental factors. Grafting high-quality sour jujube scions onto sour jujube or jujube tree stocks can result in a greater yield. However, the effects of grafting on the quality and gene expression of SZS have rarely been reported. This study used a DNA barcoding technique, high-performance liquid phase-evaporative luminescence detector (HPLC-ELSD), and transcriptomics to investigate the origin and genetic differences between grafted and wild jujube seeds. DNA barcoding identified all samples as Ziziphus jujuba var. spinosa. HPLC-ELSD analysis revealed a higher content of grafted SZS compared to that of the wild SZS. Transcriptome analysis of the metabolic pathways in SZS showed that 22 and 19 differentially expressed gene sequences encoded enzymes related to flavonoids and saponin synthesis, respectively. Weighted correlation network analysis (WGCNA) identified 15 core genes governing the differences in medicinal components between grafted and wild SZS. This study demonstrated the use of DNA barcoding and fingerprint methods to identify jujube seed species and effectively capture ingredient information of medicinal materials. Additionally, transcriptome technology provided data for identifying core differential genes, facilitating studies on quality differences between grafted and wild SZS.

DNA barcode reveals occurrence of threatened species and hidden diversity on Teleost fish trade in the Coastal Amazon

This study aimed to identify the teleost fish species sold in Bragança, a major fishing hub on the north coast of Brazil. The COI gene analysis was performed for the identification of fish species. The local market uses common names that are not accurate and do not reflect the diversity of the species. 204 sequences were obtained, with 119 haplotypes. 83 species were identified by comparing with public databases and constructing phylogenetic trees, with Carangidae being the most prevalent family. The study also found Haemulon atlanticus, Menticirrhus cuiaranensis and Hoplias misioneira, a newly described species from the Amazon basin, among the samples. Additionally, 73 commercial names were recorded, including 10 categories, and the illegal trade of Epinephelus itajara was detected. The DNA Barcode method proved to be effective for discriminating the species. The study highlights that common and commercial names are vague and underestimate the fish diversity, and that Brazil needs to revise its regulations for commercial and scientific names.