Search Results (101)

Thrips are cosmopolitan agricultural pests and important vectors of plant viruses, and the increasing coexistence of multiple morphologically similar species has intensified the demand for species-specific molecular identification. However, traditional morphological identification and PCR assays using universal primers are often inadequate for mixed-species samples and field-adaptable application. In this study, we developed a species-specific molecular identification framework targeting a polymorphism-rich region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which is more time-efficient than sequencing-based COI DNA barcoding, for four economically important thrips species in southern China, including the globally invasive Frankliniella occidentalis. By aligning COI sequences, polymorphism-rich regions were identified and used to design four species-specific primer pairs, each containing a diagnostic 3′-terminal nucleotide. These primers were combined with a PBS-based DNA extraction workflow optimized for single-insect samples that minimizes dependence on column-based purification. The assay achieved a practical detection limit of 1 ng per reaction, demonstrated species-specific amplification, and maintained reproducible amplification at DNA inputs of ≥1 ng per reaction. Notably, PCR inhibition caused by crude extracts was effectively alleviated by fivefold dilution. Although the chemical identities of the inhibitors remain unknown, interspecific variation in inhibition strength was observed, with T. hawaiiensis exhibiting the strongest suppression, possibly due to differences in lysate composition. This integrated framework balances target specificity, operational simplicity, and dilution-mitigated inhibition, providing a field-adaptable tool for thrips species identification and invasive species monitoring. Moreover, it provides a species-specific molecular foundation for downstream integration with visual nucleic acid detection platforms, such as the CRISPR/Cas12a system, thereby facilitating the future development of portable molecular identification workflows for small agricultural pests. Full article

Modern sequencing technologies have transformed the identification of medicinal plant species and varieties, overcoming the limitations of traditional approaches. To address the challenge of discriminating Toona sinensis varieties, we sequenced and compared 15 complete chloroplast genomes from five varieties in northern China. Although these genomes exhibited a highly conserved structure, we identified eight variety-specific simple sequence repeats (SSRs), two unique tandem repeats, and several hypervariable regions with elevated nucleotide diversity. Phylogenetic analysis demonstrated that whole chloroplast genomes provided the highest resolution for variety identification, outperforming conventional barcodes. Furthermore, we developed 13 specific primer pairs targeting variable regions, and PCR validation confirmed their reliable amplification across varieties. In addition, sequence-level validation by Sanger sequencing of representative SSR and tandem repeat markers revealed stable, variety-specific repeat copy number differences. These results demonstrate that the identified chloroplast markers can effectively discriminate closely related T. sinensis varieties. This study confirms that despite overall conservation, the T. sinensis plastome contains sufficient variation for reliable identification, providing a robust framework for future germplasm conservation and molecular breeding. Full article

Affordable, flexible surveillance tools are needed to detect SARS-CoV-2 and other pathogens in wildlife. Standard nucleic acid amplification tests (NAATs) are reliable but restricted to predefined targets, limiting their ability to detect co-infections or emerging pathogens. To address this, we adapted a targeted next-generation sequencing (tNGS) panel for mesocarnivores to the Oxford Nanopore Technologies (ONT) MinION platform and combined it with a SARS-CoV-2 whole-genome sequencing assay. Merging both assays before library preparation enables simultaneous SARS-CoV-2 detection, variant identification, and broader pathogen screening. The MinION platform also improves turnaround time because sequencing can begin immediately on small numbers of samples, reducing costs in low-volume workflows. We converted our validated carnivore tNGS panel from the Ion Torrent system to MinION, optimizing amplification conditions, primer pools, and barcoding for multiplexing. Analytical sensitivity was measured using contrived wildlife samples spiked with serial dilutions of SARS-CoV-2 and tested in parallel with a commercial NAAT. Diagnostic sensitivity was assessed using contrived positives, and specificity was evaluated using NAAT-negative wildlife samples and in silico analyses. All 161 wildlife samples were NAAT-negative. MinION tNGS detected SARS-CoV-2 down to Ct 34 and produced ≥ 99% genome coverage for Ct ≤ 24 while simultaneously identifying additional pathogens. Diagnostic sensitivity and specificity were 96.7% and 100%. This workflow offers a low-cost, scalable approach for comprehensive wildlife pathogen surveillance. Full article

Bats (Chiroptera) represent nearly one-fifth of all mammalian species and play vital ecological roles as pollinators, pest controllers, and reservoirs of zoonotic pathogens. Accurate identification of bat species is essential for biodiversity monitoring, conservation, and disease surveillance. Traditional methods based on morphology or acoustic calls are often limited by overlapping features, while DNA barcoding using the cytochrome oxidase I (COI) gene can be hindered by sequence variability. In this study, we developed a simple, single-step PCR assay targeting a short, variable region of the mitochondrial 12S rRNA gene. Alignment of sequences from 232 bat species allowed the design of a single primer pair producing a 203–224 bp amplicon that successfully distinguished all species analyzed. The assay achieved 100% amplification success across 241 bat samples, with 97.2% concordance between molecular and morphological identification. Two samples showed sequence divergence suggestive of an undescribed species. Overall, ten bat species from six genera were identified, with Eptesicus fuscus being the most frequent. This assay offers a practical and robust approach for bat identification, supporting biodiversity assessment and pathogen surveillance in ecological and public health research. Full article

Background/Objectives: While morphological similarity and incomplete specimens pose a challenge to the precise identification of Coelogyne orchids, accurate species and genus assignment is essential for conservation and CITES enforcement. This study evaluated the efficacy of five DNA barcode regions—rbcL, matK, trnH-psbA, atpF-atpH, and ITS2—and their combinations for species- and genus-level discrimination within the genus Coelogyne, aiming to develop a rapid and simple diagnostic tool for use by customs officers and trade inspectors. This is the first comprehensive comparative analysis of these five barcode regions specifically within Coelogyne, a genus underrepresented in molecular identification studies, and the first to propose multi-locus combinations for potential practical use. This study identified DNA barcode regions with high resolution and reliability, providing a solid basis for practical identification kits. Such tools will enhance CITES enforcement by enabling rapid detection of Coelogyne species in trade, directly supporting their conservation and contributing to the reduction in illegal orchid trade. Methods: Using a CTAB protocol, genomic DNA was extracted from leaf samples belonging to 19 Coelogyne species. Sanger sequencing was performed after PCR amplification using published primer sets for every barcode region. Sequences were modified in BioEdit, and BLASTn (accessed 15 June 2025) was used to compare them to GenBank (NCBI Nucleotide). Amplification efficiency was calculated per locus. Species and genus identification success rates were determined by the congruence of top BLAST hits with morphologically pre-identified taxa. Multi-barcode combinations (matK + rbcL, ITS2 + matK, matK + trnH-psbA, rbcL + trnH-psbA, and matK + rbcL + trnH-psbA) were also assessed. Results: With rbcL, atpF-atpH, and ITS2 yielding ≤11%, the highest single-locus species identification rates were for trnH-psbA (21%) and matK (16%). Among single-locus barcodes, matK showed the highest performance, with 84% genus assignment. ITS2 reached 27%, but genus-level resolution remained limited for the rbcL, trnH-psbA and atpF-atpH barcodes. Multi-barcode approaches maintained species resolution: matK + rbcL + trnH-psbA, matK + rbcL, and matK + trnH-psbA correctly identified 16% of species and achieved 74–79% genus assignment. Conclusions: No single locus achieves robust species discrimination in Coelogyne, but trnH-psbA, matK and atpF-atpH provide the best single-marker performance. Using the matK locus alone, in combination with either trnH-psbA or rbcL, or all three together ensures consistent genus-level identification and significantly improves taxonomic resolution. This study introduces a novel multi-locus barcode strategy tailored to Coelogyne, offering a practical solution for identification and enforcement. While promising, this approach represents a potential application that requires further validation before routine implementation. Full article

Environmental and ancient DNA are mostly present in degraded forms in nature. Plant forensics is necessary for plants like Taxus (Taxaceae), which is a medicinal, as well as poisonous, endangered plant. We designed a study to develop high-efficiency PCR mini-barcoding primers for the identification of Taxus. We collected environmental materials, fresh and old Taxus specimens from natural habitats, herbaria, and ex situ propagation sites. Taxon-specific mini-barcoding primers were prepared through primer3. All the primers were amplified onto Taxus specimens and environmental samples having Taxus DNA, while no amplification on fresh and herbarium specimens other than Taxus was noted. DNA sequencing of amplified regions of matK, ITS, and rbcL yielded lengths of 117, 175, and 200 bp. Blast taxonomy showed 100% identification power at the genus level, while 75–93% at the species level, and identified a total of 30 taxa within the genus Taxus, comprising 16 species, 5 varieties, 2 hybrids, and 7 variants. ITS was the most specific for genus identification, followed by matK and rbcL. Environmental, trade, socio-economic, and toxicological crimes were also identified. Our high-efficiency PCR mini-barcoding method can be useful in the prevention of Taxus illegal trade and habitat degradation to mitigate climate change in the Himalayan region of Pakistan. Full article

We developed and validated a rapid, cost-effective multiplex PCR assay targeting mitochondrial cytochrome c oxidase subunit I (COX1) to discriminate three commercially important sea-squirt species, Halocynthia roretzi, H. aurantium and H. hilgendorfi ritteri. Species-specific forward primers were designed from interspecific single-nucleotide polymorphisms within the barcode region and combined with a common reverse primer in a single reaction. Specificity was confirmed in all tested individuals (n = 7 per species) without cross-amplification. Sensitivity tests demonstrated consistent amplification down to 0.1 ng of template DNA, matching or surpassing detection limits reported for other food-authentication markers. Because the entire reaction including DNA extraction can be completed within three hours and requires only basic laboratory equipment, the method is well suited for quality control laboratories, border inspections and routine monitoring of processed products. The COX1 multiplex PCR set proposed here provides a reliable tool to enhance traceability, protect consumer choice, and support regulatory enforcement in the sea-squirt supply chain. Full article

Madagascar harbors a rich marine biodiversity, yet detailed knowledge of its fish species remains limited. Of the 1689 species listed in 2018, only 22% had accessible cytochrome oxidase I (COI) sequences in public databases. In response to growing pressure on fishery resources, this study aims to strengthen biodiversity monitoring tools. Its objectives were to enrich the COI database for Malagasy marine fishes, create the first 12S reference library, and evaluate the taxonomic resolution of different 12S metabarcodes for eDNA analysis, namely MiFish, Teleo1, AcMDB, Ac12S, and 12SF1/R1. An integrated approach combining morphological, molecular, and phylogenetic analyses was applied for specimen identification of fish captured using various types of fishing gear in Toliara and Ranobe Bays from 2018 to 2023. The Malagasy COI database now includes 2146 sequences grouped into 502 Barcode Index Numbers (BINs) from 82 families, with 14 BINs newly added to BOLD (The Barcode of Life Data Systems), and 133 cryptic species. The 12S library comprises 524 sequences representing 446 species from 78 families. Together, the genetic datasets cover 514 species from 84 families, with the most diverse being Labridae, Apogonidae, Gobiidae, Pomacentridae, and Carangidae. However, the two markers show variable taxonomic resolution: 67 species belonging to 35 families were represented solely in the COI dataset, while 10 species from nine families were identified exclusively in the 12S dataset. For 319 species with complete 12S gene sequences associated with COI BINs (Barcode Index Numbers), 12S primer sets were used to evaluate the taxonomic resolution of five 12S metabarcodes. The MiFish marker proved to be the most effective, with an optimal similarity threshold of 98.5%. This study represents a major step forward in documenting and monitoring Madagascar’s marine biodiversity and provides a valuable genetic reference for future environmental DNA (eDNA) applications. Full article

The molecular identification of valuable genotypes is an important problem of germplasm management. In this study, we evaluated the potential of 11 universal primer pairs for the DNA barcoding of locally derived cultivars of subtropical crops (actinidia, feijoa, citrus, and tea). A total of 47 accessions (elite cultivars, forms, and breeding lines) of these four genera were included in the study. The efficiency of the following universal primers was assessed using Sanger sequencing: ITS-p5/ITS-u4, ITS-p5/ITS-u2, ITS-p3/ITS-u4, 23S,4.5S&5S, 16S, petB/petD, rpl23/rpl2.l, rpl2 intron, rpoC1 intron, trnK intron, and trnE-UUC/trnT-GUU. Among these primers, trnE-UUC/trnT-GUU showed greater intraspecific polymorphisms, while rpl2 intron and 16S displayed the lowest polymorphism levels in all crops. In addition, the 23S,4.5S & 5S, and rpoC1 intron were efficient for intraspecific analysis of tea and actinidia species. Using five efficient chloroplast primers, a total of 22/6 SNPs/InDels were observed in tea accessions, 45/17 SNPs/InDels in actinidia, 23/3 SNPs/InDels in mandarins, and 5/4 SNPs/InDels in feijoa. These results will be useful for the further development of DNA barcodes of related accessions. Full article

DNA barcoding of intraspecific diversity of agricultural crops is important to develop the genetic passports of valuable genotypes and cultivars. The advantage of DNA-barcoding as compared to traditional genotyping of cultivars is that the procedure can be unified and applied for the broad range of accessions. This not only makes it cost efficient, but also allows to develop open access genetic databases to accumulate information of the world’s germplasm collections of different crops. In this regard, the aim of the review was to analyze the latest research in this field, including the selection of loci, universal primers, strategies of amplicons analysis, bioinformatic tools, and the development of databases. We reviewed the advantages and disadvantages of each strategy with the focus of cultivars identification. The data indicates that following chloroplast loci are the most prominent for the intraspecific diversity analysis: (trnE-UUC/trnT-GUU, rpl23/rpl2.l, psbA-trnH, trnL-trnF, trnK, rpoC1, ycf1-a, rpl32-trnL, trnH-psbA and matK). We suggest that the combination of three or four of these loci can be a sufficient DNA barcode for cultivar-level identification. This combination has to be selected for each crop. Advantages and disadvantages of different approaches of amplicons analysis are discussed. The bioinformatic tools and databases for the plant barcoding are reviewed. This review will be useful for selecting appropriate strategies for barcoding of intraspecific diversity of agricultural crops to develop genetic passports of valuable cultivars in germplasm collections worldwide. Full article

Barnyard grass is the most problematic weed in paddy fields in Ningxia. Its substantial morphological variation complicates both identification and control, yet the genetic diversity of barnyard grass infesting paddy fields in Ningxia has not been thoroughly studied. In this research, we analyzed the genetic diversity of 46 barnyard grass populations from Ningxia’s paddy fields based on the assessment of morphological traits, DNA barcoding, and SCoT-targeted gene markers. Nine morphological traits were quantitatively analyzed, among which three phenological traits, i.e., leaf length, stem diameter, and plant height, exhibited notable variations. Correlational analysis revealed a positive relationship between morphological traits and multi-herbicide resistance profiles. To assess genetic diversity, four DNA barcodes (ITS, psbA, matK, and trnL-F) were used, among which ITS demonstrated the strongest potential in single-gene barcoding for barnyard grass species identification. Cluster analysis based on ITS barcode sequences was performed to group the populations into five main categories. Additionally, SCoT marker analysis using six primers was performed to classify the 46 barnyard grass samples into five groups. The results showed that the predominant barnyard grass species in Ningxia were E. colona, E. crus-galli var. Formosensis, E. crusgalli, E. oryzoides, and E. crusgalli var. Zelayensis, with E. colona being the most prevalent. The differences observed between the morphological and molecular marker-based classifications were method-dependent. However, both SCoT molecular marker technology and DNA barcoding contributed to identifying the genetic diversity of barnyard grass. Taken together, our study revealed significant morphological and genetic variations among barnyard grass populations, which correlated with herbicide sensitivity in Ningxia’s paddy fields, underscoring the necessity for an integrated weed management approach to combat this troublesome weed species. Full article

Molecular species identification plays an increasingly important role in forensic entomology and is centered on selecting appropriate DNA barcodes, which there are not yet enough of. Such identification is decisive in discovering a better DNA barcode for the identification of necrophilous fly species. Here, we analyzed 10 common necrophilous fly species found on Hainan Island; designed 12 pairs of fly-specific primers from different mitochondrial regions; screened two fly DNA barcodes with better results than those of published studies, which were used as controls; and employed a high-resolution melting (HRM) curve to construct PCR-HRM technology systems for rapid and efficient necrophilous fly species identification. The results showed that, among the 14 DNA barcoding PCR-HRM systems, the newly designed COXII-519/COXII-615 primer was the best, which identified 10 necrophilous fly species in one test. The second-best system was the C1-J-2495/C1-N-2800 primer published in the literature, which identified six fly species in one test. Moreover, since the COXII-519/COXII-615 primer system performed successfully in both stale (stored over two years) and larval samples due to its short amplificated fragment (shorter than 97 bp), it may serve as a new efficient DNA barcode for necrophilic fly species identification. The new DNA barcoding PCR-HRM system established in this study enables the rapid and accurate identification of necrophilic fly species. Full article

Biting midges, Culicoides spp. (Diptera: Ceratopogonidae), are significant vectors capable of transmitting arboviruses, such as bluetongue virus, to livestock. New Zealand is free of Culicoides, and a national surveillance programme is in place for the early detection of an incursion. Traditionally, insect trap samples from the surveillance programme are analyzed using morphology-based diagnostics under microscopes, which is time-consuming and relies on specialized taxonomic expertise. Here, we assessed the effectiveness of DNA metabarcoding using insect bulk samples and environmental DNA (eDNA) from liquid samples collected in surveillance traps. Two Cytochrome oxidase I (COI) barcoding primer sets were employed to study biodiversity and detect exotic species. The results indicated that DNA metabarcoding with homogenized insect bulk samples had a higher overall detection accuracy rate (over 81% for both primer pairs) compared to ethanol fluid-derived eDNA samples from traps (68.42% and 55.26% for the primer sets LCO1490/HCO2198 and mlCOIintF/jgHCO2198, respectively) based on congruence with morphological identification. Detection failures were likely due to eDNA extraction issues or low target species abundance. Both approaches showed similar insect community composition and diversity in the surveillance trap samples, suggesting the potential of DNA metabarcoding for biosecurity surveillance and biodiversity assessments. Overall, DNA metabarcoding using bulk insect samples could enhance the efficiency of Culicoides surveillance, reducing workload and screening time. Full article

To learn about the yeast biodiversity of Hungarian honeys and to isolate osmotolerant yeasts, fifteen different honey varieties, beeswax, and bee bread were purchased, and samples of another, but highly osmotic material, tree sap (cherry, sour cherry, and plum), were collected from the northeastern region of the country. In total, 60 yeast strains were isolated and their taxonomic positions were determined by barcode sequences using ITS1-NL4 primers. The honey products contained mostly Zygosaccharomyces and Starmerella species. In addition, Hanseniaspora uvarum, Rhodotorula mucilaginosa and diobovata, Sporobolomyces roseus, Filobasidium magnum, Naganishia sp., and Aureobasidium pullulans were also present in smaller numbers. In contrast, tree saps contained Metschnikowia and Pichia fermentas cells. Further results suggest that some of the yeasts in honey can only “survive”, while others can propagate at high sugar levels, generally between 600 and 700 mg/g, with a predominance of fructose. Properties important for pathogenicity, such as invasive hyphae production, gelatin melting ability, and growth at 37 °C, were also examined. Hanseniaspora uvarum and Pichia fermentans representatives seemed to be negative for gelatin hydrolysis, while the other strains were able to melt gelatin. Although some of the strains could produce hyphae-like structures at 25 °C, none of them could grow at 37 °C. Full article

Seafood is a valuable commodity with increasing demand, traded for billions of USD each year. The volatility in supply chains and fluctuating prices contribute to the susceptibility of the seafood market to food fraud. Analytical methods are required to identify seafood in processed foods to ensure food authenticity and compliance with European laws. To address this need, we developed and validated a DNA metabarcoding method for the authentication of crustaceans and cephalopods in processed food samples, as both are prone to food fraud, especially in mixed products. A ~200 bp barcode of the mitochondrial 16S rDNA was selected as the marker for identification and sequenced on Illumina platforms. The DNA metabarcoding method utilizes two primer systems, one for the amplification of crustacean DNA and another for cephalopods. The crustacean primer system comprises two forward and two reverse primers, while the cephalopod primer system includes three forward and one reverse primer. DNA extracts from reference materials, model foods, processed foodstuffs, and DNA extract mixtures were investigated. Even species with a close phylogenetic relationship were successfully identified and differentiated in commercial samples, while single species were detected at amounts as low as 0.003% in model foods. However, false-negative results were obtained for certain species in DNA extract mixtures, which are most likely due to degraded or low-quality DNA and can best be prevented by optimized DNA extraction procedures. Our DNA metabarcoding method demonstrates strong potential as a qualitative screening tool in combination with other in-house DNA metabarcoding methods for food authentication in routine analysis. Full article