DNA at the whim of the water: environmental DNA as a course-based undergraduate research experience.

Course-based undergraduate research experiences (CUREs) increase student access to high impact research experiences. CUREs engage students in the scientific process by learning how to pose scientific questions, develop hypotheses, and generate data to test them. Environmental DNA (eDNA) is a growing field of research that is gaining accessibility through decreasing laboratory costs, which can make a foundation for multiple, engaging CUREs. This manuscript describes three case studies that used eDNA in an upper year undergraduate course. The first focusses on a systematic literature review of eDNA metadata reporting. The second describes the biomonitoring of brook trout in southern Ontario using eDNA. The third involves eDNA metabarcoding for freshwater fish detection in southern Ontario. Undergraduates were involved in the development and execution of experiments, scientific communication, the peer review process, and fundraising. Through this manuscript, we show the novel application of eDNA CUREs and provide a roadmap for other instructors interested in implementing similar projects. Interviews with seven students from these courses indicate the benefits experienced from taking these courses. We argue that the use of eDNA in CUREs should be expanded in undergraduate biology programs due to the benefit to students and the increasing accessibility of this technology.

Mitochondrial genome sequencing, mapping, and assembly benchmarking for Culicoides species (Diptera: Ceratopogonidae).

BACKGROUND: Mitochondrial genomes are the most sequenced genomes after bacterial and fungal genomic DNA. However, little information on mitogenomes is available for multiple metazoan taxa, such as Culicoides, a globally distributed, megadiverse genus containing 1,347 species. AIM:  Generating novel mitogenomic information from single Culicoides sonorensis and C. biguttatus specimens, comparing available mitogenome mapping and de novo assembly tools, and identifying the best performing strategy and tools for Culicoides species. RESULTS: We present two novel and fully annotated mitochondrial haplotypes for two Culicoides species, C. sonorensis and C. biguttatus. We also annotated or re-annotated the only available reference mitogenome for C. sonorensis and C. arakawae. All species present a high similarity in mitogenome organization. The general gene arrangement for all Culicoides species was identical to the ancestral insect mitochondrial genome. Only short spacers were found in C. sonorensis (up to 30 bp), contrary to C. biguttatus (up to 114 bp). The mitochondrial genes ATP8, NAD2, NAD6, and LSU rRNA exhibited the highest nucleotide diversity and pairwise interspecific p genetic distance, suggesting that these genes might be suitable and complementary molecular barcodes for Culicoides identification in addition to the commonly utilized COI gene. We observed performance differences between the compared mitogenome generation strategies. The mapping strategy outperformed the de novo assembly strategy, but mapping results were partially biased in the absence of species-specific reference mitogenome. Among the utilized tools, BWA performed best for C. sonorensis while SPAdes, MEGAHIT, and MitoZ were among the best for C. biguttatus. The best-performing mitogenome annotator was MITOS2. Additionally, we were able to recover exogenous mitochondrial DNA from Bos taurus (biting midges host) from a C. biguttatus blood meal sample. CONCLUSIONS: Two novel annotated mitogenome haplotypes for C. sonorensis and C. biguttatus using High-Throughput Sequencing are presented. Current results are useful as the baseline for mitogenome reconstruction of the remaining Culicoides species from single specimens to HTS and genome annotation. Mapping to a species-specific reference mitogenome generated better results for Culicoides mitochondrial genome reconstruction than de novo assembly, while de novo assembly resulted better in the absence of a closely related reference mitogenome. These results have direct implications for molecular-based identification of these vectors of human and zoonotic diseases, setting the basis for using the whole mitochondrial genome as a marker in Culicoides identification.

The Multiple States of Environmental DNA and What Is Known about Their Persistence in Aquatic Environments.

Increased use of environmental DNA (eDNA) analysis for indirect species detection has spurred the need to understand eDNA persistence in the environment. Understanding the persistence of eDNA is complex because it exists in a mixture of different states (e.g., dissolved, particle adsorbed, intracellular, and intraorganellar), and each state is expected to have a specific decay rate that depends on environmental parameters. Thus, improving knowledge about eDNA conversion rates between states and the reactions that degrade eDNA in different states is needed. Here, we focus on eukaryotic extraorganismal eDNA, outline how water chemistry and suspended mineral particles likely affect conversion among each eDNA state, and indicate how environmental parameters affect persistence of states in the water column. On the basis of deducing these controlling parameters, we synthesized the eDNA literature to assess whether we could already derive a general understanding of eDNA states persisting in the environment. However, we found that these parameters are often not being measured or reported when measured, and in many cases very few experimental data exist from which to draw conclusions. Therefore, further study of how environmental parameters affect eDNA state conversion and eDNA decay in aquatic environments is needed. We recommend analytic controls that can be used during the processing of water to assess potential losses of different eDNA states if all were present in a water sample, and we outline future experimental work that would help determine the dominant eDNA states in water.

Assessment of stream macroinvertebrate communities with eDNA is not congruent with tissue-based metabarcoding.

Freshwater biomonitoring programmes routinely sample aquatic macroinvertebrates. These samples are time-consuming to collect, as well as challenging and costly to identify reliably genus or species. Environmental DNA (eDNA) metabarcoding has emerged as a surrogate to traditional collection techniques and has been used in whole-community approaches across several taxa and ecosystems. However, the usefulness of eDNA-based detection of freshwater macroinvertebrates has not been extensively explored. Few studies have directly compared bulk sample and eDNA metabarcoding at a local scale to assess how effective each method is at characterizing aquatic macroinvertebrate communities. Here, we collected both eDNA and kicknet samples at the same sample transect locations across nine different streams in southern Ontario, Canada. We observed minimal overlap in community composition between these paired samples. Bulk tissue metabarcoding resulted in a greater proportion of sequences belonging to metazoan taxa (over 99%) than eDNA (12%) and had higher OTU richness for macroinvertebrate taxa. We suggest that degenerate primers are not effective for eDNA metabarcoding due to the high degree of nontarget amplification and subsequently low yield of target DNA. While both bulk sample and eDNA metabarcoding had the power to detect differences between stream communities, eDNA did not represent local communities. Bulk tissue metabarcoding thus provides a more accurate representation of local stream macroinvertebrate communities and is the preferred method if smaller-scale spatial resolution is an important factor in data analyses.

Environmental DNA bioassays corroborate field data for detection of overwintering species at risk Blanding's turtles (Emydoidea blandingii).

Environmental DNA (eDNA) is gaining traction in conservation ecology as a powerful tool for detecting species at risk. We developed a quantitative polymerase chain reaction assay to detect a DNA amplicon fragment of the mitochondrial nicotinamide adenine dinucleotide locus of the Blanding's turtle (Emydoidea blandingii) for detecting overwintering individuals. Seventy-eight water samples were collected from 17 wetland sites in Ontario, Canada. We used traditional field data to identify a priori positive and negative control sites. Fifty percent of positive control sites amplified. Detection was related to the number of individuals estimated from field observations in at least one region surveyed. Positive control sites had lower total dissolved solids and electrical conductivity in relation to negative control sites. Shedding rates were within the same order of magnitude for brumating and active turtles. We recommend collecting additional samples at a larger number of locations to maximize detection. Recommended sampling design changes may overshadow the additional effects of water chemistry and low eDNA shedding rates. eDNA offers tremendous potential to practitioners conducting species at risk assessments in environmental consulting by providing a faster, more efficient method of detection compared with traditional surveys.

Environmental DNA detection of endangered and invasive species in Kejimkujik National Park and Historic Site.

The use of environmental DNA (eDNA) allows the early detection of aquatic species at low densities (e.g., elusive and invasive species), which otherwise could be challenging to monitor using conventional techniques. Here, we assess the ability of eDNA sampling to detect the presence or absence of one species at risk (Blanding's turtle) and two invasive species (chain pickerel and smallmouth bass) in Kejimkujik National Park and National Historic Site, Nova Scotia, where the aquatic system is highly acidic and rich in organic compounds. Five replicates of 1 L water samples were taken per sampling site. Water filtration and eDNA extractions were performed on-site, while qPCR reactions were performed in the laboratory using species-specific assays. Samples were treated with an inhibition removal kit and analyzed pre- and post-inhibition removal. Despite the low pH and PCR inhibitors in water samples, our results showed positive eDNA detections in almost all expected positive sites (except in one site for Blanding's turtle). Detections of the target species were also observed at sites where their presence was previously unknown. Our study supports the advantage of eDNA to monitor species at low densities, revealing new distributions or recently invaded areas. We also demonstrate how eDNA can directly instruct management strategies in Kejimkujik.

DNA barcodes identify medically important tick species in Canada.

Medically important ticks (Acari: Ixodidae) are often difficult to identify morphologically. A standardized, molecular approach using a 658 base pair DNA barcode sequence (from the 5' region of the mitochondrial cytochrome c oxidase subunit I gene) was evaluated for its effectiveness in discriminating ticks in North America, with an emphasis on Canadian ticks. DNA barcodes were generated for 96 of 154 specimens representing 26 ixodid species. A genetic cluster analysis was performed on the barcode sequences, which separated specimens into haplogroups closely corresponding with morphologically identified species. The tree topology was further supported by a BIN analysis. COI sequences generated were found to have a mean maximum intraspecific divergence of 1.59% and a mean nearest neighbour divergence of 12.8%, indicating a significant "barcode gap". This study also revealed possible cryptic diversity among specimens morphologically identified as Ixodes soricis and Ixodes texanus. A PCR-based test for Borrelia burgdorferi determined that 18.1% of Lyme-competent ticks in this study were positive. This study is also the first to record a B. burgdorferi-positive exoskeleton. In conclusion, DNA barcoding is a powerful tool that clinicians can use to determine the identification of tick specimens which can help them to suggest whether an attached tick is a potential health risk.

DNA-based identification of invasive alien species in relation to Canadian federal policy and law, and the basis of rapid-response management.

Managing invasive alien species in Canada requires reliable taxonomic identification as the basis of rapid-response management. This can be challenging, especially when organisms are small and lack morphological diagnostic features. DNA-based techniques, such as DNA barcoding, offer a reliable, rapid, and inexpensive toolkit for taxonomic identification of individual or bulk samples, forensic remains, and even environmental DNA. Well suited for this requirement, they could be more broadly deployed and incorporated into the operating policy and practices of Canadian federal departments and should be authorized under these agencies' articles of law. These include Fisheries and Oceans Canada, Canadian Food Inspection Agency, Transport Canada, Environment Canada, Parks Canada, and Health Canada. These efforts should be harmonized with the appropriate provisions of provincial jurisdictions, for example, the Ontario Invasive Species Act. This approach necessitates that a network of accredited, certified laboratories exists, and that updated DNA reference libraries are readily accessible. Harmonizing this approach is vital among Canadian federal agencies, and between the federal and provincial levels of government. Canadian policy and law must also be harmonized with that of the USA when detecting, and responding to, invasive species in contiguous lands and waters. Creating capacity in legislation for use of DNA-based identifications brings the authority to fund, train, deploy, and certify staff, and to refine further developments in this molecular technology.

Genetic calibration of species diversity among North America's freshwater fishes.

Freshwater ecosystems are being heavily exploited and degraded by human activities all over the world, including in North America, where fishes and fisheries are strongly affected. Despite centuries of taxonomic inquiry, problems inherent to species identification continue to hamper the conservation of North American freshwater fishes. Indeed, nearly 10% of species diversity is thought to remain undescribed. To provide an independent calibration of taxonomic uncertainty and to establish a more accessible molecular identification key for its application, we generated a standard reference library of mtDNA sequences (DNA barcodes) derived from expert-identified museum specimens for 752 North American freshwater fish species. This study demonstrates that 90% of known species can be delineated using barcodes. Moreover, it reveals numerous genetic discontinuities indicative of independently evolving lineages within described species, which points to the presence of morphologically cryptic diversity. From the 752 species analyzed, our survey flagged 138 named species that represent as many as 347 candidate species, which suggests a 28% increase in species diversity. In contrast, several species of parasitic and nonparasitic lampreys lack such discontinuity and may represent alternative life history strategies within single species. Therefore, it appears that the current North American freshwater fish taxonomy at the species level significantly conceals diversity in some groups, although artificially creating diversity in others. In addition to providing an easily accessible digital identification system, this study identifies 151 fish species for which taxonomic revision is required.

Delimiting Species with Single-Locus DNA Sequences.

DNA sequences are increasingly used for large-scale biodiversity inventories. Because these genetic data avoid the time-consuming initial sorting of specimens based on their phenotypic attributes, they have been recently incorporated into taxonomic workflows for overlooked and diverse taxa. Major statistical developments have accompanied this new practice, and several models have been proposed to delimit species with single-locus DNA sequences. However, proposed approaches to date make different assumptions regarding taxon lineage history, leading to strong discordance whenever comparisons are made among methods. Distance-based methods, such as Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP), rely on the detection of a barcode gap (i.e., the lack of overlap in the distributions of intraspecific and interspecific genetic distances) and the associated threshold in genetic distances. Network-based methods, as exemplified by the REfined Single Linkage (RESL) algorithm for the generation of Barcode Index Numbers (BINs), use connectivity statistics to hierarchically cluster-related haplotypes into molecular operational taxonomic units (MOTUs) which serve as species proxies. Tree-based methods, including Poisson Tree Processes (PTP) and the General Mixed Yule Coalescent (GMYC), fit statistical models to phylogenetic trees by maximum likelihood or Bayesian frameworks.Multiple webservers and stand-alone versions of these methods are now available, complicating decision-making regarding the most appropriate approach to use for a given taxon of interest. For instance, tree-based methods require an initial phylogenetic reconstruction, and multiple options are now available for this purpose such as RAxML and BEAST. Across all examined species delimitation methods, judicious parameter setting is paramount, as different model parameterizations can lead to differing conclusions. The objective of this chapter is to guide users step-by-step through all the procedures involved for each of these methods, while aggregating all necessary information required to conduct these analyses. The "Materials" section details how to prepare and format input files, including options to align sequences and conduct tree reconstruction with Maximum Likelihood and Bayesian inference. The Methods section presents the procedure and options available to conduct species delimitation analyses, including distance-, network-, and tree-based models. Finally, limits and future developments are discussed in the Notes section. Most importantly, species delimitation methods discussed herein are categorized based on five indicators: reliability, availability, scalability, understandability, and usability, all of which are fundamental properties needed for any approach to gain unanimous adoption within the DNA barcoding community moving forward.

A Measure of the DNA Barcode Gap for Applied and Basic Research.

DNA barcoding has largely established itself as a mainstay for rapid molecular taxonomic identification in both academic and applied research. The use of DNA barcoding as a molecular identification method depends on a "DNA barcode gap"-the separation between the maximum within-species difference and the minimum between-species difference. Previous work indicates the presence of a gap hinges on sampling effort for focal taxa and their close relatives. Furthermore, both theory and empirical work indicate a gap may not occur for related pairs of biological species. Here, we present a novel evaluation approach in the form of an easily calculated set of nonparametric metrics to quantify the extent of proportional overlap in inter- and intraspecific distributions of pairwise differences among target species and their conspecifics. The metrics are based on a simple count of the number of overlapping records for a species falling within the bounds of maximum intraspecific distance and minimum interspecific distance. Our approach takes advantage of the asymmetric directionality inherent in pairwise genetic distance distributions, which has not been previously done in the DNA barcoding literature. We apply the metrics to the predatory diving beetle genus Agabus as a case study because this group poses significant identification challenges due to its morphological uniformity despite both relative sampling ease and well-established taxonomy. Results herein show that target species and their nearest neighbor species were found to be tightly clustered and therefore difficult to distinguish. Such findings demonstrate that DNA barcoding can fail to fully resolve species in certain cases. Moving forward, we suggest the implementation of the proposed metrics be integrated into a common framework to be reported in any study that uses DNA barcoding for identification. In so doing, the importance of the DNA barcode gap and its components for the success of DNA-based identification using DNA barcodes can be better appreciated.

Glacial cycles as an allopatric speciation pump in north-eastern American freshwater fishes.

Allopatric speciation may be the principal mechanism generating new species. Yet, it remains difficult to judge the generality of this process because few studies have provided evidence that geographic isolation has triggered the development of reproductive isolation over multiple species of a regional fauna. Here, we first combine results from new empirical data sets (7 taxa) and published literature (9 taxa) to show that the eastern Great Lakes drainage represents a multispecies suture zone for glacial lineages of freshwater fishes with variable levels of genetic divergence. Second, we performed amplified fragment length polymorphism analyses among four pairs of lineages. Results indicate that lineages with relatively deep levels of mtDNA 5' COI (barcode) sequence divergence (>2%) developed strong reproductive barriers, while lineages with lower levels of divergence show weaker reproductive isolation when found in sympatry. This suggests that a threshold of 2% sequence divergence at mtDNA could be used as a first step to flag cryptic species in North American freshwater fishes. By describing different levels of divergence and reproductive isolation in different co-occurring fishes, we offer strong evidence that allopatric speciation has contributed significantly to the diversification of north-eastern American freshwater fishes and confirm that Pleistocene glacial cycles can be viewed as a 'speciation pump' that played a predominant role in generating biodiversity.

Hidden diversity: DNA metabarcoding reveals hyper-diverse benthic invertebrate communities.

BACKGROUND: Freshwater ecosystems, such as streams, are facing increasing pressures from agricultural land use and recent literature stresses the importance of robust biomonitoring to detect trends in insect decline globally. Aquatic insects and other macroinvertebrates are often used as indicators of ecological condition in freshwater biomonitoring programs; however, these diverse groups can present challenges to morphological identification and coarse-level taxonomic resolution can mask patterns in community composition. Here, we incorporate molecular identification (DNA metabarcoding) into a stream biomonitoring sampling design to explore the diversity and variability of aquatic macroinvertebrate communities at small spatial scales. While individual stream reaches can be very heterogenous, most community ecology studies focus on larger, landscape-level patterns of community composition. A high degree of community variability at the local scale has important implications for both biomonitoring and ecological research, and the incorporation of DNA metabarcoding into local biodiversity assessments will inform future sampling protocols. RESULTS: We sampled twenty streams in southern Ontario, Canada, for aquatic macroinvertebrates across multiple time points and assessed local community variability by comparing field replicates taken ten meters apart within the same stream. Using bulk-tissue DNA metabarcoding, we revealed that aquatic macroinvertebrate communities are highly diverse at small spatial scales with unprecedented levels of local taxonomic turnover. We detected over 1600 Operational Taxonomic Units (OTUs) from 149 families, and a single insect family, the Chironomidae, contained over one third of the total number of OTUs detected in our study. Benthic communities were largely comprised of rare taxa detected only once per stream despite multiple biological replicates (24-94% rare taxa per site). In addition to numerous rare taxa, our species pool estimates indicated that there was a large proportion of taxa that remained undetected by our sampling regime (14-94% per site). Our sites were located across a gradient of agricultural activity, and while we predicted that increased land use would homogenize benthic communities, this was not supported as within-stream dissimilarity was unrelated to land use. Within-stream dissimilarity estimates were consistently high for all levels of taxonomic resolution (invertebrate families, invertebrate OTUs, chironomid OTUs), indicating stream communities are very dissimilar at small spatial scales.

Unveiling invasive insect threats to plant biodiversity: Leveraging eDNA metabarcoding and saturated salt trap solutions for biosurveillance.

The negative global impacts of invasive alien species (IAS) on biodiversity are second only to habitat loss. eDNA metabarcoding allows for a faster and more comprehensive evaluation of community species composition, with a higher taxonomic resolution and less taxonomic expertise required than traditional morphological-based biosurveillance. These advantages have positioned eDNA metabarcoding as the standard method for molecular-based detection of invasive alien species, where fast and accurate detectability allows prompt responses to mitigate their adverse effects. Here, eDNA metabarcoding is used for biosurveillance of invasive alien species regulated by Canada in high-risk areas with four main objectives: i) validate the effectiveness of eDNA metabarcoding of salt trap solutions as a molecular technique for IAS detection, ii) compare detection from DNA extracts obtained from filter quarters versus whole filters, iii) benchmark two different bioinformatic pipelines (MetaWorks and mBRAVE), and iv) compare canopy and ground level trapping. eDNA from up to five IAS (Agrilus planipennis, Daktulosphaira vitifoliae, Lymantria dispar, Popillia japonica, and Trichoferus campestris) were successfully detected across years from 2017 to 2022 in southern Ontario, Canada, with successful morphological validation for all except Lymantria dispar and Trichoferus campestris. Analysis of filter quarters in contrast to whole filters was demonstrated to be insufficient for effective IAS detection in each sample. All IAS were detected in only one filter quarter, suggesting a patchy eDNA distribution on the filter. The MetaWorks and mBRAVE bioinformatics pipelines proved effective in identifying IAS, with MetaWorks yielding a higher success rate when comparing molecular and morphological identifications. Ground-level and canopy-level sampling showed differential IAS recovery rates based on the molecular detection, which also varied per collection year, with all found IAS detected at the canopy level in 2022 while only one (Lymantria dispar) in 2020. The present study ratifies the efficacy and importance of eDNA-based detection in a regulatory context and the utility of adding eDNA metabarcoding of saturated salt trap solutions, a critical tool for IAS detection.

VLF: An R package for the analysis of very low frequency variants in DNA sequences.

Here, we introduce VLF, an R package to determine the distribution of very low frequency variants (VLFs) in nucleotide and amino acid sequences for the analysis of errors in DNA sequence records. The package allows users to assess VLFs in aligned and trimmed protein-coding sequences by automatically calculating the frequency of nucleotides or amino acids in each sequence position and outputting those that occur under a user-specified frequency (default of p = 0.001). These results can then be used to explore fundamental population genetic and phylogeographic patterns, mechanisms and processes at the microevolutionary level, such as nucleotide and amino acid sequence conservation. Our package extends earlier work pertaining to an implementation of VLF analysis in Microsoft Excel, which was found to be both computationally slow and error prone. We compare those results to our own herein. Results between the two implementations are found to be highly consistent for a large DNA barcode dataset of bird species. Differences in results are readily explained by both manual human error and inadequate Linnean taxonomy (specifically, species synonymy). Here, VLF is also applied to a subset of avian barcodes to assess the extent of biological artifacts at the species level for Canada goose (Branta canadensis), as well as within a large dataset of DNA barcodes for fishes of forensic and regulatory importance. The novelty of VLF and its benefit over the previous implementation include its high level of automation, speed, scalability and ease-of-use, each desirable characteristics which will be extremely valuable as more sequence data are rapidly accumulated in popular reference databases, such as BOLD and GenBank.

Environmental DNA-based biomonitoring of Cuban Crocodylus and their accompanying vertebrate fauna from Zapata Swamp, Cuba.

Crocodylians globally face considerable challenges, including population decline and extensive habitat modification. Close monitoring of crocodylian populations and their habitats is imperative for the timely detection of population trends, especially in response to management interventions. Here we use eDNA metabarcoding to identify the Critically Endangered Crocodylus rhombifer and the Vulnerable C. acutus, as well as vertebrate community diversity, in Cuba's Zapata Swamp. We tested four different primer sets, including those used previously in Crocodylus population genetic and phylogenetic research, for their efficiency at detecting crocodylian eDNA. We detected C. rhombifer eDNA in 11 out of 15 sampled locations within its historical geographic distribution. We found that data analyses using the VertCOI primers and the mBRAVE bioinformatics pipeline were the most effective molecular marker and pipeline combination for identifying this species from environmental samples. We also identified 55 vertebrate species in environmental samples across the four bioinformatics pipelines- ~ 85% known to be present in the Zapata ecosystem. Among them were eight species previously undetected in the area and eight alien species, including known predators of hatchling crocodiles (e.g., Clarias sp.) and egg predators (e.g., Mus musculus). This study highlights eDNA metabarcoding as a powerful tool for crocodylian biomonitoring within fragile and diverse ecosystems, particularly where fast, non-invasive methods permit detection in economically important areas and will lead to a better understanding of complex human-crocodile interactions and evaluate habitat suitability for potential reintroductions or recovery programs for threatened crocodylian species.

Transcriptomic Analyses of Grapevine Leafroll-Associated Virus 3 Infection in Leaves and Berries of 'Cabernet Franc'.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source-sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development.

A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use.

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries.

BACKGROUND: Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. RESULTS: We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. CONCLUSIONS: We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.