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      Environmental DNA assays for the sister taxa sauger ( Sander canadensis) and walleye ( Sander vitreus)

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          Abstract

          Sauger ( Sander canadensis) and walleye ( S. vitreus) are percid fishes that naturally co-occur throughout much of the eastern United States. The native range of sauger extends into the upper Missouri River drainage where walleye did not historically occur, but have been stocked as a sport fish. Sauger populations have been declining due to habitat loss, fragmentation, and competition with non-native species, such as walleye. To effectively manage sauger populations, it is necessary to identify areas where sauger occur, and particularly where they co-occur with walleye. We developed quantitative PCR assays that can detect sauger and walleye DNA in filtered water samples. Each assay efficiently detected low quantities of target DNA and failed to detect DNA of non-target species with which they commonly co-occur.

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          Most cited references28

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          Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

          Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.
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            The Dual Challenges of Generality and Specificity When Developing Environmental DNA Markers for Species and Subspecies of Oncorhynchus

            Environmental DNA (eDNA) sampling is a powerful tool for detecting invasive and native aquatic species. Often, species of conservation interest co-occur with other, closely related taxa. Here, we developed qPCR (quantitative PCR) markers which distinguish westslope cutthroat trout (Oncorhynchus clarkii lewsi), Yellowstone cutthroat trout (O. clarkii bouvieri), and rainbow trout (O. mykiss), which are of conservation interest both as native species and as invasive species across each other’s native ranges. We found that local polymorphisms within westslope cutthroat trout and rainbow trout posed a challenge to designing assays that are generally applicable across the range of these widely-distributed species. Further, poorly-resolved taxonomies of Yellowstone cutthroat trout and Bonneville cutthroat trout (O. c. utah) prevented design of an assay that distinguishes these recognized taxa. The issues of intraspecific polymorphism and unresolved taxonomy for eDNA assay design addressed in this study are likely to be general problems for closely-related taxa. Prior to field application, we recommend that future studies sample populations and test assays more broadly than has been typical of published eDNA assays to date.
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              Exploiting extension bias in polymerase chain reaction to improve primer specificity in ensembles of nearly identical DNA templates.

              We describe a semi-empirical framework that combines thermodynamic models of primer hybridization with experimentally determined elongation biases introduced by 3'-end mismatches for improving polymerase chain reaction (PCR)-based sequence discrimination. The framework enables rational and automatic design of primers for optimal targeting of one or more sequences in ensembles of nearly identical DNA templates. In situations where optimal targeting is not feasible, the framework accurately predicts non-target sequences that are difficult to distinguish with PCR alone. Based on the synergistic effects of disparate sources of PCR bias, we used our framework to robustly distinguish between two alleles that differ by a single base pair. To demonstrate the applicability to environmental microbiology, we designed primers specific to all recognized archaeal and bacterial genera in the Ribosomal Database Project, and have made these primers available online. We applied these primers experimentally to obtain genus-specific amplification of 16S rRNA genes representing minor constituents of an environmental DNA sample. Our results demonstrate that inherent PCR biases can be reliably employed in an automatic fashion to maximize sequence discrimination and accurately identify potential cross-amplifications. We have made our framework accessible online as a programme for designing primers targeting one group of sequences in a set with many other sequences (http://DECIPHER.cee.wisc.edu).
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                25 April 2017
                2017
                : 12
                : 4
                : e0176459
                Affiliations
                [1 ]United States Department of Agriculture, Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, Montana, United States of America
                [2 ]Montana Department of Fish, Wildlife and Parks, Region 5, Billings, Montana, United States of America
                National Cheng Kung University, TAIWAN
                Author notes

                Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: This study was completed through funding by a commercial entity – Walleyes Unlimited of Montana. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                • Conceptualization: KJC MR KSM MKY MKS.

                • Data curation: JCD.

                • Formal analysis: JCD KJC MR.

                • Funding acquisition: MR.

                • Investigation: JCD KJC MR.

                • Methodology: JCD KJC MR KSM MKY MKS.

                • Project administration: JCD KJC MR.

                • Resources: JCD KJC MR KSM MKY MKS.

                • Supervision: KJC MR KSM MKY MKS.

                • Validation: JCD.

                • Visualization: JCD KJC MR KSM MKY.

                • Writing – original draft: JCD KJC.

                • Writing – review & editing: JCD KJC MR KSM MKY MKS.

                Author information
                http://orcid.org/0000-0002-6790-7841
                Article
                PONE-D-16-48090
                10.1371/journal.pone.0176459
                5404789
                28441436
                80ffd959-003e-4108-9f17-067c4ce6dae7

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 5 December 2016
                : 11 April 2017
                Page count
                Figures: 0, Tables: 4, Pages: 9
                Funding
                Funded by: Walleyes Unlimited of Montana
                Award Recipient :
                This work was completed through funding issued to MR by Walleyes Unlimited of Montana. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Earth Sciences
                Marine and Aquatic Sciences
                Bodies of Water
                Lakes
                Ecology and Environmental Sciences
                Aquatic Environments
                Freshwater Environments
                Lakes
                Earth Sciences
                Marine and Aquatic Sciences
                Aquatic Environments
                Freshwater Environments
                Lakes
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                People and places
                Geographical locations
                North America
                United States
                Missouri
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Alignment
                Biology and Life Sciences
                Biochemistry
                Bioenergetics
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Energy-Producing Organelles
                Mitochondria
                Biology and life sciences
                Genetics
                DNA
                DNA replication
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA replication
                People and places
                Geographical locations
                North America
                United States
                Custom metadata
                All relevant data are within the paper. All genetic data used in this study were obtained from GenBank and accession numbers have been provided within the paper.

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