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Molecular Techniques for the Identification of Freshwater Fish Species for Environmental Monitoring Programs

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Molecular Techniques for the Identification of Freshwater Fish Species for Environmental Monitoring Programs Molecular techniques for the identification of freshwater fish species for environmental monitoring programs by Emily Hulley A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science (MSc) in Biology The Faculty of Graduate Studies Laurentian University Sudbury, Ontario, Canada © Emily Hulley, 2019 THESIS DEFENCE COMMITTEE/COMITÉ DE SOUTENANCE DE THÈSE Laurentian Université/Université Laurentienne Faculty of Graduate Studies/Faculté des études supérieures Title of Thesis Titre de la thèse Molecular techniques for the identification of freshwater fish species for environmental monitoring programs Name of Candidate Nom du candidat Hulley, Emily Degree Diplôme Master of Science Department/Program Date of Defence Département/Programme Biology Date de la soutenance October 26, 2018 APPROVED/APPROUVÉ Thesis Examiners/Examinateurs de thèse: Dr. Douglas Boreham (Supervisor/Directeur de thèse) Dr. John Gunn (Committee member/Membre du comité) Dr. Kabwe Nkongolo (Committee member/Membre du comité) Approved for the Faculty of Graduate Studies Approuvé pour la Faculté des études supérieures Dr. David Lesbarrères Monsieur David Lesbarrères Dr. Gustavo Ybazeta Dean, Faculty of Graduate Studies (External Examiner/Examinateur externe) Doyen, Faculté des études supérieures ACCESSIBILITY CLAUSE AND PERMISSION TO USE I, Emily Hulley, hereby grant to Laurentian University and/or its agents the non-exclusive license to archive and make accessible my thesis, dissertation, or project report in whole or in part in all forms of media, now or for the duration of my copyright ownership. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also reserve the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that this copy is being made available in this form by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. ii Abstract Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time PCR (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probe for the identification of eight freshwater fish species. The development of a fully automated species-decoder algorithm allowed for target species identification with 100% accuracy while completely removing any false-positive detection of non-target species. Furthermore, the probe-based qPCR technique utilized in this study is substantially more cost-effective and time efficient than DNA barcoding and morphological identification methods. The qPCR assays were also highly sensitive and accurately detected target species from collected environmental DNA (eDNA) samples. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification and the methodology established in this study can be utilized for various other species identification initiatives. Keywords Freshwater fish species, environmental monitoring, cytochrome oxidase I, DNA barcoding, morphological identification, TaqMan real-time PCR, multiplex PCR, environmental DNA, next generation sequencing, species identification, species detection iii Co-Authorship Statement This thesis was written with myself as the primary author, however, several coauthors were directly involved in several aspects of the research. Appendix A includes the primary co-authorship of “DNA barcoding vs. morphological identification of larval fish and embryos in Lake Huron: advantages to a molecular approach” between Emily Hulley and Natalie Taylor. Natalie Taylor conducted the original research for DNA barcoding the larval fish and embryos and prepared the original manuscript. Emily Hulley has been the corresponding author for publication with the Journal of Great Lakes Research. She has also been primarily responsible for the major revisions to the manuscript and tables from reviewers, associate editors and collaborating authors. iv Acknowledgments There are countless people I would like to thank for their help and support during my thesis. First and foremost, I would like to thank my supervisor Dr. Douglas Boreham for the opportunities and support you provided to me throughout my degree. I would also like to thank my Boreham lab mates for an amazing two years. Additionally, I would like to say a special thank you to Dr. Sujeenthar Tharmalingam and Dr. Chris Thome, who were always there, no matter how busy, to provide me with guidance and assistance whenever I needed it. I would also like to thank my family and friends for their endless love, support and encouragement. If it hadn’t been for my incredible parents and family, I would not be who or where I am today. I am so fortunate to have such an amazing and strong family for role models. v Table of Contents Abstract ............................................................................................................................. iii Co-Authorship Statement ............................................................................................... iv Acknowledgments ............................................................................................................. v Table of Contents ............................................................................................................. vi List of Tables .................................................................................................................... ix List of Figures .................................................................................................................... x List of Acronyms .............................................................................................................. xi List of Appendices ........................................................................................................... xii Appendix A: DNA barcoding vs. morphological identification of larval fish and embryos in Lake Huron: advantages to a molecular approach ............................................ xii Appendix B: Supplementary Tables ............................................................................ xii Chapter 1: Introduction ................................................................................................... 1 Chapter 2: Development and Validation of Probe-Based Multiplex Real-Time PCR Assays for the Rapid and Accurate Detection of Fish Species ...................................... 7 Abstract .......................................................................................................................... 8 Introduction .................................................................................................................... 9 Materials and Methods ................................................................................................. 12 Sample Collection & DNA Extraction ................................................................................................ 12 DNA Barcoding .................................................................................................................................. 12 qPCR ................................................................................................................................................... 13 Sequence Alignment ........................................................................................................................... 14 Results .......................................................................................................................... 15 Species of Interest and Establishment of Control Species Using Geographical and Homology Analysis............................................................................................................................................... 15 Design of Primer and Probe ................................................................................................................ 17 In Silico Verification of Primer-Probe Specificity .............................................................................. 19 Probe Specification and Primer-Probe qPCR Optimizations .............................................................. 20 Determination of Primer-Probe Sensitivity Under Single-plex vs Multiplex qPCR Conditions ........ 22 vi Determination of Primer-Probe Specificity ........................................................................................ 24 Blinded Analysis Utilizing Automated Data Decoder Algorithm ...................................................... 26 Multiplexing of Multiple Species DNA .............................................................................................. 29 Discussion ...................................................................................................................
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