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A Next-Generation Sequencing Approach for the Simultaneous A NEXT-GENERATION SEQUENCING APPROACH FOR THE SIMULTANEOUS DETECTION OF PLANT VIRUSES FOR PLANT QUARANTINE TESTING A Thesis Submitted to the Graduate Faculty In Partial Fulfillment of the Requirements For the Degree of Master of Science In the Department of Pathology and Microbiology Faculty of Veterinary Medicine University of Prince Edward Island Desmond L. Hammill Charlottetown, PE April 2019 © 2019, D.L. Hammill THESIS/DISSERTATION NON-EXCLUSIVE LICENSE Family Name: Hammill Given Name, Middle Name (if applicable): Desmond, Leslie Full Name of University: University of Prince Edward Island Faculty, Department, School: Faculty of Veterinary Medicine, Department of Pathology/Microbiology, UPEI Degree for which Date Degree Awarded: thesis/dissertation was presented: Master of Science Thesis/dissertation Title: A next-generation sequencing approach for the simultaneous detection of plant viruses for plant quarantine testing In consideration of my University making my thesis/dissertation available to interested persons, I, Desmond Hammill, hereby grant a non-exclusive, for the full term of copyright protection, license to my University, University of Prince Edward Island: a) to archive, preserve, produce, reproduce, publish, communicate, convert into any format, and to make available in print or online by telecommunication to the public for non- commercial purposes; b) to sub-licence to Library and Archives Canada any of the acts mentioned in paragraph (a). I undertake to submit my thesis/dissertation, through my University, to Library and Archives Canada. Any abstract submitted with the thesis/dissertation will be considered to form part of the thesis/dissertation. I represent that my thesis/dissertation is my original work, does not infringe any rights of others, including privacy right, and that I have the right to make the grant conferred by this non- exclusive license. If third party copyrighted material was included in my thesis/dissertation for which, under the terms of the Copyright Act, written permission from the copyright owners is required I have to obtained such permission from the copyright owners to do the acts mentioned in paragraph (a) above the full term of copyright protection. I retrain copyright ownership and moral rights in my thesis/dissertation, and may deal with the copyright in my thesis/dissertation, in any way consistent with rights granted by me to my University in this non-exclusive license. I further promise to inform any person to whom I may hereafter assign or license my copyright in my thesis/dissertation of the rights granted by me to my University in this non-exclusive license. Signature: Date: ii UNIVERSITY OF PRINCE EDWARD ISLAND FACULTY OF VETERINARY MEDICINE CHARLOTTETOWN CERTIFICATION OF THESIS WORK We, the undersigned, certify that Desmond Hammill, candidate for the degree of Master of Science has presented his/her thesis with the following title: A NEXT-GENERATION SEQUENCING APPROACH FOR THE SIMULTANEOUS DETECTION OF PLANT VIRUSES FOR PLANT QUARANTINE TESTING that the thesis is acceptable in form and content, and that a satisfactory knowledge of the field covered by the thesis was demonstrated by the candidate through an oral examination held on: Examiners Dr. Mark Fast (chair) _______________________________ Dr. Huimin Xu _______________________________ Dr. Chelsea Martin _______________________________ Dr. Russell Fraser _______________________________ Dr. Lawrence Hale _______________________________ Date: July 9, 2019 iii ABSTRACT Potato (Solanum tuberosum L.) is one of the most important vegetable crops in the world and plays a vital role in human nutrition and food security. However, potato crops have been known to be infected by at least 40 viruses and 2 viroids contributing to reduced yields. The detection of viruses and viroids is limited from a diagnostic perspective as there are very few validated procedures that exist. Globalization of agriculture has meant that crop plants are now grown further from their centres of origin and far from the pathogens that had co-evolved with them. Crops introduced to a new area may be poorly equipped to resists pathogenic organisms that are already resident. Similarly, increased trade leads to the potential of introducing pathogenic organisms that may not be endemic to a particular region leading to outbreaks having the potential for extreme economic consequences. The Potato Post-Entry Quarantine (PPEQ) program implemented by the Canadian Food Inspection Agency (CFIA) was established under the Plant Protection Act to prevent the introduction of economically devastating foreign plant diseases into Canada. The PPEQ program allows the Canadian potato industry to obtain disease-free germplasm from other countries to meet international demands for the production of seed, table, or processing potatoes. The diagnostic methods employed in the current PPEQ program are time-consuming and rely on both traditional and modern techniques. Many of these techniques are only specific for single viral targets having weak specificity and sensitivity. Recently, next-generation sequencing (NGS) platforms have been widely accepted as high-throughput, unbiased technologies that have attractive features in the field of plant diagnostics. NGS has opened the door to very sensitive and specific testing with the opportunity to detect multiple pathogens in a single sample. In this study, various RNA extraction methods were evaluated to acquire high quality non-fragmented RNA. Integrity values ranging from 7-9 were obtained using the PureLink Total RNA Mini kit and lysis buffer (Thermo Scientific, Waltham, USA). Using this method for RNA extraction, the application of NGS was explored to determine if plant pathogenic viral genomes could be generated de novo via a bioinformatic pathway when mapped to the whole host genome without a priori knowledge of their presence. Intended potato tuber imports intercepted from Bangladesh showed that viral contigs from multiple mixed infections of Potato aucuba mosaic virus (PAMV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS), Potato virus M (PVM), Potato leaf roll virus (PLRV), and Tomato chlorosis virus (ToCV) could be detected simultaneously with high specificity. Similarly, NGS was successfully applied to determine the causal agents of unknown etiology in tomato (S. lycopersicum) without a priori knowledge of their existence in the sample and confirmed the first report of Southern tomato virus (STV) in Canada. This NGS protocol will aid in the diagnosis of pathogens that were otherwise unable to be tested for within the current PPEQ program as well as identify unknown agents from other samples allowing for the development of new routine diagnostic assays and timely epidemiological and eradication strategies to be performed. iv ACKNOWLEDGEMENTS I would like to acknowledge the contributions of my co-supervisors Dr. Huimin Xu and Dr. Fred Kibenge. Thank-you, Huimin, for accepting me into your research group here at the Charlottetown Laboratory of the Canadian Food Inspection Agency and seeing the potential in me to complete my graduate studies. The time and effort it took for you to provide me with both guidance and directed studies for this effort was well beyond your requirement at the Agency. I thank you for being supportive of my idea to combine professional with academic achievements. I would also like to thank my committee members Dr. Chelsea Martin and Dr. Yingwei Wang for seeing me through this process. I would also like to thank Dr. Doug Campbell and Dr. Amanda Cockshutt of Mount Allison University who gave me an opportunity to work in their lab in 2007; something they did simply out of kindness. Their guidance provided me with many of the necessary skills, technical knowledge, and troubleshooting capabilities necessary for the future. I hope they realize how grateful I am to learn from them. I would not be in the position that I am in now without them. I was also very fortunate for Dr. Xianzhou Nie and Virginia Dickison of the Fredericton Research Centre of Agriculture Agri-Food Canada who provided me with introductory training in NGS technology which was important for my further understanding of the Illumina platform. Dr. Martin Filion and Dr. Adrien Biessy at the University of Moncton provided valuable training on the CLC Genomics Workbench platform that enabled me to create a functional NGS workflow. Erin Miller-Jones of the Canadian Food Inspection Agency provided me with outstanding help retrieving sample information at the drop of a hat. I am very fortunate to also have someone at the Charlottetown Laboratory like Joan Hardy who has the breadth of knowledge that she has in the administrative processes of government which made my experience much easier. I wish to dedicate this work to the citizens of the Canadian Federation. v TABLE OF CONTENTS Thesis/dissertation non-exclusive license……………………………………………………….ii Certification of thesis work……………………………………………………………………..iii Abstract…………………………………………………………………………………………..iv Acknowledgements………………………………………………………………………………v Table of Contents………………………………………………………………………………..vi List of Tables………………………………………………………………………………….....ix List of Figures……………………………………………………………………………………xi List of Abbreviations…………………………………………………………………………..xiii 1.0 GENERAL INTRODUCTION AND LITERATURE REVIEW……………………..2 1.1 Historical Perspective……………………………………………………………...2 1.2 Genome Characteristics of Potato…………………………………………………3 1.2.1 Potato Breeding………………………………………………………….3 1.3 Potato Industry
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