EDNA-HOST: DETECTION OF GLOBAL PLANT VIROMES USING HIGH THROUGHPUT SEQUENCING By LIZBETH DANIELA PENA-ZUNIGA Bachelor of Science in Biotechnology Escuela Politecnica de las Fuerzas Armadas (ESPE) Sangolqui, Ecuador 2014 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY May 2020 EDNA-HOST: DETECTION OF GLOBAL PLANT VIROMES USING HIGH THROUGHPUT SEQUENCING Dissertation Approved: Francisco Ochoa-Corona, Ph.D. Dissertation Adviser Committee member Akhtar, Ali, Ph.D. Committee member Hassan Melouk, Ph.D. Committee member Andres Espindola, Ph.D. Outside Committee Member Daren Hagen, Ph.D. ii ACKNOWLEDGEMENTS I would like to express sincere thanks to my major adviser Dr. Francisco Ochoa –Corona for his guidance from the beginning of my journey believing and trust that I am capable of developing a career as a scientist. I am thankful for his support and encouragement during hard times in research as well as in personal life. I truly appreciate the helpfulness of my advisory committee for their constructive input and guidance, thanks to: Dr. Akhtar Ali for his support in this research project and his kindness all the time, Dr. Hassan Melouk for his assistance, encouragement and his helpfulness in this study, Dr. Andres Espindola, developer of EDNA MiFi™, he was extremely helpful in every step of EDNA research, and for his willingness to give his time and advise; to Dr. Darren Hagen for his support and advise with bioinformatics and for his encouragement to develop a new set of research skills. I deeply appreciate Dr. Astri Wayadande thoughtful advice through my writing process, her invaluable support inspired me in unique way. I also want to thank the head of the Department of Entomology and Plant Pathology (EPP), Dr. Phil Mulderfor his support. I like to extend my gratitude to Dr. Kitty Cardwell, the director of the Institute of Biosecurity and Microbial Forensics (IBMF) at Oklahoma State University for her assistance and encouragement. I am sincerely thankful to the founding sources that supported my research, Oklahoma Department of Agriculture, Food and Forestry (ODAFF), Combating Rose Rosette Disease USDA project. I also want to thank the students, staff and professors of EPP who were incredible cooperative and friendly during these years. Finally, I would like to express my sincere love and gratitude to my family who are my biggest fans. My parents believed, encouraged and supported me all the way to accomplish each of the dreams in my life. Also, to my sister whose presence in my life gave me support and friendship. I have been extremely blessed to have them as my family and moving along with my dreams helping to make them possible. I could not accomplished this without each of you. iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: LIZBETH DANIELA PENA-ZUNIGA Date of Degree: MAY, 2020 Title of Study: EDNA-HOST: DETECTION OF GLOBAL PLANT VIROMES USING HIGH THROUGHPUT SEQUENCING Major Field: PLANT PATHOLOGY Abstract: The aim of this research was to develop the concept of host-specific virome detection methods. Three model systems were selected roses, cucurbits, and water. EDNA MiFi™ was used to generate pathogen electronic probes (E-probes) for detection of viruses in-silico and in-vitro using field samples for validation. The selected three host model allows a comprehensive detection system for specifically targeting the three pre-determined host viromes (117 plant viruses). This research also presents a validation of the analytical sensitivity determining the in-vitro limit of detection (10pg) quantified for a specific set of E-probes (ArMV-RNA2 60nt). The database of EDNA-Rose MiFi™ includes E-probes (523) specifically designed to detect 22 reported viruses infecting roses worlwide. The EDNA-Cucurbits MiFi™ includes E-probes (412) for 15 reported viruses. Finally, the MiFi™ database EDNA-Water is composed by a selection of highly specific E-probes (1730) for 80 reported viruses within the genera Potexvirus, Tobamovirus and Tombusvirus. Detection of the presence of multiple viruses in a host is challenging. Hence, the relevance of developing and applying a broad detection system to detect viruses out of a virome in a sample. The application of EDNA-Host extends to plant virus detection to all staple crops, virus-free certification programs, breeding, veterinary and life sciences. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................................... III TABLE OF CONTENTS ............................................................................................................. V LIST OF TABLES ....................................................................................................................... IX LIST OF FIGURES ................................................................................................................. XIII CHAPTER I ................................................................................................................................... 1 INTRODUCTION.......................................................................................................................... 1 REFERENCES: ............................................................................................................................. 6 CHAPTER II .................................................................................................................................. 7 REVIEW OF LITERATURE ....................................................................................................... 7 AGRICULTURAL BIOSECURITY ..................................................................................................... 7 PLANT VIRUS ORIGIN AND EVOLUTION ........................................................................................ 8 DISEASE SYMPTOMS AND HOST RANGE ....................................................................................... 9 ECONOMIC IMPACT .................................................................................................................... 10 PERSISTENCE AND DISSEMINATION ............................................................................................ 11 ECOLOGY AND EPIDEMIOLOGY .................................................................................................. 12 MANAGEMENT ........................................................................................................................... 12 HOST-PATHOGEN-VECTOR INTERACTION .................................................................................. 13 VIRUS-VIRUS INTERACTIONS AND MIXED PLANT VIRUS INFECTIONS ........................................ 14 PLANT VIROME ........................................................................................................................... 14 PLANT VIRUS VIROMICS ............................................................................................................. 15 DIAGNOSIS ................................................................................................................................. 16 POLYMERASE CHAIN REACTION ................................................................................................. 16 HIGH-RESOLUTION MELTING (HRM) ........................................................................................ 17 SEQUENCING .............................................................................................................................. 17 HIGH THROUGHPUT SEQUENCING (HTS) IN PLANT DIAGNOSTICS ............................................. 18 TRADITIONAL BIOINFORMATICS APPROACH FOR VIRUS DETECTION ......................................... 19 ELECTRONIC DIAGNOSIS NUCLEIC ACID ANALYSIS (EDNA)- MIFI TM .................................... 19 THE MODEL HOSTS SELECTED FOR THIS STUDY ......................................................................... 20 VIROMES ASSOCIATED WITH THE MODEL HOSTS ....................................................................... 23 REFERENCES .............................................................................................................................. 25 CHAPTER III .............................................................................................................................. 30 v DEVELOPMENT AND VALIDATION OF E-PROBES FOR PREDETERMINE HOST- VIRUS UNIVERSES ................................................................................................................... 30 ABSTRACT .................................................................................................................................. 30 1. INTRODUCTION ....................................................................................................................... 31 2. MATERIALS AND METHODS ................................................................................................... 32 2.1. E-PROBE DESIGN .................................................................................................................. 32 2.2 GENERATION OF SIMULATED METAGENOMES AND QUERY .................................................. 33 2.3 VALIDATION OF EDNA WATER -MIFITM WITH PUBLISHED SRA DATA ............................... 35 2.4. BLIND TEST OF EDNA ROSE-MIFITM .................................................................................