Analysis of the genetic diversity of Grapevine rupestris stem pitting-associated virus in Ontarian vineyards and construction of a full-length infectious clone by Julia C. Hooker A thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology ©Julia Hooker, April, 2017 ABSTRACT Analysis of the genetic diversity of Grapevine rupestris stem pitting-associated virus in Ontarian vineyards and construction of a full-length infectious clone Julia Catherine Hooker Advisor: University of Guelph, 2017 Dr. Baozhong Meng Advisory Committee: Dr. Peter Krell Dr. Annette Nassuth Grapevine rupestris stem pitting-associated virus (GRSPaV; Betaflexiviridae, Foveavirus) has been associated with a number of diseases including Syrah decline. Previous findings show GRSPaV sequence variants cluster into three or more main phylogroups, regardless of geographical location. Here, the genetic diversity of GRSPaV isolates from Ontarian vineyards was analyzed using broad-spectrum primers targeting the viral polymerase coding sequence. It was hypothesized that GRSPaV variants in Ontario are diverse and GRSPaV-SY variants are involved in Syrah decline symptoms. In total, 169 cDNA clones from 21 Vitis sources were used for phylogenetic analysis. Similarly to previous reports, four major lineages were observed; GRSPaV-PN, -SG1, -SY, and –GG. Variants of the GRSPaV–SY lineage were confirmed in all 14 sources tested with SY-specific primers. Syrah cultivars expressing red canopy decline symptoms had more clones clustering with the GRSPaV-SY lineage than those without observable decline symptoms. GRSPaV clones from 8 hybrid sources mostly clustered with GRSPaV-SY, followed distantly by GRSPaV-BS. A full-length infectious cDNA clone of a GRSPaV-SY-related variant is being constructed to further investigate this relationship. ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Baozhong Meng, for providing me with the opportunity to delve into the world of grapevine virology and for helping me pursue my passions in molecular virology. I would like to thank my committee members, Dr. Peter Krell and Dr. Annette Nassuth, for their knowledge and guidance throughout my research. I would also like to thank NSERC for funding through the Discovery grant and the NSERC Engage program. A big thank you to all my lab members, past and present. Thank you for years of laughter, dancing, and most importantly for going through the experience of this learning process with me. A special thank you to Dr. Huogen Xiao for all his time, patience, and lab lessons, in addition to assistance in collecting grapevine source material and RNA extractions. Thank you to Sunny Li for all the years of guidance and encouragement, and for help with troubleshooting for the full-length clone and sequencing work. Thank you to Mehdi Shabanian for his help in source material collection and providing cDNA from hybrid sources. A very special thank you to Deb Flett and Paula Russell for their guidance through each TA-ship and thesis writing. I truly do not think I could have made it without you two! Thank you to Erin and Diana at Student Counselling Services for their personal help. I would like to say a special thank you to my loving parents, siblings, friends, and cats for their continued love, encouragement, and support. A special shout out to my partner Nick for being my number 1 fan and for being such an important emotional support through all my research woes and breakthroughs iii iv TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................................ xi LIST OF VIRUS ABBREVIATIONS .......................................................................................... xii CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1 Grapevine history: Cultivation and exchange of viral disease .............................................. 1 1.2 Grapevine viruses .................................................................................................................. 2 1.2.1 Fanleaf degeneration ....................................................................................................... 3 1.2.2 Grapevine leafroll disease complex ................................................................................ 4 1.3 GRSPaV implications in disease ........................................................................................... 6 1.4 GRSPaV taxonomy, genome and virion structure .............................................................. 12 1.5 Replication and life cycle .................................................................................................... 17 1.6 GRSPaV genetic diversity and phylogeny .......................................................................... 21 1.7 GRSPaV phylogroup disease associations .......................................................................... 30 1.8 Full-length infectious clones ............................................................................................... 31 1.9 FLCs in literature ................................................................................................................ 34 1.10 Rationale............................................................................................................................ 37 1.11 Hypotheses ........................................................................................................................ 38 1.12 Objectives .......................................................................................................................... 38 CHAPTER 2: MATERIALS AND METHODS .......................................................................... 40 2.1 GRSPaV Diversity .............................................................................................................. 40 2.1.1 Plant Total RNA extraction .......................................................................................... 40 2.1.2 RT-PCR of RdRp fragments for sequencing ................................................................ 40 2.1.3 Cloning of RdRp PCR products into pGEM-T-Easy ................................................... 41 2.1.4 Sanger sequencing of plasmid DNA ............................................................................ 42 2.1.5 Phylogenetic analysis of sequenced fragments ............................................................ 43 2.1.6 CP diversity .................................................................................................................. 43 2.2 Full-Length Clone ............................................................................................................... 45 2.2.1 Primers .......................................................................................................................... 45 2.2.2 PCR Amplification and Cloning of CaMV 35S Promoter ........................................... 45 v 2.2.3 RNA Extraction and RT-PCR of FLC Fragments ........................................................ 49 2.2.4 Cloning FLC fragments into pGEM-T-Easy ................................................................ 50 2.2.5 Overlap PCR of F2 and F3 ........................................................................................... 51 2.2.6 Sanger sequencing of FLC fragments in pGEM-T-Easy.............................................. 53 2.2.7 Cloning F4 and F3 into pBS35S ................................................................................... 56 CHAPTER 3: RESULTS .............................................................................................................. 57 3.1 GRSPaV Diversity .............................................................................................................. 57 3.1.1 Genetic diversity of GRSPaV from Vitis varieties ....................................................... 57 3.1.2 RSP35/RSP36 amino acid sequence alignment............................................................ 64 3.1.3 RSP35-SY/RSP36 to specifically target GRSPaV-SY ................................................. 66 3.1.4 Syrah cultivars with and without red canopy ............................................................... 68 3.1.6 CP Diversity sequencing outcomes .............................................................................. 74 3.2 Full-Length Clone ............................................................................................................... 75 3.2.1 Sequence analysis of CaMV 35S promoter and HDVnos terminator sequences ......... 75 3.2.2 Sequence analysis of pGEM-F2a and pGEM-F2b ....................................................... 76 3.2.3 Sequence analysis of pGEM-RSP13-36, pGEM-F3a and pGEM-F3b ......................... 77 3.2.4 Sequence analysis of pGEM-F4 ................................................................................... 79 3.2.5 Attempt to clone F4 into pBS-35S failed ..................................................................... 79 3.2.6 Attempt to clone F3 into pBS-35S failed ....................................................................