Genome sequence and variation in the Australian native Velvet tobacco mottle virus by Kieren Arthur MSc (Hons), The University of Auckland, NZ A thesis submitted for the degree of Doctor of Philosophy at The University of Adelaide, School of Agriculture, Food and Wine February, 2011 i - Table of Contents - Thesis Summary ....................................................................................................................... vii Statement .................................................................................................................................... ix Acknowledgements .................................................................................................................... x Dedication .................................................................................................................................... xi List of Abbreviations ................................................................................................................. xii Chapter 1: General Introduction ................................................................................................. 1 1.1 Velvet tobacco mottle virus ...................................................................................... 1 1.1.1 VTMoV taxonomy ........................................................................................ 1 1.1.2 The genus Sobemovirus ............................................................................. 2 1.2 Unique features of VTMoV ........................................................................................ 5 1.2.1 VTMoV virusoid ........................................................................................... 6 1.2.2 VTMoV Transmission .................................................................................. 6 1.2.3 VTMoV RNA................................................................................................ 7 1.3 Sequence variation in VTMoV .................................................................................. 8 1.4 Genetic bottlenecks during vector transmission ................................................... 8 1.5 Scope of this thesis .................................................................................................. 9 Chapter 2: General Materials & Methods ................................................................................. 10 2.2 Materials................................................................................................................... 10 2.2.1 Virus .......................................................................................................... 10 2.2.2 Plants ........................................................................................................ 10 2.2.3 Solutions, buffers, media ........................................................................... 10 2.2.4 Primers ...................................................................................................... 10 2.3 Methods ................................................................................................................... 10 2.3.1 Inoculation of plants .................................................................................. 10 2.3.2 Harvest and storage of leaf ....................................................................... 11 2.3.3 Partial purification of VTMoV ..................................................................... 11 2.3.4 Serological testing - DIBA ......................................................................... 11 2.3.5 Total nucleic acid extraction ...................................................................... 12 2.3.6 Virus RNA extraction ................................................................................. 12 ii 2.3.7 Polyacrylamide Gel Electrophoresis .......................................................... 13 2.3.8 cDNA synthesis ......................................................................................... 13 2.3.9 PCR Amplification ..................................................................................... 14 2.3.10 Agarose gel electrophoresis .................................................................... 14 2.3.11 Purification of PCR products ................................................................... 14 2.3.12 Cloning .................................................................................................... 14 2.3.13 Sequence analysis .................................................................................. 16 Chapter 3: Sequencing the VTMoV genome ............................................................................ 17 3.1 Introduction ............................................................................................................. 17 3.2 Materials and Methods............................................................................................ 17 3.2.1 Sequence fragment collection ................................................................... 17 3.2.2 Sequence analysis .................................................................................... 18 3.3 Results ..................................................................................................................... 19 3.3.1 VTMoV genome ........................................................................................ 19 3.3.2 Virusoid sequence ..................................................................................... 22 3.3.3 ORF analysis ............................................................................................. 23 3.4 Discussion ............................................................................................................... 27 Chapter 4: Annotation of the VTMoV genome ......................................................................... 29 4.1. Introduction ............................................................................................................ 29 4.2. Materials and methods .......................................................................................... 29 4.3. Results .................................................................................................................... 30 4.3.1 Nucleotide sequence annotation ............................................................... 30 4.3.2 Annotation of putative translation products ............................................... 34 4.3.3. Tertiary structure and function predictions of VTMoV proteins ................. 36 4.3.4 Phylogenetic analysis ................................................................................ 38 4.4. Discussion .............................................................................................................. 41 4.4.1 Annotation of nucleotide sequence ........................................................... 41 4.4.2 VTMoV proteins ........................................................................................ 42 4.4.3 Phylogenetic analysis ................................................................................ 44 Chapter 5: VTMoV isolate R17 .................................................................................................. 45 5.1 Introduction ............................................................................................................. 45 5.2 Materials and Methods............................................................................................ 45 5.2.1 Sequence fragment collection ................................................................... 45 5.2.2 Sequence analysis .................................................................................... 46 iii 5.3 Results ..................................................................................................................... 46 5.3.1 Nucleotide sequence ................................................................................. 46 5.3.2 Amino acid sequence ................................................................................ 48 5.3.3. Sequence similarities between isolates of VTMoV ................................... 50 5.3.4 Sequence differences between isolates of VTMoV ................................... 51 5.4 Discussion ............................................................................................................... 52 Chapter 6: Sequence variation in the RdRp gene of various isolates of VTMoV .................. 54 6.1 Introduction ............................................................................................................. 54 6.2 Materials and Methods............................................................................................ 54 6.2.1 Collection of sequences from isolates ....................................................... 54 6.2.2 Analysis of RdRp sequence ...................................................................... 56 6.2.3 Estimation of sequence error rate ............................................................. 56 6.2.4 Detection of Recombination ...................................................................... 57 6.2.5 Assessing evolutionary mechanisms ........................................................ 58 6.2.6 Determining relationships between VTMoV isolates ................................. 58 6.3 Results ..................................................................................................................... 59 6.3.1 Estimation of error rate .............................................................................