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Genome Sequence and Variation in the Australian Native Velvet Tobacco Mottle Virus

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Genome Sequence and Variation in the Australian Native Velvet Tobacco Mottle Virus 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 .............................................................................
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