University of Alberta A functional approach to profiling candidate genes in non model Brassicales by Peter James Mankowski A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Systematics and Evolution Biological Sciences ©Peter James Mankowski Spring 2013 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Dedication This thesis is for everyone who struggles to pause and enjoy the view. Abstract Obtaining functional data is an essential component in understanding mechanisms underlying morphological variation. Virus-induced gene silencing (VIGS) is a reverse genetic technique for identifying the function of target loci through viral mediated transcript knockdown. Here we introduce this technique to two plant systems Cleome violacea (Cleomaceae) and Erucaria erucarioides (Brassicaceae) chosen for their morphological traits and phylogenetic position. In both species, a construct with a heterologous sequence and a construct with an endogenous sequence of the visual marker PHYTOENE DESATURASE (PDS) were incorporated into separate viral constructs for downregulation. Downregulation using a heterologous sequence produced a comparable fold change in transcript abundance to the endogenous sequence in both species however C. violacea was more susceptible to this process. Additional targets FRUITFULL (FUL) and a TCP1 homologue were targeted for knockdown within C. violacea to extend the application of this technique. A protocol for VIGS application is now available to both species for optimization and future functional analysis. Acknowledgements I would like to thank my supervisor Jocelyn Hall who has always supported my academic career and with her guidance helped me to achieve my ambitious pursuits. I would also like to thank both of my committee members, Janice Cooke and Mike Deyholos who offered continuous concrete advice during the entirety of this project. I would like to thank Walid El Kayal whose unconditional mentoring and kindness served as a beacon during this journey. I am truly indebted to both Kelsey Bernard and Melanie Patchell whose encouragement abilities are rivaled by no one. It has been an honor to work with them both. Thanks to my family who never fail to provide ongoing support in everything I set out to do. Table of Contents Chapter 1: Introduction to the methodology of virus-induced gene silencing (VIGS) and its profile as a transient RNAi technique ................................ 1 RNAi approach: Virus-induced gene silencing .......................................................................... 2 Overview of VIGS technique ............................................................................................................ 6 Benefits to using VIGS ..................................................................................................................... 13 Limitations to using VIGS............................................................................................................... 14 Using VIGS to target Non Model Systems ................................................................................. 16 Figures .................................................................................................................................................. 22 Literature Cited ................................................................................................................................. 27 Chapter 2 Virus-induced gene silencing as a tool for functional studies in relatives of Arabidopsis thaliana ................................................................................. 37 Introduction ....................................................................................................................................... 38 Methods................................................................................................................................................ 43 Construct design ............................................................................................................................................ 43 Preparation of Agrobacterium tumefaciens ........................................................................................ 47 Plant materials ............................................................................................................................................... 47 Vacuum infiltration ...................................................................................................................................... 47 RNA extraction and cDNA synthesis ..................................................................................................... 50 Expression analysis of VIGS-treated plants........................................................................................ 51 Scanning electron microscopy of C. violacea fruit ........................................................................... 53 Results .................................................................................................................................................. 53 Downregulation of PDS using heterologous and endogenous constructs ............................. 53 Downregulation of C. violacea FRUITFULL using hybrid construct.......................................... 58 Discussion ........................................................................................................................................... 61 Downregulation of PDS in two emerging models using both heterologous and endogenous constructs ............................................................................................................................... 62 Dowregulation of valve margin identity homologue FUL within C. violacea ........................ 64 Silencing of valve margin identity homologue FUL with E. erucarioides ............................... 66 Conclusion ........................................................................................................................................... 68 Figures .................................................................................................................................................. 70 Chapter 3. Functional assessment of the homologue TCP1 and its contribution to monosymmetry in Cleome violacea ............................................... 101 Introduction ....................................................................................................................................... 102 Methods.............................................................................................................................................. 107 Construct design and VIGS ..................................................................................................................... 107 Morphological characterization ........................................................................................................... 109 Results ................................................................................................................................................ 110 Discussion ......................................................................................................................................... 112 Conclusion ......................................................................................................................................... 117 Figures ................................................................................................................................................ 118 Literature Cited ............................................................................................................................... 122 Chapter 4 Conclusion ......................................................................................................... 128 Functional analysis and VIGS contribution to Evo-devo research ..................... 129 Fruit development in heteroarthrocarpic species Erucaria erucarioides ................. 133 Floral development in monosymmetric Cleome violacea ................................................ 135 Conclusion ......................................................................................................................................... 136 Literature Cited ............................................................................................................................... 138 Appendix ................................................................................................................................ 142 List of Tables Table 2.1. Primers used for construct assembly.. .................................................................... 70 Table 2.2. Collection records of VIGS trails for Cleome violacea. ....................................... 72 Table 2.3. Statistical