INVESTIGATION OF THE GENOMICS OF GENDER REGULATION IN POPULUS TRICHOCARPA by Nyssa A. Temmel B.Sc. (Hons) University of Victoria, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2011 © Nyssa A. Temmel, 2011 Abstract This thesis reports the findings of four projects conducted to study the genomics of gender regulation in Populus trichocarpa. Sex-linked markers previously discovered in Salix vimilanis were tested to determine if they were also sex-linked in other Salix species and P. trichocarpa. It was found that the DNA sequence of the SCAR 354 marker, and its position at the 5’ end of a gene encoding an Ssu72-like protein, was conserved with some SFP variability in species of Salix and P. trichocarpa. While this marker may be useful for phylogenetic or population studies in Salix, this marker was not sex-linked in the species investigated in this study. An investigation of genes located on the telomeric end of chromosome 19, the putative sex chromosome in P. trichocarpa, was conducted to look for gender-biased SNPs that would indicate recombination suppression in the region on a sex locus. A large variability in the number of SNPs was observed in the gene sequences studied, but no SNPs that segregated with gender were discovered so a genetic marker that could be used to sex P. trichocarpa individuals of unknown gender could not be developed. Using a microarray approach, gender-biased gene-expression was studied in leaf tissue of P. trichocarpa. While some gender-biased gene-expression was observed in vegetative tissues the differences observed were statistically insignificant due to biological variation in the samples tested, the small sample size used in this study, and changes in the genome annotation between version 1.1 and 2.0 of the poplar genome. This study could not verify the microarray results using rtPCR in a larger sample of male and female leaf tissue. MADS-box genes involved in floral development were identified as having gender- biased gene-expression using a microarray approach. Thirteen putative MADS-box genes ii that showed gender-biased expression in male and female inflorescences were discovered. Novel expression patterns for nine floral MADS-box genes were identified with this microarray data, and the expression patterns of three of these genes were investigated in further detail using reverse-transcription PCR. iii Preface A version of chapter two has been published. Temmel, Nyssa A., Rai, Hardeep S., and Cronk, Quentin C.B. (2007) Sequence characterization of the putative sex-linked Ssu72- like locus in willow and its homologue in poplar. Canadian Journal of Botany 85(11):1092- 1097 I wrote the majority of the manuscript and was responsible for all the laboratory work, while Dr. Rai contributed the DNA sequence editing and alignment, and Dr. Cronk assisted with the writing and the research was conducted in his laboratory. In chapter three and four, Dr. Rick White, the Managing Director of the Statistical Consulting and Research Laboratory at the University of British Columbia, did the statistical analysis of the microarray data. Collection of the P. trichocarpa leaf and floral buds from trees on the UBC Vancouver campus that were used for RNA, DNA and cDNA preparation was assisted by Collin Varner, the Horticulturist/Arborculturist with UBC Plant Operations and Gregg Doughty, the UBC Plant Operations arborist. iv Table of Contents Abstract .............................................................................................................................. ii Preface ................................................................................................................................iv Table of Contents ................................................................................................................v List of Tables................................................................................................................... xiii List of Figures...................................................................................................................xiv List of Abbreviations........................................................................................................xvi Acknowledgements..........................................................................................................xvii Dedication.........................................................................................................................xix Chapter 1: Introduction.....................................................................................................1 1.1 Sexual systems in plants ...........................................................................................1 1.2 Evolution of dioecy ..................................................................................................3 1.2.1 Gynodioecious pathway .....................................................................................3 1.2.2 Heterostyly pathway ..........................................................................................4 1.2.3 Monoecious pathway .........................................................................................5 1.2.4 Evolution of dioecy in Populus ..........................................................................5 1.3 Sex chromosomes in plants.......................................................................................6 1.3.1 Identification and description of sex chromosomes ............................................7 1.3.2 A brief history of the discovery of sex chromosomes .........................................7 1.3.3 Discovery of sex chromosomes in plants............................................................8 1.3.4 Evolution of sex chromosomes...........................................................................9 1.3.5 Proposed mechanism for the evolution of sex chromosomes ............................10 v 1.3.6 Degeneration of the Y chromosome .................................................................12 1.3.7 The absence of sex chromosomes in Populus ...................................................14 1.4 The genetics of gender............................................................................................14 1.4.1 Gender-determining genes ...............................................................................15 1.4.2 Gender-differentiation and development in plants ............................................16 1.4.3 Gender-differentiation in Zea mays ..................................................................17 1.4.4 Gender-differentiation in Cucumis sativus and C. melo ....................................18 1.4.5 The role of MADS box genes in gender differentiation ....................................21 1.4.6 Gender-associated genes ..................................................................................23 1.4.7 Differences in genetic control of gender-determination between monoecy and dioecy …………………………………………………………………………………. 24 1.5 Populus trichocarpa as a model organism for studying gender-determination in plants...............................................................................................................................25 1.5.1 Life history of Populus trichocarpa .................................................................25 1.5.2 Characteristics of Populus trichocarpa as a model species ...............................26 1.5.3 Genomic markers linked to sex-differentiation in Populus and Salix ................27 1.5.4 Gender-determination and chromosome 19 in Populus.....................................28 1.6 Research objectives ................................................................................................30 Chapter 2: Characterization of sex-linked genetic markers developed in Salix viminalis in other Salix species and Populus trichocarpa.................................................................31 2.1 Introduction............................................................................................................31 2.1.1 Sex-linked genetic markers in Salix..................................................................32 2.2 Objectives...............................................................................................................33 vi 2.3 Methods..................................................................................................................34 2.3.1 Collection and preparation of plant materials ...................................................34 2.3.2 PCR conditions and sequencing of samples......................................................35 2.3.3 Sequencing alignment for the SCAR sequences amplified in Salix...................36 2.3.4 Novel primer design based on SCAR sequence and associated coding gene regions in P. trichocarpa .............................................................................................36 2.3.5 Cloning, sequencing and phylogenetic analysis of S. arctica and S. reticulata samples .......................................................................................................................37 2.4 Results....................................................................................................................38 2.4.1 Amplification and characterization of the SCAR 354 marker ...........................38 2.4.2 Gene-anchored amplification