Genes that underlie natural variation in growth rate and flowering time in local accessions of Arabidopsis thaliana

i Lay summary. iii Declaration . iv Acknowledgements . v Table of contents . vi List of figures . xii List of tables . xv Abbreviations . xviii 1.0 INTRODUCTION . 1 1.1 Growth and growth rate . 1 1.1.1 Phenotyping aims . 2 1.1.2 Tools for phenotyping . 2 1.2 Plant growth parameters. 3 1.2.1 Plant height and shoot length in Arabidopsis. 3 1.2.2 Measuring the rosette areas of plants . 4 1.2.3 Single time point rosette area as proxy for growth rate . 5 1.3 Natural variation and evolution. 10 1.4 Quantitative traits and quantitative trait loci . 11 1.4. 1 QTL analysis in plants . 12 1.4. 2 Natural variation in plant growth rate . 13 1.4. 3 Usefulness of RILs in QTLs study . 17 1.5 Flowering . 18 1.5. 1 Genetic control of flowering in Arabidopsis. 19 vii 1.5. 2 FLOWERING LOCUS C (FLC) . 19 1.5. 2.1 Regulation of FLC by FRIGIDA (FRI) . 20 1.5. 2.2 The Autonomous pathway . 21 1.5. 2.3 The vernalization pathway . 22 1.5. 3.4 Histone Deacetylase (HDAC) co-repressor complex . 23 1.6 Natural variation in flowering in cereals crops . 26 1.7 Natural variation in flowering in Brassicaceae . 28 1.8 Natural genetic variation in flowering time in Arabidopsis . 29 1.9 Summary of previous work in Hudson lab . 30 2.0 MATERIALS AND METHODS . 32 2.1 Seeds sterilization and growth conditions of plants . 32 2.2 Adobe Photoshop . 32 2.3 Arabidopsis collection sites in and around Edinburgh. 35 2.4 Measurements of rosette areas of plants in Image Tool. 38 2.5 Flowering time measurements . 38 2.6 Scoring of anthocyanin production . 39 2.7 Herbivory estimates . 39 2.8 Quantitative Trait Loci (QTLs) analysis . 40 2.9 Bulk –Segregant analysis . 40 2.10 Heritability estimates . 40 2.11 Collection of tissues for DNA extraction . 41 2.11.1 Genomic DNA extraction by CTAB (Hexadecyltrimethyl ammonium bromide) method . 41 2.11.2 Genomic DNA extraction by Edwards method . 44 2.11.3 10x TE buffer recipe . 46 viii 2.12 Simple Sequence Length Polymorphism (SSLP) markers . 47 2.12.1 SSLP analysis . 52 2.12.2 PCR reactions . 53 2.13 Agarose Gel Electrophoresis . 56 2.13.1 Materials. 56 2.13.2 Loading and running agarose gels . 58 2.14 Cloning, bacterial transformation and sequencing . 58 2.14. 1 Preparation of KCM competent cells . 62 2.14. 2 DH5α transformation . 62 2.14. 3 Bacterial transformation using di-methyl sulphoxide (DMSO) and polyethylene glycol (PEG) . 63 2.14. 5 PCR reactions with pJET primers . 65 2.14. 6 Gel purification of DNA amplicons . 67 2.14. 7 DNA Sequencing . 68 2.15 Reverse transcription polymerase chain reaction (RT-PCR) for gene expression analysis . 68 2.15. 1 Trizol method of RNA extraction . 68 2.15. 2 cDNA synthesis. 69 2.15. 3 RT buffer, dNTPs and Oligo dT primer mix . 70 2.16 5’-RACE (rapid amplification of cDNA ends) . 74 2.16. 1 Purification of cDNA free of Oligos using Qiaquick gel extraction kit . 79 2.17 Gateway Cloning. 80 2.17. 1 To Create a Gateway Entry Clone . 80 2.17. 2 LR Clonase Reaction . 81 2.17. 3 Transformation . 82 ix 2.17. 4 Agrobacterium strain GV3101: . 84 2.18 Transformation of A. tumefaciens with plasmid DNA (binary vector system) . 84 2.18. 1 Freeze/thaw shock transformation . 84 2.18. 2 Floral Dip of Arabidopsis transformation . 85 3.0 RESULTS AND DISCUSSION . 87 3.1 Growth rate variation, anthocyanin synthesis and herbivory in plants of F3 and F5 populations. 87 3.2 Introduction . 87 3.3 Growth rate variations in control conditions. 90 3.4 Mapping of growth rate genes . 92 3.4. 1 Estimation of plant growth in a greenhouse (controlled conditions) of F3 population. 94 3.4. 2 Estimation of plant growth in outside garden (natural conditions) of F3 population. 94 3.4. 3 Estimation of plant growth in an outside garden (natural conditions) of F5 population.