Physical mapping of genes on plant chromosomes Ilia V. Kirov This PhD thesis is dedicated to my wife Nadya, daughter Angelika and son Luka Promoters: Prof. dr. Nadine Van Roy, Ghent University, Ghent, Belgium Dr. ir. Katrijn Van Laere, Institute for Agricultural and Fisheries Research, Melle, Belgium Prof. dr. Ludmila I. Khrustaleva, Russian Timiryazev Agricultural Academy, Moscow, Russia Members of examination committee: Prof. dr. Jan Gettemans, (chairman) Ghent University, Gent, Belgium Prof. dr. ir. Paul Coucke, Ghent University, Gent, Belgium Prof. dr. ir. Danny Geelen, Ghent University, Gent, Belgium Prof. dr. ir. Winnok De Vos, Ghent University, Gent, Belgium Dr. Mohammed Bendahmane, Ecole normale supérieure de Lyon, Lyon, France Dr. Tom Ruttink, Institute for Agricultural and Fisheries Research, Melle, Belgium Ghent University Faculty of Medicine and Health Sciences Center for Medical Genetics Physical mapping of genes on plant chromosomes Ilia Kirov Email address: [email protected] This thesis is submitted to fulfill of the requirements for the degree of doctor in Health Sciences, 2017 Please refer to this work as follows: Kirov I (2017) Physical mapping of genes on plant chromosomes. PhD Thesis, Ghent University, Gent, Belgium The author and the promoters give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. TABLE OF CONTENT Abbreviations ........................................................................................................................................... 8 Summary................................................................................................................................................... 9 Samenvatting.......................................................................................................................................... 10 PART I - INTRODUCTION AND RESEARCH OBJECTIVES........................................................................... 12 1. Physical mapping of plant genomes ................................................................................................... 13 1.1 Relevance ................................................................................................................................................................... 13 1.2 Physical maps versus genetic maps.......................................................................................................................... 14 1.3 Physical mapping using fluorescence in situ hybridization .................................................................................... 15 2. Physical mapping of individual genes by FISH ................................................................................... 18 2.1 Chromosome preparation......................................................................................................................................... 20 2.2 Detection system ....................................................................................................................................................... 21 2.3 Chromosome markers ............................................................................................................................................... 22 3. Tyramide-FISH – a highly sensitive technique for cytogenetic mapping of short dna probes on plant chromosomes .................................................................................................................................... 24 4. Applications of FISH based physical maps.......................................................................................... 27 4.1 Assistance in genome assembly ........................................................................................................................................ 27 4.2 Comparative genomics ...................................................................................................................................................... 28 4.3 Genetic and physical map integration .............................................................................................................................. 30 5. Cytogenetics in Rosa and Allium ........................................................................................................ 31 5.1 Rosa .................................................................................................................................................................................... 31 5.2 Allium .................................................................................................................................................................................. 32 Research objectives ................................................................................................................................ 34 Research outline ..................................................................................................................................... 36 PART II - RESULTS.................................................................................................................................... 38 Chapter 1 Development of a highly efficient chromosome preparation method suitable for species with small and large chromosomes................................................................................................... 40 Paper 1: An easy “SteamDrop” method for high quality plant chromosome preparation................................................ 41 Chapter 2 Evaluation of direct and indirect detection systems for efficient physical .............................. mapping of genes ............................................................................................................................ 41 Paper 2: Anchoring linkage groups of the Rosa genetic map to physical chromosomes with Tyramide-FISH and EST-SNP markers........................................................................................................................................................ 53 Chapter 3 Development of cytogenetic markers for Rosa and Allium chromosome identification...... 63 Paper 3: Towards a FISH-based karyotype of Rosa L.. ......................................................................................................4364 Paper 4: Tandem repeats of Allium fistulosum associated with major chromosomal landmarks .................................... 77 6 Chapter 4 Physical mapping of target genes on chromosomes of Allium and Rosa species ............... 103 Paper 5: High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa ..... 104 Paper 6: The chromosome organization of genes and some types of extragenic DNA in Allium.. .................................. 114 PART III: General discussion and future perspectives .......................................................................... 124 1. Chromosome preparation quality as the key factor in Tyramide-FISH reproducibility ................... 125 2. Increasing signal frequency is still the biggest challenge for Tyramide-FISH................................... 127 3. Efficient probe design and detection system enhance Tyramide-FISH efficiency ........................... 128 4. Increased spatial resolution (SR) of physical mapping by Tyramide-FISH ....................................... 129 5. Guidelines for Tyramide-FISH optimization and application in other plant species........................ 130 6. Limitations of Tyramide-FISH ........................................................................................................... 132 7. Tyramide-FISH in the post-genomic era ........................................................................................... 133 7.1. Physical mapping in Allium ............................................................................................................................................. 134 7.1.1 New cytogenetic markers for Allium chromosomes ............................................................................................... 135 7.1.2 Allium comparative genomics based on single-copy gene physical mapping....................................................... 136 7.1.3 Allium breeding can benefit from physical mapping .............................................................................................. 137 7.2 Rosa wichurana can be a model species for molecular cytogenetics within the Rosa genus .................................... 138 7.3 An integrated approach for genome assembly and its application for R. wichurana genome sequencing .............. 140 References .............................................................................................................................................................................. 148 CURRICULUM VITAE ............................................................................................................................. 167 ACKNOWLEDGEMENTS ........................................................................................................................ 173 7 ABBREVIATIONS BSA Bovine Serum Albumin CY3 Cyanine-3 EST Expressed Sequence Tag FISH Fluorescence In Situ Hybridization FITC Fluoresceine Isothiocyanate HRM High Resolution Melting HRP Horse Radish Peroxidase NGS Next generation sequencing OOMT Orcinol O-MethylTransferase PAL Phenylalanine Ammonia Lyase P5CS Pyrroline-5-Carboxylate