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Cytogenetic Analysis of Crop Genomes

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Cytogenetic Analysis of Crop Genomes Palacký University Olomouc Faculty of Science Department of Botany And Institute of Experimental Botany Centre of Region Haná for Biotechnological and Agricultural Research Olomouc Miroslava Karafiátová Cytogenetic analysis of crop genomes Ph.D. Thesis Supervisor: Mgr. Jan Bartoš, Ph.D. Olomouc 2014 1 Acknowledgements: First, I would like to thank to the head of laboratory, prof. Ing. Jaroslav Doležel, DrSc., for the great opportunity to work in his famous and successful group. It was my pleasure to be a part of his excellent team for these several years. My cordial thanks also go to my supervisor, Mgr. Jan Bartoš, Ph.D., for his stimulating ideas, kindness and professional guidance. Further, I would like to express my thanks to all my colleagues and friends in Centre of Plant Structural and Functional Genomics for their never-ending encouragements and friendly atmosphere, which make my Ph.D. study unforgettable period of my life. Finally, I would like to thanks to my family for their patience and all their support. 2 Declaration: I hereby declare that I elaborated this Ph.D. thesis independently under the supervision of Mgr. Jan Bartoš, Ph.D. using only information sources referred in the Reference chapter. ………………………. 3 This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic, the European Regional Development Fund (Operational Programme Research and Development for Innovations No. ED0007/01/01), Centre of Region Haná for Biotechnological and Agricultural Research and the grant LO1204 from the National Program of Sustainability I and Internal Grant Agency of Palacký University (grant award No. IGA PrF/2012/001). 4 BIBLIOGRAPHIC IDENTIFICATION Author’s name: Mgr. Miroslava Karafiátová Title: Cytogenetic analysis of crop genomes Type of Thesis: Ph.D. thesis Department: Botany Supervisor: Mgr. Jan Bartoš, Ph.D. The Year of Presentation: 2014 Abstract: Crops represent highly variable plant group accompanying the human society from beginning of time and having an essential impact also on recent human living. These plants contribute to the standard of living with various aspects, nevertheless the crops used as a food and fodder have influenced the human life most significantly. Wheat, maize, rice, soybean, sugarcane and further barley and chickpea represent the fundamental nutrition source for enormous number of people all over the world. Reduced crop production or their total absence results in serious malnutrition, what substantially stimulate the crop plant research. Cytogenetics is a versatile tool for plant genome analysis. It provides the number of fundamental characteristics of each species, which are considered to be the essential background for more extended studies. In the first part of the thesis, potential of cytogenetics in plant genome mapping was emphasised on cultivated barley (Hordeum vulgare L.). Mapping of short, low-copy full length cDNA clones on barley metaphase chromosomes has proved that even DNA fragments of limited length can be reliably localize using fluorescence in situ hybridization (FISH), what provide an alternative approach for physical map construction or verifying the contigs assembly in problematic region like centromere. Further, the second part of the thesis was focused on developing of chromosome painting protocol intended primarily for the plant species with large genome and considerable portion of repetitive sequences. The protocol was applied on barley metaphase chromosomes with the aim to paint barley chromosome 1H. Highly innovative approach employs the procedure of gene-capture and flow cytometry in combination with FISH. Our results represent very important leap forward the study of chromosome organization in interphase nuclei in species with large and complex genetic information. 5 Key words: crop, cytogenetics, FISH, cytogenetic mapping, probe, single-copy region, chromosome painting Number of pages/Apendices: 75/XI Language: English 6 BIBLIOGRAFICKÁ IDENTIFIKACE Jméno: Mgr. Miroslava Karafiátová Název: Cytogenetická analýza genomů hospodářsky významných plodin Typ práce: Disertační práce Obor: Botanika Školitel: Mgr. Jan Bartoš, Ph.D. Rok obhajoby: 2014 Abstrakt: Hospodářsky významné plodiny představují velice rozmanitou skupinu rostlin, které jsou od prvopočátku neodmyslitelně spjaty s utvářením lidské civilizace a které ve značné míře ovlivňují i život dnešního člověka. Tyto plodiny přispívají ke kvalitě života různými aspekty, přičemž největší dopad mají plodiny, které slouží jako zdroj potravy buď pro lidstvo samotné, nebo jsou využívány jako krmivo pro hospodářská zvířata. Mezi plodiny tvořící esenciální složku potravy obyvatel v různých částech světa patří hlavně pšenice, kukuřice, rýže, sója, cukrová třtina a v neposlední řadě i ječmen či cizrna. Jejich nedostatek se odráží v podvýživě obyvatel, což významně podněcuje výzkum v této oblasti. Velmi široké možnosti pro studium rostlinných genomů nabízí cytogenetické techniky. Cytogenetika umožňuje řadu analýz, jež poskytují základní charakteristiku všech druhů a jsou nutným základem pro většinu komplexnějších studií. Potenciál cytogenetiky při mapování rostlinných genomů byl zdůrazněn v první části disertační práce na ječmeni setém (Hordeum vulgare L.). Mapování krátkých a nízkokopiových fl-cDNA klonů na metafázní chromozómy ječmene prokázalo, že i DNA fragmenty s omezenou délkou mohou být spolehlivě lokalizovány pomocí fluorescenční in situ hybridizace (FISH), což nabízí alternativní variantu při sestavování fyzických map a ověřování správnosti uspořádání jednotlivých kontigů především v problematických oblastech jako jsou např. centromery. Další podstatná část práce byla zaměřena na vytvoření protokolu tzv. malování chromozómů (chromosome painting) určeného především pro druhy s komplexními genomy s vyšším obsahem repetitivních sekvencí. Protokol byl testován na metafázních chromozómech ječmene se sondou pro chromozóm 1H. Vysoce inovativní přístup využívá metodiku tzv. vychytávání genů a průtokové cytometrie v kombinaci s FISH. Dosažené výsledky představují velmi důležitý krok ke studiu uspořádání chromozómů v interfázních jádrech. 7 Klíčová slova: hospodářsky významné plodiny, cytogenetika, FISH, cytogenetické mapování, sonda, jednokopiové oblasti, „malování“ chromozómů Počet stránek/ příloh: 75/XI Jazyk: anglický 8 CONTENT 1 INTRODUCTION ........................................................................................................ 11 2 LITERATURE OVERVIEW ..................................................................................... 12 2.1 CROPS AND THEIR IMPORTANCE .............................................................................................. 12 2.1.1 Cereal crops ............................................................................................................................ 12 2.1.2 Forage crops ............................................................................................................................ 13 2.1.3 Grain legumes ......................................................................................................................... 15 2.2 PLANT NUCLEAR GENOME........................................................................................................ 17 2.2.1 Genome size ............................................................................................................................ 17 2.2.2 Genome structure and organization ........................................................................................ 19 2.2.2.1 Chromosomes .................................................................................................................. 19 2.2.2.2 Interphase nucleus ........................................................................................................... 21 2.2.3 Composition of plant nuclear DNA ........................................................................................ 24 2.3 CYTOGENETIC ANALYSIS OF PLANT NUCLEAR GENOMES .......................................................... 27 2.3.1 Banding ................................................................................................................................... 27 2.3.2 Fluorescence in situ hybridization (FISH) .............................................................................. 28 2.3.2.1 Probe labelling procedures .............................................................................................. 31 2.3.2.2 Probes for fluorescence in situ hybridization .................................................................. 32 2.3.2.3 Cytological targets for FISH ............................................................................................ 36 2.3.2.4 FISH modifications and technical developments ............................................................ 40 2.3.2.4.1 Chromosome painting .............................................................................................. 40 2.3.2.4.2 Primed in situ labelling (PRINS).............................................................................. 41 2.3.2.4.3 Fluorescence in situ hybridization in suspension (FISHIS) ..................................... 42 2.3.2.5 Other FISH variants ......................................................................................................... 43 2.3.2.6 FISH-based karyotyping and chromosome identification ............................................... 45 2.4 REFERENCES ............................................................................................................................
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