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Physical Mapping and Evolution of Banana Genome (Musa Spp.)

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Physical Mapping and Evolution of Banana Genome (Musa Spp.) Palacký University Olomouc Faculty of Science Department of Botany and Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany Olomouc Jana Čížková Physical Mapping and Evolution of Banana Genome (Musa spp.) Ph.D. Thesis Supervisor: prof. Ing. Jaroslav Doležel, DrSc. Olomouc 2013 Acknowledgements: I would like to thank to my supervisor prof. Ing. Jaroslav Doležel, DrSc. for the opportunity to be a member of the Laboratory of Molecular Cytogenetics and Cytometry and the Centre of Plant Structural and Functional Genomics. I am very grateful for his guidance, good advice and kindness. I would like to express my thanks to my second supervisor Mgr. Eva Hřibová, Ph.D. for her continuous support and friendship. Her help and great patience at all times have been invaluable. My sincere thanks also go to my colleagues and friends in the Laboratory of Molecular Cytogenetics and Cytometry and the Centre of Plant Structural and Functional Genomics for making my Ph.D. studies an unforgettable experience. Lastly, I would like to thank to my family for all their love and encouragement. 2 Declaration: I hereby declare that I elaborated this Ph.D. thesis independently under the supervision of prof. Ing. Jaroslav Doležel, DrSc. and Mgr. Eva Hřibová, Ph.D., using only information sources refered in the Literature chapter. .................................................. 3 This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic and the European Regional Development Fund (Operational Programme Research and Development for Innovations No. ED0007/01/01, Ministry of Education, Youth and Sports of the Czech Republic (grant award No. LG12021), Bioversity International, Montpellier, France (LoA CfL 2010/58, LoA CfL 2011/51, LoA CfL 2012/43) and Internal Grant Agency of Palacký University (grant award No. IGA PrF/2012/001). 4 BIBLIOGRAPHIC IDENTIFICATION Author’s name: Jana Čížková Title: Physical Mapping and Evolution of Banana Genome (Musa spp.) Type of Thesis: Ph.D. thesis Department: Botany Supervisor: prof. Ing. Jaroslav Doležel, DrSc. Second Supervisor: Mgr. Eva Hřibová, Ph.D. The Year of Presentation: 2013 Abstract: Bananas (Musa spp., family Musaceae) are one of the top export commodities and an essential nutrition source for millions of people, especially in developing countries of tropical and subtropical regions. Despite the unquestionable importance of this crop, there is a lack of knowledge about the genetic diversity, phylogenetic relationships and the structure, organization and evolution of banana genome. This study employed several molecular approaches with the aim to contribute to the characterization of the genetic diversity and evolution of species belonging to the family Musaceae. Cytogenetic mapping of specific sequences on mitotic chromosomes of bananas was used to shed more light on the structure and organization of banana genome. In the first part of this thesis, analysis of the ITS region of nrDNA was used to study the phylogenetic relationships within the family Musaceae. The study provides a plausible picture of the evolution of Musaceae species which can be further utilized in improving the classification within the genus. The second part of the work was aimed at molecular analysis and cytogenetic mapping of two main banana DNA satellites. Their genomic organization and molecular diversity was estimated in a set of nineteen Musa accessions. A high potential of these satellites as cytogenetic markers was shown. Their use as probes for FISH significantly increases the number of chromosomes which can be identified in Musa. In the third part of this thesis, genetic diversity of a set of wild Musa species was analysed. The study provides a novel data on nuclear genome size and genomic distribution of rRNA genes in banana and increases the knowledge about the genome structure of wild Musa species. For molecular characterization of studied accessions, SSR genotyping 5 and analysis of the ITS region were used confirming their usefulness for assessing the genetic diversity and evolutionary relationships among banana species. Keywords: Musa, banana, genome, physical cytogenetic mapping, evolution Number of Pages/Apendices: 191/0 Language: English 6 BIBLIOGRAFICKÁ IDENTIFIKACE Jméno: Jana Čížková Téma: Fyzické mapování a evoluce genomu banánovníku (Musa spp.) Typ práce: Disertační práce Obor: Botanika Školitel: prof. Ing. Jaroslav Doležel, DrSc. Školitel specialista: Mgr. Eva Hřibová, Ph.D. Rok obhajoby: 2013 Abtrakt: Banánovník (Musa spp, čeleď Musaceae) je jednou z nejdůležitějších exportních plodin a nezastupitelný zdroj obživy pro miliony lidí žijících v rozvojových zemích tropických a subtropických oblastí. Navzdory nezpochybnitelné důležitosti této plodiny je zde nedostatek informací o genetické diverzitě, fylogenetických vztazích v rámci čeledi a struktuře, organizaci a evoluci genomu banánovníku. Tato disertační práce si kladla za cíl přispět k charakterizaci genetické diverzity a evoluce druhů čeledi Musaceae za pomoci molekulárních metod. Struktura a organizace genomu banánovníku byla studována pomocí cytogenetického mapování specifických sekvencí na mitotické chromozómy. V první části práce byla pro studium fylogenetických vztahů v rámci čeledi Musaceae využita analýza ITS oblasti ribozomální DNA. Tato studie poskytla přesvědčivý pohled na evoluci druhů čeledi Musaceae, který může být dále využitelný pro zpřesnění klasifikace jednotlivých druhů. Druhá část této práce byla zaměřena na molekulární analýzu a cytogenetické mapování dvou hlavních DNA satelitů vyskytujících se v genomu banánovníku. V souboru devatenácti zástupců rodu Musa byla určena genomická organizace a molekulární diverzita těchto DNA satelitů. Použití těchto satelitních sekvencí jako sond pro FISH výrazně zvýšilo počet chromozómů, které mohou být genomu banánovníku identifikovány, což dokazuje jejich vysoký potenciál jakožto cytogenetických markerů. Třetí část této disertační práce se zabývala charakterizací genetické diverzity planě rostoucích druhů rodu Musa. Tato studie přináší nové poznatky o velikosti jaderného genomu a distribuci rRNA genů v genomu banánovníku. Zvyšuje tak znalosti o struktuře genomu planě rostoucích druhů rodu Musa. Pro molekulární charakterizaci studovaných položek bylo použito SSR genotypování a analýza ITS oblasti. Tento přístup se ukázal jako 7 užitečný a vhodný pro stanovení genetické diverzity a evolučních vztahů mezi jednotlivými druhy banánovníku. Klíčová slova: Musa, banánovník, genom, fyzické cytogenetické mapování, evoluce Počet stran/příloh: 191/0 Jazyk: anglický 8 CONTENT 1 INTRODUCTION ............................................................................................... 11 2 LITERATURE REVIEW ................................................................................... 12 2.1 CHARACTERISTICS OF BANANAS ......................................................... 12 2.1.1 Morphological characteristics ......................................................................... 12 2.1.2 Taxonomic classification and distribution ...................................................... 14 2.1.3 Socio-economic importance ........................................................................... 18 2.1.4 Diseases and breeding of resistant cultivars ................................................... 20 2.1.5 Banana germplasm .......................................................................................... 21 2.2 PLANT NUCLEAR GENOME ..................................................................... 22 2.2.1 Nuclear genome size ....................................................................................... 22 2.2.1.1 Methods for nuclear genome size estimation ........................................................ 22 2.2.1.2 Nuclear genome size of banana ............................................................................. 24 2.2.2 Structure and organization of plant genomes .................................................. 26 2.2.2.1 Composition of plant nuclear DNA ....................................................................... 27 2.2.2.1.1 Repetitive DNA sequences ........................................................................ 27 2.2.2.1.1.1 Tandemly repeated DNA ................................................................... 28 2.2.2.1.1.2 Dispersed DNA sequences ................................................................. 29 2.2.2.2 Structure and organization of banana genome ...................................................... 32 2.2.2.2.1 Composition of nuclear DNA of banana .................................................... 33 2.3 PHYSICAL MAPPING OF PLANT GENOMES .......................................... 35 2.3.1 Cytogenetic mapping of plant genomes .......................................................... 36 2.3.1.1 Fluorescence in situ hybridization ......................................................................... 37 2.3.1.2 Cytogenetic mapping of banana genome .............................................................. 39 2.4 PLANT MOLECULAR PHYLOGENETICS ................................................ 42 2.4.1 Molecular DNA markers ................................................................................ 42 2.4.1.1 Molecular DNA markers based on hybridization .................................................. 42 2.4.1.2 Molecular DNA markers based on PCR ............................................................... 43 2.4.1.3 Molecular
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