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4.2. Chromosome Work in Compositae

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4.2. Chromosome Work in Compositae DIPLOMARBEIT Titel der Diplomarbeit “Phylogenetic relationship of the genus Scorzoneroides (Compositae) inferred from plastid and low-copy nuclear markers, and karyological data” Verfasser Reinhold Stockenhuber Angestrebter akademischer Grad Magister der Naturwissenschaften (Mag.rer.nat.) Wien, im Juni 2011 Studienkennzahl laut Studienblatt: A438 Studienrichtung laut Studienblatt: Diplomstudium Botanik Betreuerin: Ao. Univ.-Prof. Dr. Rosabelle Samuel ERKLÄRUNG Ich versichere hiermit, dass ich die vorliegende Arbeit selbstständig und ohne fremde Hilfe verfasst, andere als die angegebenen Quellen und Hilfsmittel nicht benutzt und die entnommenen Stellen als solche gekennzeichnet habe. I confirm to have conceived and written this paper in English all by myself. Quotations from other authors and any ideas borrowed and/or passages paraphrased from other authors are all clearly marked within the text and acknowledged in the bibliographical references. Wien, im Juni 2011 Reinhold Stockenhuber DANKSAGUNG Ao. Univ.-Prof. Dr. Rosabelle Samuel für das Vertrauen, das Sie in mich setzte und mich in ihr Projekt aufnahm, und natürlich für die Unterstützung bei dieser Diplomarbeit in vielen Treffen und Gesprächen. Mag. Michael Barfuß für die ständige, geduldige Hilfe im Labor und vor dem Computer, die vielen hilfreichen Antworten auf meine Fragen, und Gespräche, die mich zu wichtigen Erkenntnissen gebracht haben. O. Univ.-Prof. Dr. Tod Stuessy dafür, dass er mein Interesse an der Evolutionsbiologie weckte, für seine hilfreiche Literatur, die mich während der letzten Jahre begleitete, Dr. Walter Gutermann für die zur Verfügung gestellten Herbarmaterialien aus seinen eigenen Sammlungen. Ing. Elfriede Grasserbauer, Verena Klejna, Mag. Gudrun Kohl, Mag. Stefan Safer, Mag. Barbara Turner, Dr. Eva Temsch und vielen anderen für die Unterstützung, die Hilfe im Labor, die Aufmunterungen und Gespräche. Meiner Freundin, die immer an meiner Seite war, und auch in den schwierigen Phasen zu mir hielt. Und natürlich auch meinen Eltern, die viel Geduld aufwiesen, und stets an mich geglaubt haben. Ihr habt mir diese Chance gegeben, und mein Studium erst ermöglicht. ACKNOWLEDGEMENTS Ao. Univ.-Prof. Dr. Rosabelle Samuel for her trust in me, when she employed me in her project, for her patience with me, and also for her aid, for the many meetings where we were talking about the project and my thesis. Mag. Michael Barfuss for his all-time helpfulness in the lab as well as in front of the computer, the answers that he always gave the many times I was asking questions and for the introduction to the labwork. O. Univ.-Prof. Dr. Tod Stuessy for the interest he awoke in me concerning evolutionary biology or evolution on the whole, and for his helpful literature which accompanied me through the last years. Dr. Walter Gutermann For the help with plant material he provided for the study from his own collections Ing. Elfriede Grasserbauer, Verena Klejna, Mag. Gudrun Kohl, Mag. Stefan Safer, Mag. Barbara Turner, Dr. Eva Temsch and many others, who helped me with my labwork, who supported me in so many ways, and with whom I had nice and helpful talks. My girlfriend, who was on my side whenever I needed her, who always encouraged me to carry on, even in hard times. And of course my parents, who had so much patience in me, always believed in me and made it possible for me to study. TABLE OF CONTENTS KURZFASSUNG .............................................................................................................................. II ABSTRACT ................................................................................................................................... III 1.) INTRODUCTION ......................................................................................................................... 1 1.1. Compositae ......................................................................................................................... 1 1.2. Cichorieae ........................................................................................................................... 6 1.3. Hypochaeridinae ................................................................................................................. 9 1.4. Scorzoneroides ................................................................................................................... 9 1.4.1. Morphology of Scorzoneroides ................................................................................. 10 1.4.2. Distribution & Ecology of Scorzoneroides ............................................................... 12 1.4.3. Recent changes in the classification of Scorzoneroides ............................................ 14 1.5. Molecular Markers ........................................................................................................... 16 1.5.1. Plastid markers .......................................................................................................... 17 1.5.2. Nuclear markers ........................................................................................................ 19 2.) PLASTID MARKERS IN SCORZONEROIDES ................................................................................ 22 2.1. Introduction ...................................................................................................................... 22 2.2. Material & Methods ......................................................................................................... 23 2.2.1. Sampling .................................................................................................................... 23 2.2.2. DNA Extraction ......................................................................................................... 23 2.2.3. Primer selection ......................................................................................................... 23 2.2.4. PCR amplification and sequencing ........................................................................... 27 2.2.5. Phylogenetic analysis ................................................................................................ 29 2.3. Results .............................................................................................................................. 30 2.3.1. Primer Choice ............................................................................................................ 30 2.3.2. rpl16-Intron ............................................................................................................... 31 2.3.3. ndhF-rpl32 intergenic spacer .................................................................................... 32 2.3.4. atpH-atpI intergenic spacer ....................................................................................... 33 2.3.5. Combined analysis .................................................................................................... 33 2.4. Discussion ........................................................................................................................ 34 3.) NUCLEAR MARKERS ............................................................................................................... 44 3.1. Introduction ...................................................................................................................... 44 3.2. Material & Methods ......................................................................................................... 45 3.2.1. Primers and PCR conditions ...................................................................................... 45 3.2.3. Cloning ...................................................................................................................... 53 3.2.4. Cycle Sequencing ...................................................................................................... 55 3.2.5. Phylogenetic analysis ................................................................................................ 55 3.3. Results .............................................................................................................................. 56 3.3.1. GAPDH ..................................................................................................................... 56 3.3.2. A39 locus ................................................................................................................... 57 3.3.3. Combination of low-copy nuclear markers ............................................................... 59 3.3.4. Combination of plastid and low-copy nuclear markers ............................................. 59 3.4. Discussion ........................................................................................................................ 60 4.) CHROMOSOME COUNTS AND GENOME SIZE MEASUREMENTS ................................................ 71 4.1. Introduction ...................................................................................................................... 71 4.2. Chromosome work in Compositae ................................................................................... 73 4.2.1. Chromosome work in Hypochaeridinae .................................................................... 73 4.2.1. Chromosome work in Scorzoneroides ...................................................................... 74 4.2.2. Genome size measurements in Scorzoneroides ........................................................ 74 4.3. Material & Methods ........................................................................................................
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