Analysis of phylogenetic relationships of Hordeum (Poaceae) polyploids Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von Herrn Jonathan Brassac geb. am 05.11.1986 in Lyon, Frankreich Gutachter/in 1. Dr. Frank R. Blattner 2. Prof. Dr. Martin Röser 3. Prof. Dr. Christoph Oberprieler Halle (Saale), verteidigt am 06. Mai 2015 CONTENTS 1. Introduction .................................................................................................................................................... 5 The Genus Hordeum ...................................................................................................................................... 5 Taxonomy ................................................................................................................................................ 5 Polyploidy ................................................................................................................................................ 8 Previous Work: Hordeum and the Development of Phylogenetic Markers ........................................... 10 Objectives of This Study .............................................................................................................................. 13 Methods ....................................................................................................................................................... 14 In Vitro vs In Silico Cloning ................................................................................................................. 14 Phylogenetic Inference Methods ............................................................................................................ 16 2. A Single Locus Analysis: Cloning ............................................................................................................... 23 Introduction .................................................................................................................................................. 23 Materials and Methods ................................................................................................................................. 24 Plant Materials ...................................................................................................................................... 24 Molecular Methods ................................................................................................................................ 26 Data Analyses ........................................................................................................................................ 27 Results .......................................................................................................................................................... 28 Recombinant Clones of TOPO6 and Comparison of Two Polymerases ................................................ 34 Discussion .................................................................................................................................................... 35 Inference of Extinct Diploid Progenitors of Allopolyploids .................................................................. 36 Polyploid Species of the Old World ....................................................................................................... 37 Hordeum roshevitzii, a Key Species in the Evolution of Tetraploid Hordeum Species ......................... 38 The Extinct californicum-like Taxon, a Key Species in the Evolution of American Polyploids ............. 39 Hordeum depressum, an All-American Tetraploid ................................................................................ 40 Hordeum brevisubulatum, a Complex Group ........................................................................................ 41 PCR Recombination Results in Chimerical Sequences.......................................................................... 42 Conclusions ........................................................................................................................................... 43 3. A Multilocus Analysis: Next-Generation-Sequencing ................................................................................. 45 Introduction .................................................................................................................................................. 45 Materials and Methods ................................................................................................................................. 46 Plant Materials ...................................................................................................................................... 46 Molecular Methods ................................................................................................................................ 48 Quality Control and Haplotype Phasing ............................................................................................... 50 Phylogenetic Analyses ........................................................................................................................... 51 Results .......................................................................................................................................................... 55 Sequencing and Sequence Assembly ...................................................................................................... 55 Phylogenetic Relationships .................................................................................................................... 57 Incongruences Among Loci and Between Methods ............................................................................... 58 Ages of Clades ....................................................................................................................................... 60 Inference of Parental Progenitors of Polyploids ................................................................................... 62 Discussion .................................................................................................................................................... 64 Combining PCR Amplification with Second-Generation Sequencing.................................................... 64 Phylogeny of Hordeum .......................................................................................................................... 66 Incongruences Among Loci and Between Methods ............................................................................... 67 Conclusions ............................................................................................................................................ 68 4. Synthesis ...................................................................................................................................................... 69 A Shift of Paradigm in Phylogenetics: Multilocus Analyses ....................................................................... 70 Progenitors of Polyploid Taxa ...................................................................................................................... 70 Origin of Allopolyploids .............................................................................................................................. 71 Phylogenetic Relationships and Evolutionary Time Frame ......................................................................... 72 Abstract .................................................................................................................................................................. 75 Zusammenfassung ................................................................................................................................................. 76 References ............................................................................................................................................................. 77 Acknowledgments ................................................................................................................................................. 95 Abbreviations ......................................................................................................................................................... 96 Figures ................................................................................................................................................................... 98 Tables .................................................................................................................................................................... 98 Supplementary Information ................................................................................................................................... 99 Curriculum Vitae & Publications ........................................................................................................................ 134 1. INTRODUCTION THE GENUS HORDEUM TAXONOMY Hordeum L. belongs to the grass tribe Hordeeae. The name Hordeeae (Martinov, 1820), recently rediscovered, has priority over the widely used term Triticeae (Dumortier, 1824). The genus consists of 33 species and altogether 45 taxa are described (Bothmer et al., 1995; Blattner, 2009). The key character to recognize Hordeum within the tribe is the possession of three single-flowered spikelets at each rachis node of the inflorescence