The Evolutionary Dynamics of Genes and Genomes: Copy Number Variation of the Chalcone Synthase Gene in the Context of Brassicaceae Evolution Dissertation submitted to the Combined Faculties for Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Liza Paola Ding born in Mosbach, Baden-Württemberg, Germany Oral examination: 22.12.2014 Referees: Prof. Dr. Marcus A. Koch Prof. Dr. Claudia Erbar Table of contents INTRODUCTION ............................................................................................................. 18 1 THE MUSTARD FAMILY ....................................................................................... 19 2 THE TRIBAL SYSTEM OF THE BRASSICACEAE ........................................... 22 3 CHALCONE SYNTHASE ........................................................................................ 23 PART 1: TROUBLE WITH THE OUTGROUP............................................................ 27 4 MATERIAL AND METHODS ................................................................................. 28 4.1 Experimental set-up ......................................................................................................................... 28 4.1.1 Plant material and data composition .............................................................................................. 28 4.1.2 DNA extraction and PCR amplification ......................................................................................... 29 4.1.3 DNA cloning and sequencing .......................................................................................................... 31 4.1.4 Internal validation with ITS ............................................................................................................ 32 4.2 Bioinformatical data analysis .......................................................................................................... 32 4.2.1 Sequence editing and alignment ..................................................................................................... 32 4.3 Comparative phylogenetic reconstructions and analyses ............................................................. 33 4.3.1 Tests applied on data set prior to phylogenetic analyses .............................................................. 33 4.3.2 Phylogenetic analysis ....................................................................................................................... 34 4.3.2.1 Divergence time estimates ............................................................................................................... 35 4.4 Sequence analysis ............................................................................................................................. 37 4.4.1 Alignment analysis of the complete data ........................................................................................ 37 4.4.2 Identifying gene regions ................................................................................................................... 37 4.4.2.1 Trinucleotide frequency (k-mers) ................................................................................................... 38 5 RESULTS .................................................................................................................... 38 5.1 Comparative phylogenetic reconstructions and analyses ............................................................. 38 5.1.1 Tests applied on data set prior to phylogenetic analysis ............................................................... 38 5.1.2 Comparative phylogenetic reconstructions.................................................................................... 39 5.1.3 Divergence time estimates ............................................................................................................... 43 5.2 Sequence analysis ............................................................................................................................. 45 5.2.1 Alignment analysis of complete data .............................................................................................. 45 5.2.2 Alignment analysis of complete data set with focus on tribes ...................................................... 47 5.2.3 Alignment analysis of complete data set with focus on sequences ............................................... 49 5.2.4 Identifying gene regions ................................................................................................................... 52 5.2.4.1 Promoter ........................................................................................................................................... 52 5.2.4.2 Intron ................................................................................................................................................ 54 5.2.4.3 Exon 1 ................................................................................................................................................ 55 5.2.4.4 Exon 2 ................................................................................................................................................ 55 5.2.5 Conserved residues of the complete gene ....................................................................................... 56 5.2.6 Trinucleotide frequency (k-mers) ................................................................................................... 58 6 DISCUSSION ............................................................................................................. 60 PART 2: TROUBLE WITH THE TRIBAL ARRANGEMENT .................................. 64 7 MATERIAL AND METHODS ................................................................................. 65 7.1 Comparative phylogenetic reconstructions and analyses ............................................................. 65 7.1.1 Test for recombination .................................................................................................................... 66 7.1.2 Divergence time estimates ............................................................................................................... 66 7.1.3 Lineage through time plots (LTT) .................................................................................................. 66 7.2 Sequence analysis ............................................................................................................................. 67 7.2.1 Alignment analysis of the complete data ........................................................................................ 67 7.2.2 Identifying gene regions ................................................................................................................... 67 7.2.3 DNA motif search among gene ........................................................................................................ 67 7.2.4 Transition/transversion rate bias .................................................................................................... 68 7.3 Compositional heterogeneity among DNA ..................................................................................... 68 7.3.1 GC content ........................................................................................................................................ 68 7.3.2 Base composition .............................................................................................................................. 69 7.3.3 Trinucleotide frequency .................................................................................................................. 69 7.4 Adaptive evolution ........................................................................................................................... 69 7.4.1 Origin of purifying selection ........................................................................................................... 70 7.4.2 Synonymous substitution rate ......................................................................................................... 71 7.4.3 Ancestral sequence reconstruction ................................................................................................. 71 8 RESULTS .................................................................................................................... 71 8.1 Comparative phylogenetic reconstructions and analyses ............................................................. 71 8.1.1 Test for recombination .................................................................................................................... 74 8.1.2 Divergence time estimates ............................................................................................................... 75 8.1.3 Lineage through time plots .............................................................................................................. 76 8.2 Identifying gene regions ................................................................................................................... 77 8.2.1 Promoter ........................................................................................................................................... 77 8.2.2 Intron ................................................................................................................................................ 78 8.2.3 Exon 1 ...............................................................................................................................................