Genetic Basis and Adaptive Significance of Repeated Scent Loss in Selfing Capsella Species Friederike Jantzen Dissertation zur Erlangung des akademischen Grades „doctor rerum naturalium“ (Dr. rer. nat.) in der Wissenschaftsdisziplin Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät Institut für Biochemie und Biologie der Universität Potsdam Potsdam, April 2019 Hauptbetreuer: Prof. Dr. Michael Lenhard Gutachter: Prof. Dr. Alexander Vainstein Prof. Dr. Florian P. Schiestl Published online at the Institutional Repository of the University of Potsdam: https://doi.org/10.25932/publishup-43525 https://nbn-resolving.org/urn:nbn:de:kobv:517-opus4-435253 Contents Summary ....................................................................................................................................................... 1 Zusammenfassung ........................................................................................................................................ 3 List of figures ................................................................................................................................................. 5 List of tables .................................................................................................................................................. 6 Abbreviations ................................................................................................................................................ 7 1. Introduction .............................................................................................................................................. 9 1. 1 Evolution of the Selfing Syndrome ..................................................................................................... 9 1. 2 The role of floral scent (loss) ............................................................................................................ 16 1. 3 Evolution of flower size .................................................................................................................... 20 1. 4 Evolutionary history of Capsella ...................................................................................................... 22 1. 5 Aims of this work .............................................................................................................................. 26 2. Manuscript: Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella ................................................................................................................... 27 2. 1 Summary .......................................................................................................................................... 29 2. 2 Introduction ..................................................................................................................................... 30 2. 3 Material and Methods ..................................................................................................................... 33 2. 3. 1 Plant material and growth conditions ...................................................................................... 33 2. 3. 2 Scent phenotyping ................................................................................................................... 33 2. 3. 3 Molecular cloning and plant transformation ........................................................................... 34 2. 3. 4 In vitro CNL enzyme assays ...................................................................................................... 34 2. 3. 5 Transient expression assay in petunia flowers ........................................................................ 35 2. 3. 6 QTL mapping ............................................................................................................................ 36 2. 3. 7 Population-genetic analyses .................................................................................................... 36 2. 3. 8 Statistical analysis .................................................................................................................... 36 2. 4 Results .............................................................................................................................................. 37 2. 4. 1 Loss of BAld emission in C. rubella Cr1504 is due to a single amino-acid exchange in CNL1 .. 37 2. 4. 2 Evolutionarily independent amino-acid exchanges in CNL1 underlie the loss of BAld emission in additional C. rubella accessions ...................................................................................................... 40 2. 4. 3 Evolutionary history of the CNL1 mutations in C. rubella ........................................................ 41 2. 4. 4 Loss of BAld emission in C. orientalis is not due to a mutation in CNL1 .................................. 43 2. 4. 5 Loss of BAld emission in C. orientalis results from polygenic downregulation of the benzenoid pathway .............................................................................................................................................. 45 2. 5 Discussion ......................................................................................................................................... 47 2. 5. 1 CNL1 as a target for parallel evolution of BAld loss ................................................................. 47 2. 5. 2 Evolutionary history of CNL1 mutations in C. rubella .............................................................. 48 2. 5. 3 The molecular basis of the loss of BAld emission in C. orientalis ............................................ 48 2. 6 Acknowledgements .......................................................................................................................... 50 2. 7 Author Contributions ....................................................................................................................... 50 2. 8 References ........................................................................................................................................ 51 2. 9 Supporting information .................................................................................................................... 53 2. 9. 1 Supplemental Figures ............................................................................................................... 53 2. 9. 2 Supporting Tables ..................................................................................................................... 60 2. 10 Transcriptome analysis of genes involved in the benzenoid pathway .......................................... 63 3. Manuscript: A high-throughput amplicon-based method for estimating outcrossing rates.................. 66 3. 1 Abstract ............................................................................................................................................ 68 3. 2 Background ...................................................................................................................................... 69 3. 3 Methods ........................................................................................................................................... 71 3. 3. 1 Reagents ................................................................................................................................... 71 3. 3. 2 Equipment ................................................................................................................................ 71 3. 3. 3 Plant material and growth conditions ...................................................................................... 72 3. 3. 4 DNA extraction from pooled seeds .......................................................................................... 72 3. 3. 5 Design of PCR primers .............................................................................................................. 73 3. 3. 6 PCR amplification and library generation ................................................................................ 75 3. 3. 7 Amplicon sequencing ............................................................................................................... 79 3. 3. 8 Sequence analysis and estimation of outcrossing rates .......................................................... 80 3. 4 Results .............................................................................................................................................. 81 3. 5 Discussion ......................................................................................................................................... 88 3. 6 References ........................................................................................................................................ 91 3. 7 Declarations ..................................................................................................................................... 93 3. 8 Supplemental Information ............................................................................................................... 94 4. Identifying a candidate gene underlying PAQTL1 ................................................................................... 96 4. 1 Introduction ..................................................................................................................................... 96 4. 2 Material and Methods ....................................................................................................................