Evolutionary analyses of orphan genes in mouse lineages in the context of de novo gene birth Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Rafik Tarek Neme Garrido Plön, April, 2013 Erstgutachter: Prof. Dr. Diethard Tautz Zweitgutachter: Prof. Dr. Thomas C. G. Bosch Tag der mündlichen Prüfung: 07.07.2014 Zum Druck genehmigt: 07.07.2014 gez. Prof. Dr. Wolfgang Duschl (Dekan) 2 Contents Contents .......................................................................................................................................... 3 Summary of the thesis .................................................................................................................... 6 Zusammenfassung der Dissertation............................................................................................... 7 Acknowledgements ....................................................................................................................... 10 General introduction...................................................................................................................... 12 A brief historic perspective on the concepts of gene birth .................................................... 12 Gene duplication is the main source of new genes .............................................................. 12 Orphan genes and the genomics era .................................................................................... 14 Phylostratigraphy and the continuous emergence of new genes ......................................... 16 Not all genes come from other genes ................................................................................... 17 Considering gene birth from molecular and evolutionary perspectives ................................... 19 Overprinting: true innovation from existing genes .................................................................... 20 The life cycle of genes .............................................................................................................. 22 Overview.................................................................................................................................... 24 Chapter 1: Phylogenetic patterns of emergence of new genes support a model of frequent de novo evolution ............................................................................................................................... 26 Introduction................................................................................................................................ 26 Results....................................................................................................................................... 27 Phylostratigraphy of mouse genes ........................................................................................ 27 Genomic features across ages.............................................................................................. 29 Chromosomal distribution ...................................................................................................... 33 Association with transcriptionally active sites ....................................................................... 33 Testis expressed genes......................................................................................................... 35 Alternative reading frames..................................................................................................... 36 Discussion ................................................................................................................................. 39 De novo evolution versus duplication-divergence ................................................................ 40 Regulatory evolution .............................................................................................................. 40 Overprinting ........................................................................................................................... 41 Conclusion................................................................................................................................. 42 Methods ..................................................................................................................................... 43 Phylostratigraphy ................................................................................................................... 43 3 Gene structure analyses........................................................................................................ 43 Transcription associated regions........................................................................................... 44 Expression data for testis ...................................................................................................... 44 Secondary reading frames .................................................................................................... 44 Acknowledgements ................................................................................................................... 45 Chapter 2: Sequencing of genomes and transcriptomes of closely related mouse species....... 46 Introduction................................................................................................................................ 46 Using wild mice to understand gene birth at the transcriptome level ................................... 46 Phylogeographic distribution of the samples ........................................................................ 47 Methods ..................................................................................................................................... 49 Biological material.................................................................................................................. 49 Transcriptome sequencing .................................................................................................... 49 Genome sequencing.............................................................................................................. 49 Raw data processing ............................................................................................................. 50 Transcriptome read mapping, annotation and quantification................................................ 50 Genome read mapping .......................................................................................................... 51 Available resources ................................................................................................................... 51 Chapter 3: Differential selective constrains across phylogenetic ages and their impact on the turnover of protein-coding genes. ................................................................................................. 53 Introduction................................................................................................................................ 53 Methods ..................................................................................................................................... 53 Transcriptome assembly ....................................................................................................... 53 Generation of ortholog pairs and rate analyses .................................................................... 54 Overlapping genes................................................................................................................. 54 Reading frame polymorphism detection and annotation ...................................................... 55 Statistical analyses ................................................................................................................ 55 Results....................................................................................................................................... 55 Rate differences between genes of different ages ............................................................... 55 Overlapping genes are an unlikely source of bias ................................................................ 57 Impact of reading frame polymorphisms across phylogenetic time...................................... 59 Discussion ................................................................................................................................. 64 Acknowledgements ................................................................................................................... 66 Chapter 4: A transcriptomics approach to the gain and loss of de novo genes in mouse lineages ....................................................................................................................................................... 67 Introduction................................................................................................................................ 67 4 How is a gene made? ............................................................................................................ 67 The early phase of new gene emergence............................................................................. 69 Pervasive transcription and junk-DNA as raw material for new genes ................................ 70 Methods ..................................................................................................................................... 71 Transcriptome presence/absence matrix and mapping of gains and losses ......................