Immunology going wild: genetic variation and immunocompetence in the mallard (Anas platyrhynchos) Doctoral thesis for obtaining the academic degree Doctor of Natural Science submitted by Elinor Jax at the Faculty of Sciences Department of Biology Konstanz, 2019 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-1ehyozma1kf2n5 Date of the oral examination: 19.12.2018 1. Reviewer: Prof. Dr. Martin Wikelski 2. Reviewer: Prof. Dr. Katharine Magor 3. Reviewer: PD Dr. Elizabeth Yohannes Table of Contents Summary .................................................................................................................................... 9 Zusammenfassung ................................................................................................................... 11 General introduction ............................................................................................................... 15 Chapter 1 .................................................................................................................................. 21 Abstract ............................................................................................................................................. 21 1. Background .................................................................................................................................... 22 2. RNA analysis of birds before NGS, a brief history ......................................................................... 24 3. Ornithology in the age of *omics .................................................................................................. 26 3.1 RNA-seq, a brief introduction .................................................................................................. 28 4. Applications of transcriptomics for non-model bird species ........................................................ 28 4.1 Characterisation of transcriptomes ......................................................................................... 29 4.2 Functional studies (gene expression) ...................................................................................... 32 4.3 Gene interactions and function ............................................................................................... 36 4.4 Genetic marker discovery ........................................................................................................ 37 4.5 Genetic variation ..................................................................................................................... 40 4.6 Alternative splicing / transcriptome isoforms ......................................................................... 42 5. Planning of an RNA-seq study - quick guide .................................................................................. 48 5.1 Sample collection .................................................................................................................... 48 5.2 RNA-isolation and library preparation .................................................................................... 50 5.3 Sequencing .............................................................................................................................. 53 5.4 Transcriptome assembly or alignment .................................................................................... 56 5.5 Analysis .................................................................................................................................... 57 6. Challenges with RNA-seq and its utility for non-model ornithology ............................................. 59 6.1 Library preparation, we might be introducing bias in the data .............................................. 59 6.2. Working with large datasets (“Big Data”), a new era for biology .......................................... 59 6.3 Transcriptomics, from the lab to the field ............................................................................... 60 6.4 Experimental proof as a last step to ascribe biological function ............................................ 60 7. Future ............................................................................................................................................ 61 7.1 Single Cell Sequencing ............................................................................................................. 61 7.2 Spatially resolved *omics ........................................................................................................ 62 7.3 On-site sequencing platforms ................................................................................................. 62 Acknowledgements ........................................................................................................................... 63 Chapter 2 .................................................................................................................................. 65 Abstract ............................................................................................................................................. 65 Introduction ....................................................................................................................................... 66 Methods ............................................................................................................................................ 69 Birds and housing .......................................................................................................................... 69 Treatments .................................................................................................................................... 70 Experimental setup ....................................................................................................................... 70 Analysis of body temperature, heart rate and activity data ......................................................... 72 Leukocyte counting and differentiation ........................................................................................ 73 Genome wide gene expression profiling ....................................................................................... 74 Gene expression of target genes ................................................................................................... 77 Results ............................................................................................................................................... 78 Body temperature, heart rate and activity ................................................................................... 78 Genome wide gene expression profiling ....................................................................................... 83 Gene expression of target genes ................................................................................................... 92 Discussion .......................................................................................................................................... 92 Body temperature, heart rate and activity ................................................................................... 92 Leukocyte counting and differentiation ........................................................................................ 93 Gene expression profiling .............................................................................................................. 94 Acknowledgements ........................................................................................................................... 97 Chapter 3 .................................................................................................................................. 99 Abstract ............................................................................................................................................. 99 Introduction ..................................................................................................................................... 101 Materials and Methods ................................................................................................................... 103 Birds ............................................................................................................................................. 103 Bait design ................................................................................................................................... 103 Library preparation and enrichment ........................................................................................... 103 Reference-based assembly and retrieval of immune genes ....................................................... 104 Genetic variation in immune genes in mallards .......................................................................... 104 Evidence of Natural Selection ..................................................................................................... 105 Immune pathway function .......................................................................................................... 107 Results and Discussion .................................................................................................................... 107 Reference-based assembly and retrieval of immune genes ....................................................... 107 Genetic variation in immune genes in mallards .......................................................................... 108 Evidence of Natural