Transcriptomic Analysis of the Gray short-tailed opossum (Monodelphis domestica) B-cell Genes Photo credit: J. Old Andrea L. Schraven A thesis presented as fulfilment of the requirements for the degree of Master of Research Western Sydney University, Hawkesbury School of Science and Health Date: December 2019 Table of Contents Table of Contents ......................................................................................................................... 2 Table of Figures ............................................................................................................................ 4 Table of Tables ........................................................................................................................... 10 Acknowledgements .................................................................................................................... 11 Statement of Authenticity .......................................................................................................... 12 Preface ......................................................................................................................................... 13 Publications and Prepared Manuscripts................................................................................... 14 Conference Presentations .......................................................................................................... 15 Abstract ........................................................................................................................................ 16 Introduction ............................................................................................................................ 18 1.1 Mammalian Evolution ......................................................................................................... 19 1.1.1 Marsupial Reproductive Strategies ........................................................................... 20 1.2 Marsupial Immunology ....................................................................................................... 22 1.3 Marsupial Genomics ............................................................................................................ 27 1.3.1 Transcriptomics ........................................................................................................... 29 1.4 Model Marsupial: The gray short-tailed opossum (Monodelphis domestica) ............. 32 1.5 Research Aim ....................................................................................................................... 34 1.6 Thesis Outline ...................................................................................................................... 35 1.7 Chapter 1 References........................................................................................................... 36 Immunogenetics of Marsupial B-cells .................................................................................. 44 2.1 Chapter Outline and Authorship ........................................................................................ 45 2.2 Introduction .......................................................................................................................... 46 2.3 Mammalian Evolution ......................................................................................................... 48 2.4 Marsupial Immunology ....................................................................................................... 49 2.5 B-cells .................................................................................................................................... 51 2.5.1 Immunoglobulins ......................................................................................................... 58 2.5.2 Cluster of Differentiation Markers ............................................................................ 66 2.5.3 Signal Transduction Molecules ................................................................................. 69 2.5.4 Transcriptional Regulators ........................................................................................ 71 2.6 Conclusions .......................................................................................................................... 72 2.7 Conflicts of Interest ............................................................................................................. 72 2.8 Chapter 2 References........................................................................................................... 73 Single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica) ................. 83 2 3.1 Chapter Outline and Authorship ........................................................................................ 84 3.2 Introduction .......................................................................................................................... 85 3.3 Materials and Methods ........................................................................................................ 87 3.3.1 Ethics Statement........................................................................................................... 87 3.3.2 Tissue Collection, Cell Sorting and RNA Extraction .............................................. 87 3.3.3 cDNA Synthesis, Sequencing and Alignment ........................................................... 87 3.3.4 Single-cell Identification and Statistical Analyses .................................................. 88 3.4 Results ................................................................................................................................... 89 3.4.1 Heavy and Light Chain Immunoglobulin Usage ..................................................... 89 3.4.2 Immunoglobulin Variable and Joining Domains .................................................... 90 3.4.3 VH and VL Gene Segment Pairing ............................................................................ 93 3.5 Discussion ............................................................................................................................. 95 3.6 Acknowledgements ............................................................................................................. 99 3.7 Conflicts of Interest ............................................................................................................. 99 3.8 Chapter 3 References......................................................................................................... 100 3.9 Chapter 3 Supplementary Figures ................................................................................... 103 Single-cell transcriptomic analysis of marsupial B-cells, the gray short-tailed opossum (Monodelphis domestica) ...................................................................................................... 105 4.1 Chapter Outline and Authorship ...................................................................................... 106 4.2 Introduction ........................................................................................................................ 107 4.3 Materials and Methods ...................................................................................................... 109 4.3.1 Ethics Statement......................................................................................................... 109 4.3.4 Transcriptome Annotation and Gene Ontology .................................................... 110 4.3.5 Statistical Analyses .................................................................................................... 111 4.3.6 Phylogenetic Analyses and Motif Comparison ...................................................... 111 4.4 Results ................................................................................................................................. 112 4.4.1 Overview of the Transcriptome ............................................................................... 112 4.4.2 Highly Expressed Genes ........................................................................................... 114 4.5 Discussion ........................................................................................................................... 126 4.6 Acknowledgements ........................................................................................................... 130 4.7 Conflicts of Interest ........................................................................................................... 130 4.8 Chapter 4 References......................................................................................................... 131 Concluding Summary and Future Directions ................................................................... 135 5.1 Concluding Summary ........................................................................................................ 136 5.2 Future Directions ............................................................................................................... 140 5.3 Chapter 5 References......................................................................................................... 142 3 Table of Figures Figure 2.1 Phylogenetic tree based on B-cell relationship in vertebrate groups and divergence of marsupials referred to in this review, constructed using the NJ method. Figure 2.2 Unrooted phylogenetic tree based on amino acid