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Functional Genetic Approaches to Provide Evidence for the Role of Toolkit Genes in the Evolution of Complex Color Patterns in Drosophila Guttifera

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Functional Genetic Approaches to Provide Evidence for the Role of Toolkit Genes in the Evolution of Complex Color Patterns in Drosophila Guttifera Michigan Technological University Digital Commons @ Michigan Tech Dissertations, Master's Theses and Master's Reports 2021 FUNCTIONAL GENETIC APPROACHES TO PROVIDE EVIDENCE FOR THE ROLE OF TOOLKIT GENES IN THE EVOLUTION OF COMPLEX COLOR PATTERNS IN DROSOPHILA GUTTIFERA Mujeeb Olushola Shittu Michigan Technological University, [email protected] Copyright 2021 Mujeeb Olushola Shittu Recommended Citation Shittu, Mujeeb Olushola, "FUNCTIONAL GENETIC APPROACHES TO PROVIDE EVIDENCE FOR THE ROLE OF TOOLKIT GENES IN THE EVOLUTION OF COMPLEX COLOR PATTERNS IN DROSOPHILA GUTTIFERA", Open Access Dissertation, Michigan Technological University, 2021. https://doi.org/10.37099/mtu.dc.etdr/1174 Follow this and additional works at: https://digitalcommons.mtu.edu/etdr Part of the Biology Commons, Developmental Biology Commons, Evolution Commons, Molecular Genetics Commons, and the Other Cell and Developmental Biology Commons FUNCTIONAL GENETIC APPROACHES TO PROVIDE EVIDENCE FOR THE ROLE OF TOOLKIT GENES IN THE EVOLUTION OF COMPLEX COLOR PATTERNS IN DROSOPHILA GUTTIFERA By Mujeeb Olushola Shittu A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Biochemistry and Molecular Biology MICHIGAN TECHNOLOGICAL UNIVERSITY 2021 ©2021 Mujeeb Olushola Shittu This dissertation has been approved in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY in Biochemistry and Molecular Biology. Department of Biological Sciences Dissertation Advisor: Dr. Thomas Werner Committee Member: Dr. Chandrashekhar Joshi Committee Member: Dr. Stephen Techtmann Committee Member: Dr. Hairong Wei Department Chair: Dr. Chandrashekhar Joshi Table of Contents List of figures .......................................................................................................................4 List of tables .........................................................................................................................7 Author Contribution Statement ............................................................................................8 Acknowledgements ............................................................................................................10 Abstract ..............................................................................................................................12 1 Introduction ...............................................................................................................14 1 Chapter 1 ...................................................................................................................30 1.1 Abstract ..........................................................................................................31 1.2 Introduction ....................................................................................................32 1.3 Experimental design .......................................................................................34 1.3.1 Required materials and equipment....................................................34 1.3.1.1 Materials .........................................................................34 1.3.1.2 Equipment .......................................................................35 1.4 Methods ..........................................................................................................36 1.4.1 The Egg-laying cage .........................................................................36 1.4.2 Egg collection and preparation for microinjections ..........................39 1.4.3 Lining up the eggs for microinjections .............................................43 1.4.4 Preparation of transgene DNA ..........................................................45 1.4.5 Preparation of the injection cocktail .................................................46 1.4.6 Needle preparation and microinjections ...........................................47 1.4.7 Post-injection treatment and heat shock of the eggs .........................49 1.4.8 After-care and fly crosses .................................................................50 1.4.9 Screening for transgenic larvae .........................................................52 1.5 Expected results ..............................................................................................53 1.6 Troubleshooting ..............................................................................................54 1.7 Reagents .........................................................................................................55 1.7.1 Anti-fungus paper .............................................................................55 1.7.2 Halocarbon oil mixture .....................................................................56 1.8 Conclusions ....................................................................................................56 1.9 Supplementary Materials ................................................................................57 1.10 Acknowledgments ..........................................................................................57 1.11 Author Contributions ......................................................................................57 1 2 Chapter 2 ...................................................................................................................61 2.1 Abstract ..........................................................................................................62 2.2 Introduction ....................................................................................................62 2.2.1 Overview ...........................................................................................64 2.2.2 Application of this protocol ..............................................................64 2.2.3 Advantages and limitations ...............................................................65 2.3 Experimental Design ......................................................................................66 2.3.1 Probe design and synthesis ...............................................................66 2.3.2 Pupal staging .....................................................................................67 2.3.3 Abdominal epidermis preparation.....................................................67 2.3.4 Pupal wing preparation .....................................................................67 2.3.5 ISH of Drosophila abdomens and wings ..........................................68 2.4 Materials .........................................................................................................69 2.4.1 Reagents ............................................................................................69 2.4.2 Equipment and supplies ....................................................................72 2.4.3 Reagent setup ....................................................................................74 2.5 Procedure ........................................................................................................76 2.5.1 A-tail genomic PCR ..........................................................................76 2.5.2 Gel extraction and purification of PCR products ..............................76 2.5.3 Ligation .............................................................................................78 2.5.4 Transformation of DH-5α cells and colony PCR ..............................78 2.5.5 Culturing the positive colonies .........................................................80 2.5.6 Plasmid extraction from a positive clone (mini-prep) ......................80 2.5.7 Insertion direction PCR.....................................................................81 2.5.8 RNA probe synthesis ........................................................................83 2.5.9 RNA probe quality check and preparation........................................84 2.5.10 Drosophila pupa collection and processing for abdominal ISH .......86 2.5.11 ISH of Drosophila abdomens and wings ..........................................89 2.6 Troubleshooting guide ....................................................................................94 2.7 Anticipated results ..........................................................................................96 2.8 Supplementary information ..........................................................................109 3 Chapter 3A ..............................................................................................................116 3.1 Abstract ........................................................................................................117 3.2 Introduction and results ................................................................................118 3.3 Materials and Methods .................................................................................128 3.3.1 Molecular procedures......................................................................128 3.3.2 Drosophila stocks ............................................................................128 3.3.3 PCR primer sequences ....................................................................129 3.4 Acknowledgments ........................................................................................132 2 3.5 Author Contributions ....................................................................................132 Chapter 3B .......................................................................................................................139
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