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DIPTERA: CALLIPHORIDAE) by Sarah E

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DIPTERA: CALLIPHORIDAE) by Sarah E THE GENOMICS OF DEVELOPMENT RATE VARIATION IN COCHLIOMYIA MACELLARIA (DIPTERA: CALLIPHORIDAE) by Sarah E. Lewis A Thesis Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Master of Science Department of Chemistry Indianapolis, Indiana August 2018 ii THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Christine Picard, Chair Department of Biological Sciences Dr. Aaron Tarone Department of Entomology Dr. Randall Roper Department of Biological Sciences Approved by: Dr. John Goodpaster Head of the Graduate Program iii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii ABSTRACT ................................................................................................................................. viii 1. INTRODUCTION ................................................................................................................... 1 What is Forensic Entomology? ........................................................................................... 1 1.1.1 Two approaches for estimating PMI............................................................................ 1 How is Forensic Entomology typically used? .................................................................... 2 1.2.1 Blow fly life cycle ....................................................................................................... 3 What data types are used in Forensic Entomology? ........................................................... 4 What contributes to development rate variation? ............................................................... 4 1.4.1 Seasonal temperatures ................................................................................................. 5 1.4.2 Ambient temperature ................................................................................................... 6 1.4.3 Larval masses ............................................................................................................... 7 1.4.4 Type of carrion ............................................................................................................ 8 1.4.5 Toxins present in carrion ............................................................................................. 8 1.4.6 Blow fly species present on carrion ............................................................................. 9 1.4.7 Predation behavior of certain blow fly larvae ............................................................ 10 What about the genetic contribution to development rate variation? ............................... 10 How can the study determine what genetic components influence development rate? .... 12 1.6.1 Pooled sequencing method ........................................................................................ 13 Developmental process and involved genes ..................................................................... 14 1.7.1 Hormones and development ...................................................................................... 14 1.7.2 Nutrient signaling pathways ...................................................................................... 14 1.7.3 Prothoracicotropic hormone signaling pathways ....................................................... 15 1.7.4 Activin signaling pathway ......................................................................................... 15 1.7.5 Nitric oxide pathway .................................................................................................. 16 1.7.6 Two approaches for identifying variants between genomes...................................... 16 The blow fly of interest and related model organism ....................................................... 17 What does this study plan to accomplish? ........................................................................ 18 iv 2. DEVELOPMENT RATE GENETIC MARKERS ................................................................ 19 Introduction ....................................................................................................................... 19 Materials and Methods ...................................................................................................... 21 2.2.1 Identifying variants potentially associated with development rate ............................ 21 2.2.1.1 Amplification and sequencing of developmental genes ....................................... 23 2.2.1.2 DNA amplification ............................................................................................... 24 2.2.1.3 Sequencing amplification and genotyping ........................................................... 24 2.2.2 Wild fly sequencing ................................................................................................... 25 2.2.2.1 Preparing replicates from wild C. macellaria ...................................................... 25 2.2.2.2 Development of wild offspring ............................................................................. 26 2.2.2.3 DNA extraction ..................................................................................................... 26 Results ............................................................................................................................... 27 2.3.1 Selection of genes ...................................................................................................... 27 2.3.2 Wild offspring development and sequencing ............................................................ 28 Discussion ......................................................................................................................... 33 2.4.1 Enzymes in MAPK pathway effect development rate ............................................... 34 2.4.2 Enzymes in activin pathway effect development rate ............................................... 34 2.4.3 Ecdysone-induced proteins effect development rate ................................................. 35 2.4.4 Novel variants identified in sequencing .................................................................... 36 Conclusion ........................................................................................................................ 37 3. GENOMIC REGIONS EFFECTING DEVELOPMENT RATE .......................................... 38 Introduction ....................................................................................................................... 38 Materials and Methods ...................................................................................................... 40 3.2.1 Description of genomes used ..................................................................................... 40 3.2.2 Variant discovery ....................................................................................................... 41 3.2.3 Genome wide association study................................................................................. 41 3.2.4 Functional characterization ........................................................................................ 42 Results ............................................................................................................................... 43 3.3.1 Selection strain genome comparison ......................................................................... 43 3.3.2 Functional characterization of sequences containing significant variants ................. 43 3.3.2.1 Biological processes, molecular functions, and enzyme pathways effected ........ 44 v Discussion ......................................................................................................................... 50 3.4.1 Regulation of RNA polymerase II ............................................................................. 50 3.4.2 Protein dimerization ................................................................................................... 51 3.4.3 Neural development transcription factors .................................................................. 52 3.4.4 Cytochrome P450 genes ............................................................................................ 53 3.4.5 Metabolic pathways ................................................................................................... 54 Conclusion ........................................................................................................................ 54 4. CONCLUSION AND FUTURE STUDIES .......................................................................... 56 REFERENCES ............................................................................................................................. 58 APPENDIX A ............................................................................................................................... 74 APPENDIX B ............................................................................................................................... 76 APPENDIX C ............................................................................................................................... 79 vi LIST OF TABLES Table 1. Drosophila melanogaster genes and their associated involvement in development rate ...........................................................................................................
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