University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2018 Genomic Regulation Of Abiotic Stress Response In The oiS l Nematode Oscheius Tipulae Riley Mcglynn Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Mcglynn, Riley, "Genomic Regulation Of Abiotic Stress Response In The oS il Nematode Oscheius Tipulae" (2018). Theses and Dissertations. 2417. https://commons.und.edu/theses/2417 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. GENOMIC REGULATION OF ABIOTIC STRESS RESPONSE IN THE SOIL NEMATODE OSCHEIUS TIPULAE by Riley D. McGlynn Bachelor of Arts, Concordia College, Moorhead, MN, 2012 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota December 2018 PERMISSION Title Genomic Regulation of Abiotic Stress Response in the Soil Nematode Oscheius tipulae Department Biology Degree Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work, or in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Riley D. McGlynn December 6, 2018 iii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….. viii LIST OF TABLES……………………………………………………………….... x ACKNOWLEDGMENTS…..…………………………………………………….. xiv ABSTRACT………………………………………………………………………... xv CHAPTER I. LITERATURE REVIEW………………………………………… 1 Introduction..……………………………………………… 1 Phylum Nematoda…………………………………………. 1 Caenorhabditis elegans…………………………………….. 2 Oscheius tipulae...…………………………………………. 4 Heat Stress: Threats and Response..………………………. 4 Desiccation Stress: Threats and Response ………………… 6 Freezing Stress: Threats and Response …………………… 10 Genetic Regulation…...…………………………………… 12 Genome Sequencing.……...……………………………….. 17 Nematode Genomics……………………………………… 19 DNA Methylation………………………………………… 22 Research Goals……………………………………………. 23 References………………………………………………… 28 iv II. DRAFT GENOME OF THE KJO STRAIN OF THE SOIL NEMATODE OSCHEIUS TIPULAE ……………………………. 37 Abstract……………………………………………………. 37 Introduction.…………………………….…………………. 38 Materials and Methods.……………………………………. 39 Nematode Preparation..…………………………….. 39 Genomic DNA Extraction and Genome Assembly... 40 Mitochondrial Genome.……………………………. 42 Synteny.………………………...………………….. 43 Genome Annotation..…………………………….. 43 Positive Selection..…………...…………………….. 44 Results and Discussion..…………………………………… 45 Genome Assembly…...…………………………….. 46 Genome Annotation……..………………………… 46 Gene Content………....………………………….... 48 Mitochondrial Genome.....……...………………….. 49 Synteny.………………………...………………….. 51 Positive Selection..…………...……………………. 54 Conclusions...…………..………………………………….. 56 References……………..…………………………………… 58 III. ABIOTIC STRESS RESPONSE TRANSCRIPTION PROFILES IN THE SOIL NEMATODE OSCHEIUS TIPULAE……………… 61 Abstract…………………………………………………….. 61 Introduction.…………………………….………………….. 62 v Materials and Methods…………………………………….. 67 Nematode Preparation……………………………… 67 Stress Treatments..………..……………………….. 68 RNA Extraction....………………………………… 69 Sequencing……...………………………………….. 70 RNA-Seq………..………………………………….. 70 Positive Selection.…………………………………. 72 Results and Discussion..…………………………………… 72 Transcription Profiles...……………………………. 72 Upregulated Genes………………………………… 76 Downregulated Genes…...………………………… 83 Positive Selection.………………………………….. 90 Conclusions………………………………………………… 93 References……………..…………………………………… 96 IV. PRESENCE AND LOCATION OF DNA METHYLATION IN THE GENOMES OF THE SOIL NEMATODES OSCHEIUS TIPULAE AND CAENORHABDITIS ELEGANS…….. 100 Abstract…………………………………………………….. 100 Introduction.…………………………….…………………. 101 Materials and Methods………………….………………….. 104 Nematode Growth and DNA Extraction.....……….. 104 Library Preparation and Sequencing……………….. 105 Whole-Genome Bisulfite Sequencing Analysis….…106 MethylCap-Seq Analysis…………………...……… 107 vi Methyltransferase Presence….……………...…….. 109 Role of Methylation on Abiotic Stress Response…. 109 Results and Discussion..…………………………………… 110 Whole-Genome Bisulfite Sequencing………………110 MethylCap-Seq………..…………….……...……... 111 Methyltransferase Presence....……………...……… 116 Role of Methylation on Abiotic Stress Response….. 117 Conclusions...…………..………………………………….. 119 References……………..…………………………………… 121 V. EPILOGUE………….….………………………………………….. 124 The Oscheius tipulae Genome…..………..……………….. 125 Genomic Regulation of Abiotic Stress in O. tipulae……..... 125 The Presence and Role of DNA Methylation…………..….. 126 Conclusion………….....…………………………………… 127 APPENDICES………….....………………………………………. 129 vii LIST OF FIGURES Figure Page 1.1. Simplified organizational models for genomic stress response centered on the one-gene-one-polypeptide hypothesis…………………………………. 13 1.2. Simplified organizational model for genomic stress response centered on the idea of pleiotropy …………………………………………………..……. 15 2.1. Synteny comparison between the O. tipulae and C. elegans genomes..……… 52 2.2. Synteny comparison between the KJO draft genome scaffolds and the CEW1 draft genome scaffolds ………............................................................... 53 2.3. Blast2GO Gene Ontology analysis of the predicted O. tipulae genes................ 55 2.4. Histogram of the dN/dS ratios of the nematode reciprocal orthologs……….... 57 3.1. Heatmap of relative transcription levels across all treatment replicates......….. 73 3.2. Multi-dimensional analysis of transcription pattern replicates…………………75 3.3 Appendix. RNA-Seq dispersion estimates………………………...…………… 130 3.4 Appendix. RNA-Seq fitted dispersions …………………………………….…. 131 3.5. Venn diagram of genes upregulated in at least one of the three treatments.….. 82 3.6. Venn diagram of genes downregulated in at least one of the three treatments……………………………………………………………………… 88 3.7 Appendix. Differentially expressed genes under the heat treatment……..……. 132 3.8 Appendix. Differentially expressed genes under the desiccation treatment…… 133 3.9 Appendix. Differentially expressed genes under the freezing treatment..…….. 134 3.10 Histogram of the dN/dS ratios for upregulated genes……………………..…. 91 3.11 Venn diagrams of the positively selected genes…………………….……….. 92 viii 3.12 Histogram of the dN/dS ratios for the downregulated genes…...……………. 94 ix LIST OF TABLES Table Page 2.1 Genome assembly statistics for this Oscheius tipulae genome and various other nematode species……………………………….......…………… 47 2.2 Genomic content makeup of the assembled KJO O. tipulae genome…………. 50 3.1 Lists of a subset of the known molecular and physiological responses nematodes have to three abiotic stresses: heat, desiccation and freezing…..….. 66 3.2 Appendix. DAVID functional annotation results for all genes upregulated under at least one abiotic stress……………………………………………..….. 135 3.3 Appendix. DAVID functional annotation results for all genes downregulated under at least one abiotic stress……………………………….. 138 3.4 Appendix. DAVID functional annotation results for all genes upregulated under heat stress……………………………………………………………….. 141 3.5 Appendix. DAVID functional annotation results for all genes upregulated under desiccation stress…………………………………….………………….. 144 3.6 Appendix. DAVID functional annotation results for all genes upregulated under freezing stress…….………………………………….………………….. 147 3.7 Appendix. DAVID functional annotation results for all genes upregulated under only heat stress…….………………………………….…………………. 150 3.8 Appendix. DAVID functional annotation results for all genes upregulated under only desiccation stress…………………………………………………... 153 x 3.9 Appendix. DAVID functional annotation results for all genes upregulated under only freezing stress……………………………………………….…….. 156 3.10 Appendix. DAVID functional annotation results for all genes upregulated under both heat and desiccation stresses………………………… 159 3.11 Appendix. DAVID functional annotation results for all genes upregulated under both heat and freezing stresses…………………………… 162 3.12 Appendix. DAVID functional annotation results for all genes upregulated under both freezing and desiccation stresses……………………. 164 3.13 Appendix. DAVID functional annotation results for all genes upregulated under heat, freezing, and desiccation stresses………………....... 167 3.14 Appendix. DAVID functional annotation results for all genes downregulated under heat stress…………………………………………....... 170 3.15 Appendix. DAVID functional annotation results for all genes downregulated under desiccation stress…………………………………........ 173 3.16 Appendix. DAVID functional annotation results for all genes downregulated under freezing stress………………………………………….. 176 3.17 Appendix. DAVID functional annotation results