Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations 2013 Understanding Osmoregulation and Immunity in Heteromyid Rodents Through Insights Gained from RNA-Seq Nicholas John Marra Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Part of the Genetics Commons Recommended Citation Marra, Nicholas John, "Understanding Osmoregulation and Immunity in Heteromyid Rodents Through Insights Gained from RNA- Seq" (2013). Open Access Dissertations. 86. https://docs.lib.purdue.edu/open_access_dissertations/86 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Nicholas John Marra Entitled UNDERSTANDING OSMOREGULATION AND IMMUNITY IN HETEROMYID RODENTS THROUGH INSIGHTS GAINED FROM RNA-SEQ Doctor of Philosophy For the degree of Is approved by the final examining committee: J. Andrew DeWoody Chair Brian P. Dilkes Krista M. Nichols Michael J. Zanis To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________J. Andrew DeWoody ____________________________________ Approved by: Christine A. Hrycyna 12/02/2013 Head of the Graduate Program Date i UNDERSTANDING OSMOREGULATION AND IMMUNITY IN HETEROMYID RODENTS THROUGH INSIGHTS GAINED FROM RNA-SEQ A Dissertation Submitted to the Faculty of Purdue University by Nicholas John Marra In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2013 Purdue University West Lafayette, Indiana ii For my loving family, friends, and especially for my grandfather, Alexander Patterson, who always encouraged me to pursue learning at all times and in all ways possible iii ACKNOWLEDGEMENTS Reaching this point would have been impossible without the guidance, assistance, and support of a great number of people whom I cannot thank in this space; however, there are several that I would especially like to mention here. First I’d like to thank my advisor, Dr. Andrew DeWoody for all of his guidance, support, and mentorship. His honest approach and ability to help me recognize my strengths and also identify and sharpen my areas of weakness has truly helped me grow as a scientist, researcher, and teacher. I want to thank my graduate committee: Dr. Brian Dilkes, Dr. Krista Nichols, and Dr. Michael Zanis for their advice and guidance. I would also like to thank all of the DeWoody and Genetics lab members past and present for their support and help throughout the years, especially: Kendra Abts, Jennifer Antonides, Dr. Jackie Doyle, Dr. Anna Fahey (my mentor and friend), Yanzhu Ji, Dr. Steve Kimble, Bhagya Wijayawardena, Janna Willoughby, Dr. Soo Hyung Eo, Jennifer McCreight, Jamie Nogle, and Joe Ruhl. Andrew strives for a lab that works together and cares for one another like a family and I truly have felt that level of support from everyone. In particular, I would like to thank Ruby Liu and Sabrina Schuchard for their help working as undergraduates on the project, this work would have been impossible without their assistance. My project would have impossible without the help and guidance I received from Dr. Peter Waser as well as the kindness of Mary Sue Waser during two separate iv collection trips to Arizona. Peter’s questions, knowledge about kangaroo rats, and discussions about the project helped me gain an appreciation for the natural history of the Heteromyids and I would like to thank him for his significant contributions to this project. I also need to thank Andrea Romero and her field crew at La Selva biological station for their help in collecting H. desmarestianus. I would also like to thank Purdue Genomics and Bioinformatics cores for their aid in sequencing and advice with regards to sequence assembly. I would like to thank Dr. Matt Hale for his advice, support, and friendship over cups of coffee, career and research questions, and time at the gym. I would also like to thank my friends in PULSe and the students who worked with me in the Science in Schools program for providing an outlet to relax and enjoy talking about science. In addition, I would like to thank Annie Murray for all of her support and assistance in the final couple months in making sure that this dissertation was completed. Finally, I would like to thank my parents, Margaret and Charles Marra for all of their love and support as well as my sister Michelle for being by my side throughout my life and especially during my time at Purdue University. v TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ............................................................................................................ x ABSTRACT ............................................................................................................. xii CHAPTER 1. A PRIORI AND A POSTERIORI APPROACHES FOR FINDING GENES OF EVOLUTIONARY INTEREST IN NON-MODEL SPECIES: OSMOREGULATORY GENES IN THE KIDNEY TRANSCRIPTOME OF THE DESERT RODENT DIPODOMYS SPECTABILIS ......................................................... 1 1.1 Abstract ......................................................................................................2 1.2 Introduction ................................................................................................3 1.3 Methods ......................................................................................................6 1.3.1 Sample Collection, RNA Extraction, and cDNA Synthesis ...............6 1.3.2 454 Sequencing and Transcriptome Assembly ...................................7 1.3.3 A Priori Candidate Genes Identified via Model Species ....................8 1.3.4 A Posteriori Candidate Genes Identified via Differential Expression 9 1.3.5 GO Terms and Molecular Markers Present in Candidates................11 1.3.6 Testing for Positive Selection ...........................................................11 1.4 Results ......................................................................................................14 1.4.1 Sequence Assembly and BLAST Annotation ...................................14 1.4.2 A Priori Candidate Genes Identified via Model Species ..................15 1.4.3 A Posteriori Candidate Genes Identified via Differential Expression . ...........................................................................................................15 1.4.4 GO Terms and Molecular Markers Present in Candidates................16 1.4.5 Testing for Positive Selection ...........................................................17 vi Page 1.5 Discussion ................................................................................................18 1.5.1 A Priori Candidate Genes Identified via Model Species ..................19 1.5.2 A Posteriori Candidate Genes Identified via Differential Expression . ...........................................................................................................22 1.5.3 GO Terms and Molecular Markers Present in Candidates................23 1.5.4 Testing for Positive Selection ...........................................................24 1.6 Conclusions and Future Directions ..........................................................25 1.7 References ................................................................................................27 CHAPTER 2. NATURAL SELECTION AND THE GENETIC BASIS OF OSMOREGULATION IN HETEROMYID RODENTS AS REVEALED BY RNA-SEQ ............................................................................................................. 45 2.1 Abstract ....................................................................................................46 2.2 Introduction ..............................................................................................47 2.3 Methods ....................................................................................................50 2.3.1 Sample Collection .............................................................................50 2.3.2 RNA Extraction and Sequencing ......................................................51 2.3.3 Transcriptome Asemblies .................................................................52 2.3.4 Test for Differential Expression (DE) ...............................................53 2.3.5 Test for Positive Selection ................................................................55 2.4 Results ......................................................................................................57 2.4.1 Sequencing, Assembly, and Annotation ...........................................57 2.4.2 Differential Expression (DE) ............................................................57 2.4.3 Selection Tests ..................................................................................58 2.4.4 A Priori Candidates ...........................................................................59 2.5 Discussion ................................................................................................59