Transcriptomic analysis of ovarian development in parasitic Ichthyomyzon castaneus (chestnut lamprey) and non-parasitic Ichthyomyzon fossor (northern brook lamprey) By Nisha Ajmani A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of MASTER OF SCIENCE Department of Biological Sciences University of Manitoba Winnipeg, Manitoba Copyright © 2017 by Nisha Ajmani i Abstract Lampreys are primitive jawless fishes that diverged over 550 million years ago. As adults, they are either parasitic or non-parasitic. In non-parasitic species, sexual differentiation and oocyte development generally occur earlier than in parasitic species; fecundity is reduced and sexual maturation is accelerated following metamorphosis. The genes controlling ovarian differentiation and maturation in lampreys are poorly understood. This study used RNA-Seq data in the parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzon fossor to identify suites of genes expressed during different stages of ovarian development that show different developmental trajectories with respect to ovarian differentiation and sexual maturation. For this, reference-guided and de novo assembly pipelines were designed for studying a non-model species. To test and explore the relative advantages of the pipelines, expression of insulin superfamily genes was used. This research helps to identify genes involved in lamprey ovarian development and provides insight into evolution of the insulin superfamily in vertebrates. ii Acknowledgements Firstly, I would like to express my sincere gratitude to my advisors, Prof. Dr. Margaret Docker & Prof. Dr. Sara Good for their generous guidance, continuous support and motivation throughout my Master’s and related research. I sincerely appreciate all their contributions of time, inestimable support and ideas to make my research experience productive and stimulating. The joy, optimism, and enthusiasm they have for research were inspiring, motivational and contagious. This tenure has been a life changing experience for me and has been a source of friendships and collaboration. Your help in every aspect was imperative to my completion of this project. I would also like to thank my committee, Dr. Colin Garroway and Dr. Mike Domaratzki for their advice, encouragement and insightful comments. I am really thankful to NSERC Discovery Grant (MFD, SVG), Great Lakes Fishery Commission (MFD), University of Manitoba Faculty of Science Graduate Enhancement of Tri- Council Stipends (MFD), Faculty of Graduate Studies, Faculty of Science and Department of Biological Sciences (travel award) for financially supporting this project. I have cherished every single day of my research in these last two years and it has given me a positive direction towards life and I feel it is the start of a new beginning. I strongly believe in Kaizen philosophy and have tried my best to improve myself every day. The cooperation, experience, and assistance, of my fellow graduate students were essential for completion of my project and I would like to extend thanks to Biological Sciences, Faculty of Graduate Studies and my lab mates for making my research experience a memorable one. It has been an honor for me to be a part of this wonderful team and project. iii My final words go equally to me family and I want to thank them for their tremendous love and support. My loving husband Adishail, I promise that all your efforts will gain something awe-inspiring in the near future. iv Dedication I would like to dedicate this M.Sc. degree to my loving Husband, Adishail and would like to rephrase the proverb, “Behind every successful woman there is a man”. Without your love and support this would not have been possible. I am thankful to my parents especially to my sister, Manisha who encouraged me and were there with me in my thick and thin. I would also like to dedicate this degree to my little baby who is growing inside me and by the time I graduate, you will be here in this beautiful world. Thank you God for making me what I am today and giving me the courage to face all the challenges with an open heart and smile. I owe this thesis to all of you. v Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements ....................................................................................................................... iii Dedication ....................................................................................................................................... v List of Tables ...................................................................................................................................x List of Figures .............................................................................................................................. xiii Chapter 1: General introduction.......................................................................................................1 1.1 Lamprey biology ....................................................................................................................7 1.1.1 Paired species concept .....................................................................................................7 1.1.2. Morphological differences..............................................................................................9 1.1.3. Gonadal development in lampreys and other vertebrates .............................................10 1.1.3.1 Ovarian differentiation and sexual maturation in lampreys ...................................11 1.2. Gene based studies in lampreys and other vertebrates ........................................................13 1.2.1. Genes of interest involved in gonadal development in lampreys and other vertebrates ..........................................................................................................................15 1.2.1.1. Gonadotropin releasing hormone ..........................................................................16 1.2.1.2. Gonadotropin ........................................................................................................17 1.2.1.3. Growth hormone ...................................................................................................18 1.2.1.4. The insulin superfamily of peptides and receptors ..............................................19 1.2.1.5. Insulin like growth factors ....................................................................................22 1.3. Lampreys as a model species ..............................................................................................24 1.4. Different methods for gene expression data analysis ..........................................................26 1.5. Goals and objectives ............................................................................................................28 1.6. Significance of proposed research .......................................................................................33 1.7. References ...........................................................................................................................34 1.8. Tables and Figures ..............................................................................................................54 Chapter 2: RNA-Seq pipeline for mapping and counting genes expressed during ovarian development in lampreys: a genome-guided approach ..................................................................62 2.1. Abstract ...............................................................................................................................62 2.2. Introduction .........................................................................................................................63 2.3. Material and methods ..........................................................................................................68 2.3.1. Sample collection .....................................................................................................68 vi 2.3.2. RNA sequencing ......................................................................................................68 2.4. Pipeline designed for transcriptomic data analysis in the presence of a reference genome ...............................................................................................................................70 2.4.1. Mapping and counting the number of times a gene is expressed using a reference genome – Galaxy platform.................................................................................................70 2.4.1.1. Gene annotation .......................................................................................71 2.5. RNA-Seq analysis tools ......................................................................................................71 2.5.1. Preparation of raw data ............................................................................................72 2.5.2. Removal of adaptor sequences.................................................................................73 2.5.3. Format conversion ...................................................................................................74 2.5.4. Mapping and assembling the reads ..........................................................................74 2.5.5. Counting the number