Genomic and phylogenetic assessment of sea lamprey (Petromyzon marinus) Hox genes and analysis of Hox genes in association with myomeres across multiple lamprey genera By Darcy Childs A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In Partial fulfillment of the requirements of the degree of MASTER OF SCIENCE Department of Biological Science University of Manitoba Winnipeg Copyright© 2013 by Darcy Childs Abstract Lampreys are an important model for the study of early vertebrate development due to their unique evolutionary position as one of only two extant jawless vertebrates. In this study, 12 new putative Hox gene fragments were identified within the recently available Petromyzon marinus (sea lamprey) genome. These and the other previously- identified Hox genes were analyzed phylogenetically, which enabled the assignment of many of the new sequences to distinct paralogous gene clusters and showed distinctions between gnathostome and lamprey Hox sequences. An examination of Hox genes in other lamprey species was conducted using genomic PCR-based detection methods and identified 26 putative Hox gene homeobox fragments from multiple Hox genes across nine lamprey species. A study of Hox10 coding sequences in different lamprey species failed to find any correlation with variable numbers of trunk myomeres in lampreys, which suggests that other sequences or factors regulate the number of myomeres in different species. i Acknowledgments I would like to acknowledge a great number of people for their parts taken in the completion of my MSc thesis. First and foremost, I would like to thank my supervisors Dr. Steve Whyard and Dr. Margaret Docker for their support, guidance and encouragement during my stay in the Department of Biological Sciences. I would also like to recognize the members of both the Whyard/Docker labs for their help and expertise, which helped me greatly. A special thanks to: George Heath for his collection of lamprey samples at the Hammond Bay Biological Station; Cass Erdelyan for his in silico knowledge and expertise; and Craig McFarlane and Erin Spice for their roles in RNA sequencing. I would also like to thank all of the other students and who helped me during field seasons and who helped me finish my work. I thank my advisory committee: Dr. Mark Belmonte and Dr. Mojgan Rastegar for providing me the guidance and advice needed to help complete my thesis. I would also like to thank the Animal Holding staff for their expertise and assistance. I greatly acknowledge the financial assistance provided to me through the Faculty of Science Graduate Studentship. I would lastly like to thank my friends and family for their huge amount of support that they provided me. Especially, I thank Kris Fleet who was encouraging, supportive and always there to help me through. ii Glossary and Abreviations Monophyletic - A group that includes all organisms descended from a single common ancestor Myomere - A block of segmented skeletal muscle fibres Orthologous genes - Two genes separated by a speciation event Paralogous genes - Two genes separated by gene duplication event Paraphyletic - A group that includes the most recent common ancestor, but not all of its descendents Synapomorphy - A derived trait that is shared between two or more taxa MYA - Million years ago RACE - Rapid amplification of cDNA ends ML - Maximum likelihood MP - Maximum parsimony ORF - Open reading frame PG - Paralogous group iii Table of Contents ABSTRACT ............................................................................................................................................. I ACKNOWLEDGMENTS ....................................................................................................................... II GLOSSARY AND ABREVIATIONS ................................................................................................... III TABLE OF CONTENTS ....................................................................................................................... IV LIST OF FIGURES .............................................................................................................................. VII 1. INTRODUCTION ............................................................................................................................... 1 1.1 BIOLOGY OF LAMPREYS ......................................................................................................................... 1 1.1.1 Lamprey Taxonomy ....................................................................................................................... 1 1.1.2 Lamprey Life Cycle ........................................................................................................................ 3 1.1.3 Paired Species ............................................................................................................................... 4 1.1.4 Lamprey Embryonic Development ................................................................................................. 7 1.2 MYOMERES ............................................................................................................................................ 8 1.2.1 Myomeres in Lampreys .................................................................................................................. 8 1.2.2 Myomere Variation ........................................................................................................................ 9 1.3 HOX GENES .......................................................................................................................................... 10 1.3.1 Hox Gene Overview ..................................................................................................................... 10 1.3.2 Hox Expression ............................................................................................................................ 11 1.3.3 Hox Structure ............................................................................................................................... 12 1.3.4 Anterior Hox Genes ..................................................................................................................... 12 1.3.5 Posterior Hox Genes .................................................................................................................... 13 1.3.6 Petromyzon marinus Genome Assembly ...................................................................................... 14 1.4 RESEARCH OBJECTIVES ....................................................................................................................... 15 1.4.1 Analysis of Full Length Coding Region of Posteriorly Expressed Hox Genes ............................ 16 1.4.2 Hox Gene Identification Within P. marinus Genome ................................................................... 16 1.4.3 Identification of Hox10s Homeobox Orthologs and Relationship to Myomere Development ..... 16 1.4.4 Identification of Novel Lamprey Hox Genes in Lamprey Species ................................................ 17 2. MATERIALS AND METHODS....................................................................................................... 18 2.1 POSTERIOR HOX GENE AMPLIFICATION ............................................................................................... 18 2.1.1 Isolation of Lamprey Genomic DNA ........................................................................................... 18 2.1.2 Primer Design .............................................................................................................................. 20 2.1.3 Polymerase Chain Reaction Amplification .................................................................................. 22 2.1.4 Cloning and Verification ............................................................................................................. 23 2.2 - RAPID AMPLIFICATION OF CDNA ENDS ............................................................................................ 24 2.2.1 - Lamprey Handling ..................................................................................................................... 24 2.2.2 - Isolation of Petromyzon marinus (Sea Lamprey) Embryo RNA ............................................... 25 2.2.3 - cDNA Synthesis and PCR Verification ...................................................................................... 26 2.2.4 - Rapid Amplification of cDNA Ends (RACE) ............................................................................. 26 2.3.2 – Next Generation RNA Sequencing ............................................................................................ 30 2.3.3 - Hox Phylogenetic Trees ............................................................................................................. 30 2.4 - PETROMYZON MARINUS HOX GENE VERIFICATION .............................................................................. 31 2.5 - IDENTIFICATION OF LAMPREY HOX GENES IN MULTIPLE LAMPREY GENERA .................................... 32 iv 2.5.1 - Amplification of Exon1 in Posterior Hox Genes ....................................................................... 32 2.5.2 - Hox10 Gene Analysis Across Multiple Lamprey Species .......................................................... 36 2.5.3 - General Hox Gene Screening in Multiple Lamprey Genera ..................................................... 37 3. RESULTS .........................................................................................................................................