COMPARATIVE GENOMICS OF ENDOSYMBIOTICALLY-DERIVED ORGANELLES IN CRYPTOPHYTE ALGAE by Christa E. Moore Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Dalhousie University Halifax, Nova Scotia June 2013 © Copyright by Christa E. Moore, 2013 DALHOUSIE UNIVERSITY DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY The undersigned hereby certify that they have read and recommend to the Faculty of Graduate Studies for acceptance a thesis entitled “COMPARATIVE GENOMICS OF ENDOSYMBIOTICALLY-DERIVED ORGANELLES IN CRYPTOPHYTE ALGAE” by Christa E. Moore in partial fulfilment of the requirements for the degree of Doctor of Philosophy. Dated: June 6, 2013 External Examiner: _________________________________ Research Supervisor: _________________________________ Examining Committee: _________________________________ _________________________________ Departmental Representative: _________________________________ ii DALHOUSIE UNIVERSITY DATE: June 6, 2013 AUTHOR: Christa E. Moore TITLE: COMPARATIVE GENOMICS OF ENDOSYBIOTICALLY-DERIVED ORGANELLES IN CRYPTOPHYTE ALGAE DEPARTMENT OR SCHOOL: Department of Biochemistry and Molecular Biology DEGREE: Ph.D. CONVOCATION: October YEAR: 2013 Permission is herewith granted to Dalhousie University to circulate and to have copied for non-commercial purposes, at its discretion, the above title upon the request of individuals or institutions. I understand that my thesis will be electronically available to the public. The author reserves other publication rights, and neither the thesis nor extensive extracts from it may be printed or otherwise reproduced without the author’s written permission. The author attests that permission has been obtained for the use of any copyrighted material appearing in the thesis (other than the brief excerpts requiring only proper acknowledgement in scholarly writing), and that all such use is clearly acknowledged. _______________________________ Signature of Author iii DEDICATION PAGE This thesis is dedicated to my husband, my loving family, and to my supervisor for pushing me to see just how far I could go. While I’ve enjoyed the ride, I’m happy to say that I’ve reached the end and am ready for the next adventure. iv TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... vii LIST OF FIGURES ....................................................................................................... viii ABSTRACT ....................................................................................................................... x LIST OF ABBREVIATIONS USED .............................................................................. xi ACKNOWLEDGEMENTS .......................................................................................... xiii CHAPTER 1 INTRODUCTION .................................................................................. 1 CHAPTER 2 NUCLEOMORPH COMPARATIVE GENOMICS ........................ 13 2.1 CRYPTOPHYTE NUCLEOMORPH GENOME BIOLOGY AND EVOLUTION ................. 13 2.2 METHODS ........................................................................................................... 16 2.2.1 Cell Culture and Pulsed-Field Gel Electrophoresis ................................... 16 2.2.2 Nucleomorph DNA Isolation ...................................................................... 18 2.2.3 DNA Sequencing and Genome Assembly ................................................... 19 2.2.4 RNA Extraction and Transcriptome Sequencing ........................................ 21 2.2.5 Genome Annotation .................................................................................... 22 2.2.6 Reverse-Transcription PCR and Intron Verification .................................. 23 2.2.7 Significance Testing and Data Deposition ................................................. 24 2.3 RESULTS AND DISCUSSION ................................................................................. 25 2.3.1 Genome Architecture and Size Variation ................................................... 25 2.3.2 Proteins of Known Function ....................................................................... 35 2.3.3 Proteins of Unknown Function ................................................................... 40 2.3.4 Spliceosomal Introns .................................................................................. 42 2.3.5 Synteny and Recombination ........................................................................ 48 2.4 CHAPTER SUMMARY ........................................................................................... 52 CHAPTER 3 PLASTID COMPARATIVE GENOMICS ........................................ 55 3.1 CRYPTOPHYTE PLASTID GENOME BIOLOGY AND EVOLUTION ............................. 55 3.2 METHODS ............................................................................................................ 59 3.2.1 Genome Assembly and Annotation ............................................................. 59 v 3.2.2 Phylogenetic Analyses ................................................................................ 60 3.3 RESULTS AND DISCUSSION ................................................................................. 61 3.3.1 Architectural Features of Cryptophyte Plastid Genomes ........................... 61 3.3.2 Pseudogenes in Cryptophyte Plastid Genomes .......................................... 66 3.3.3 Cryptophyte and Red Algal Group II IEPs ................................................. 68 3.3.4 Synteny in Cryptophyte and Red Algal Plastid Genomes ........................... 75 3.3.5 Gene Content of ‘Red’ Plastids .................................................................. 81 3.4 CHAPTER SUMMARY ........................................................................................... 85 CHAPTER 4 ORIGIN OF THE CRYPTOPHYTE PLASTID ............................... 88 4.1 INTRODUCTION ................................................................................................... 88 4.2 METHODS ........................................................................................................... 92 4.3 RESULTS AND DISCUSSION ................................................................................. 95 4.3.1 Phylogeny of Cryptophyte and Red Algal Plastid Genes ........................... 96 4.3.2 Nucleomorph and Red Algal Nuclear Gene Phylogeny ............................. 98 4.4 CHAPTER SUMMARY ......................................................................................... 100 CHAPTER 5 CONCLUSIONS ................................................................................. 103 REFERENCES .............................................................................................................. 107 APPENDIX A LIST OF PRIMERS USED FOR PROBES .................................... 117 APPENDIX B CONSERVED CRYPTOPHYTE NUCLEOMORPH GENES ..... 118 APPENDIX C COPYRIGHT PERMISSION ........................................................... 126 vi LIST OF TABLES Table 2.1 Comparison of cryptophyte nucleomorph genome features ............................ 31 Table 2.2 Comparison of average intergenic spacer size for different gene orientations ..................................................................................................... 34 Table 2.3 Spliceosomal intron-containing genes and intron sequences in the nucleomorph genome of Chroomonas mesostigmatica .................................. 44 Table 3.1 Comparison of the main features of cryptophyte plastid genomes .................. 63 Table 3.2 Presence/absence of protein genes and tRNAs in the plastid genomes of photosynthetic cryptophyte species ................................................................ 65 Table 3.3 Presence/absence of pseudogenes in cryptophyte and red algal plastid genomes .......................................................................................................... 67 Table 3.4 Distribution of group II introns in red algal and cryptophyte plastid genomes .......................................................................................................... 69 Table 4.1 List of taxa used for phylogenetic analyses ..................................................... 93 vii LIST OF FIGURES Figure 1.1 Primary and secondary endosymbiosis ............................................................ 3 Figure 1.2 Nucleomorph-containing algae ........................................................................ 5 Figure 2.1 Chroomonas mesostigmatica CCMP1168 cultures ........................................ 16 Figure 2.2 Schematic of the various genetic compartments in cryptophyte cells ........... 17 Figure 2.3 Separation of Chroomonas mesostigmatica CCMP1168 total DNA by Hoechst dye-CsCl density gradient centrifugation and Southern blot hybridizations ............................................................................................... 18 Figure 2.4 Chroomonas mesostigmatica nucleomorph chromosomes separated by pulsed-field gel electrophoresis and contig mapping using Southern blot hybridizations ............................................................................................... 20 Figure 2.5 Verification of spliceosomal intron removal .................................................. 24 Figure 2.6 Chroomonas mesostigmatica nucleomorph genome map .............................. 27 Figure 2.7 Percentage of all cryptophyte nucleomorph open reading