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A Genomic Survey of Two Dinotoms

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A Genomic Survey of Two Dinotoms A GENOMIC SURVEY OF TWO DINOTOMS by Behzad Imanian MSc., The University of British Columbia, 2006 BSc., The University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2013 © Behzad Imanian, 2013 Abstract Endosymbiosis has played a major role in shaping eukaryotic cells, their success and diversity. At the base of the eukaryotic tree, an α-proteobacterium endosymbiont in a protoeukaryotic cell was converted into the mitochondrion through its reductive evolution, endosymbiotic gene transfer (EGT) and the development of a protein targeting system to direct the products of the transferred genes to this organelle. Similar events mark the plastid evolution from a cyanobacterium. However, the primary endosymbiosis of plastid, unlike the mitochondrion, was followed by the secondary and tertiary movement of this organelle between eukaryotes through analogous endosymbiotic reduction, EGT and evolution of a protein targeting system and many subsequent independent losses from different eukaryotic lineages. The obligate tertiary diatom endosymbiont in a small group of dinoflagellates called ‘dinotoms’ is exceptional in that it retains most of its ancestral characters including a large nucleus, its own mitochondria, plastids and many other eukaryotic organelles and structures in a large cytoplasm all enclosed in and separated from its dinoflagellate host by a single membrane. This level of conservation of ancestral features in the endosymbiont suggests an early stage of integration. In order to investigate the impacts of endosymbiosis on the organelle genomes and to determine the extent of EGT and the contribution of the host nuclear genome to the proteomes of the organelles, I conducted mass pyrosequencing of the A+T-rich portion of the DNA extracted from two dinotoms, Durinskia baltica and Kryptoperidinium foliaceum, and the SL cDNA library constructed for D. baltica. The plastid and mitochondrial genomes of these two dinotoms were sequenced, and the results indicated that, despite the permanent symbiosis between the host and its endosymbiont in dinotoms and in spite of small variations, the dinotom organelle genomes have changed very ii little from those of free-living diatoms and dinoflagellates. There was also no sign of EGT to the host in D. baltica, suggesting a strict compartmentalization in which the host mitochondria remain reliant on the host nucleus while the endosymbiont organelles, mitochondria and plastids, stay entirely dependent on the endosymbiont nucleus with no genetic exchange between the host and endosymbiont. iii Preface A version of chapter 2 has been published. Imanian B, Pombert J-F, Keeling PJ. 2010. The complete plastid genomes of the two “dinotoms” Durinskia baltica and Kryptoperidinium foliaceum. PloS ONE. 5:e10711. doi: 10.1371/journal.pone.0010711. The project was conceived by PJK, J-FP and I. J-FP worked on the genome assembly, annotation and Sanger sequencing of the plastid genome of K. foliaceum. I conducted the culturing, DNA and RNA extractions, CsCl gradient density centrifugations, DNA amplifications, PCRs, RT-PCRs, Sanger sequencing, final chromosome walking steps, genome assembly, base calling, annotation and finishing of both plastid genomes. J-FP and I analyzed the data. I wrote the first draft. J-FP, PJK and I contributed in editing and writing the final draft. A version of chapter 3 has been published as well. Imanian B, Pombert J-F, Dorrell RG, Burki F, Keeling PJ. 2012. Tertiary endosymbiosis in two dinotoms has generated little change in the mitochondrial genomes of their dinoflagellate hosts and diatom endosymbionts. PLoS ONE. 7:e43763. doi: 10.1371/journal.pone.0043763. PJK and I conceived and designed the experiments. J-FP and RGD helped with the PCRs, RT-PCRs and Sanger sequencing for the endosymbiont mitochondrial genomes. FB prepared the PolyA cDNA library of K. foliaceum. I conducted culturing, DNA and RNA extractions, CsCl gradient density centrifugations, PCRs, RT-PCRs, cDNA amplifications, genome assemblies, base callings, chromosome walking, Sanger sequencing and annotation of all the four mitochondrial genomes. I analyzed the data and wrote the first draft. J-FP, PJK and I contributed in editing and writing the final draft. iv Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi Acknowledgements ......................................................................................................................xv Dedication .................................................................................................................................. xvii Chapter 1: Introduction ...............................................................................................................1 The mitochondrion and plastid endosymbioses .......................................................................... 1 Secondary endosymbioses .......................................................................................................... 4 Tertiary endosymbiosis in dinoflagellates with a cryptophyte or a haptophyte endosymbiont .. 7 Tertiary endosymbiosis in dinotoms ......................................................................................... 10 Research objectives ................................................................................................................... 13 Chapter 2: The plastid genomes of two dinotoms ....................................................................18 Introduction ............................................................................................................................... 18 Results ....................................................................................................................................... 21 Genome structure, gene repertoire, and GC content of the D. baltica and K. foliaceum genomes ................................................................................................................................ 21 Compactness of dinotom plastid genomes ............................................................................ 23 Conserved ordered gene blocks ............................................................................................ 23 Low gene density regions of the Kryptoperidinium foliaceum plastid genome ................... 25 The tyrC gene in K. foliaceum and Heterosigma akashiwo ................................................. 26 v Similarity between the K. foliaceum plastid genome and pCf1 and pCf2 plasmids in Cylindrotheca fusiformis ....................................................................................................... 26 Discussion ................................................................................................................................. 28 The divergent evolution of two tertiary plastid genomes of diatom origin .......................... 28 The ancestral state of the tertiary endosymbiont genome ..................................................... 31 Conclusions ............................................................................................................................... 32 Materials and methods .............................................................................................................. 32 Strains and culture conditions ............................................................................................... 32 DNA and RNA extractions, PCR, RT-PCR, and DNA fractionation and precipitation ....... 32 Genome sequencing .............................................................................................................. 33 Genome annotation and analysis .......................................................................................... 34 Chapter 3: The mitochondrial genomes of the endosymbiont and host in two dinotoms ....39 Introduction ............................................................................................................................... 39 Results ....................................................................................................................................... 42 The endosymbiont mitochondrial genomes of D. baltica and K. foliaceum ........................ 42 General features of the endosymbiont mitochondrial genomes of D. baltica and K. foliaceum ............................................................................................................................... 43 Gene fission .......................................................................................................................... 45 An in-frame insertion ...........................................................................................................
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