Theses and Dissertations from UMD
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Item Dinoflagellate Genomic Organization and Phylogenetic Marker Discovery Utilizing Deep Sequencing Data(2016) Mendez, Gregory Scott; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Dinoflagellates possess large genomes in which most genes are present in many copies. This has made studies of their genomic organization and phylogenetics challenging. Recent advances in sequencing technology have made deep sequencing of dinoflagellate transcriptomes feasible. This dissertation investigates the genomic organization of dinoflagellates to better understand the challenges of assembling dinoflagellate transcriptomic and genomic data from short read sequencing methods, and develops new techniques that utilize deep sequencing data to identify orthologous genes across a diverse set of taxa. To better understand the genomic organization of dinoflagellates, a genomic cosmid clone of the tandemly repeated gene Alchohol Dehydrogenase (AHD) was sequenced and analyzed. The organization of this clone was found to be counter to prevailing hypotheses of genomic organization in dinoflagellates. Further, a new non-canonical splicing motif was described that could greatly improve the automated modeling and annotation of genomic data. A custom phylogenetic marker discovery pipeline, incorporating methods that leverage the statistical power of large data sets was written. A case study on Stramenopiles was undertaken to test the utility in resolving relationships between known groups as well as the phylogenetic affinity of seven unknown taxa. The pipeline generated a set of 373 genes useful as phylogenetic markers that successfully resolved relationships among the major groups of Stramenopiles, and placed all unknown taxa on the tree with strong bootstrap support. This pipeline was then used to discover 668 genes useful as phylogenetic markers in dinoflagellates. Phylogenetic analysis of 58 dinoflagellates, using this set of markers, produced a phylogeny with good support of all branches. The Suessiales were found to be sister to the Peridinales. The Prorocentrales formed a monophyletic group with the Dinophysiales that was sister to the Gonyaulacales. The Gymnodinales was found to be paraphyletic, forming three monophyletic groups. While this pipeline was used to find phylogenetic markers, it will likely also be useful for finding orthologs of interest for other purposes, for the discovery of horizontally transferred genes, and for the separation of sequences in metagenomic data sets.Item Inferring dinoflagellate genome structure, function, and evolution from short-read high-throughput mRNA-Seq(2015) Gibbons, Theodore Robert; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Dinoflagellates are a diverse and ancient lineage of globally abundant algae that have adapted to fill a diverse array of important ecological roles. Despite their importance, dinoflagellate genomes remain relatively poorly understood because of their enormous size. It is suspected that dinoflagellate genomes have expanded through rampant gene duplication, possibly using a lineage-specific mechanism that involves reinsertion of mature transcripts back into the genome, and that may rely on spliced leader trans-splicing for reactivation and processing of recycled transcripts. Draft genomes have recently been published for two extremely small endosymbiotic species. These genomes confirm expansion of nearly 10k gene families, relative to other eukaryotes. In the more complete genome, evidence for transcript recycling based on relict spliced leader sequences was found in over 5,500 genes. Genomic efforts in larger dinoflagellates have focused instead on transcriptome sequencing, but transcriptomes assembled from short-read HTS data contain very little evidence for rampant gene duplication, or for trans-splicing. I have shown that apparent disagreement with hypotheses related to ubiquitous trans-splicing and widespread gene duplication are the result of technological limitations. By leveraging the statistical power of high-throughput sequencing, I found that spliced leader suffixes as short as six nucleotides are sufficient for positive identification. I also found that isoform sequences from families of conserved paralogs are systematically collapsed during assembly, but that many of these consensus sequences can be identified using a custom SNP-calling procedure that can be combined with traditional clustering based on pairwise sequence alignment to obtain a more complete picture of gene duplication in dinoflagellates. Efficient, automated homology detection based on pairwise sequence alignment is an equally challenging problem for which there is much room for improvement. I explored alternative metrics for scoring alignments between sequences using a popular procedure based on BLAST and Markov clustering, and showed that simplified metrics perform as well or better than more popular alternatives. I also found that Markov clustering of protein sequences suffers from a serious false positive problem when compared against manual curation, suggesting that it is more appropriate for pre-clustering of very large data sets than as a complete clustering solution.Item Genomic studies of the evolution of haptophytes and dinoflagellates with emphasis on the chromalveolate hypothesis(2006-06-08) Sanchez Puerta, Maria Virginia; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)All photosynthetic eukaryotes rely, partially or totally, on their plastids to live. The plastids, which ultimately are highly modified cyanobacteria, were acquired through a process of primary, secondary, or tertiary endosymbiosis. Four photosynthetic lineages, including haptophytes, dinoflagellates, cryptophytes, and heterokonts, contain secondary plastids with chlorophyll c as a main photosynthetic pigment. These four lineages were grouped together, along with their heterotrophic relatives, on the basis of their pigmentation and called chromalveolates by Cavalier-Smith. However, the phylogenetic relationships among these algae are unknown and the chromalveolate hypothesis remains very controversial. This study focuses on increasing the amount of genomic data from a poorly studied chromalveolate lineage, the haptophytes, and understanding plastid evolution in chromalveolates. Both the chloroplast and mitochondrial genomes of the haptophyte <em>Emiliania huxleyi</em> were sequenced and examined to describe basic genomic properties, as well as perform comparative studies. Phylogenetic analyses, including data acquired from haptophytes, support a monophyletic chl c containing plastid clade derived from the red algae, after the divergence of Cyanidiales, with the cryptophyte plastid basal or sister to the haptophyte plastid. In addition, phylogenetic analyses using mitochondrial data suggest a relationship of haptophytes and cryptophytes. The chromalveolate clade as a whole is not recovered nor rejected by the data. Analysis of an EST project from the heterotrophic dinoflagellate <em>Crypthecodinium cohnii</em> indicates that <em>C. cohnii</em> is not only derived from a photosynthetic ancestor, but very likely retains a non-photosynthetic plastid. Analyses of putative gene function suggest that heme biosynthesis, non-mevalonate isoprenoid biosynthesis, amino-acid metabolism, and Fe-S cluster assembly may occur in the plastid. These observations are also consistent with the chromalveolate hypothesis, which proposes that several major groups of eukaryotes, including alveolates, haptophytes, cryptophytes, and heterokonts, may form a monophyletic group with a photosynthetic common ancestor, and that nonphotosynthetic members are secondarily so.Item SWIMMING FOR SULFUR: ANALYSIS OF THE ROSEOBACTER-DINOFLAGELLATE INTERACTION(2005-01-06) Miller, Todd Rex; Belas, Robert; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Marine algae are some of the most productive organisms on earth, and their survival is dependent upon a diverse community of bacteria that consume algal products. The identity of these bacteria and mechanisms used to interact with their algal partner are not well understood. Recently it has been shown that -Proteobacteria of the Roseobacter clade are the primary consumers of the algal osmolyte, dimethylsulfoniopropionate (DMSP). In addition, their production and activity is highly correlated with DMSP producing algal blooms, especially those containing dinoflagellates. To understand more about this relationship, I have studied Roseobacter-dinoflagellate interactions in laboratory cultures of Pfiesteria dinoflagellates, a ubiquitous group of estuarine, heterotrophic dinoflagellates. The results show that cultures of P. piscicida and a similar dinoflagellate, Cryptoperidiniopsis sp., harbor a robust DMSP degrading bacterial community that contains members of the Roseobacter clade. One of these bacteria, Silicibacter sp. TM1040 degrades DMSP by demethylation producing 3-methymercaptopropionate (MMPA). Interestingly, this bacterium senses and actively moves toward P. piscicida cells. It is highly chemotactic toward amino acids, especially methionine, and DMSP metabolites, including DMSP and MMPA. Chemotaxis of TM1040 toward P. piscicida cells is mediated in part by the presence of these compounds in the dinoflagellates. Using a fluorescent tracer dye, this bacterium was found attached and/or within P. piscicida cells. The apparent intracellular occurence of Silicibacter sp. TM1040 requires both flagella and motility since mutants lacking motility and/or flagella are not found within the dinoflagellate, although they can be found attached. The presence of Silicibacter sp. TM1040 in axenic dinoflagellate cultures enhances dinoflagellate growth, a process that does not require the bacteria to be intracellular. The genome sequence of Silicibacter sp. TM1040 indicates that this bacterium contains a large number (20) of chemoreceptors and a full complement of flagellar and other chemotaxis genes. In addition, this bacterium contains all of the genes necessary to produce a type IV secretion system similar to the vir pilus of Agrobacterium tumefaciens. Taken together, the data suggest that Silicibacter sp. TM1040 is an attached and/or intracellular symbiont of P. piscicida. The significance of this study to microbial and algal bloom ecology is discussed.