The transcriptome represents the entirety of RNA molecules within a cell or tissue at a given time. Recent advances have facilitated the production of large-scale, global interrogations of transcriptomes, finding that genomes are extensively transcribed and contain diverse classes of RNAs (Dinger et al., 2009). Information generated by high-throughput analyses of mRNA transcription start sites (TSSs) such as CAGE (Cap Analysis of Gene Expression) indicate that eukaryotic genomes have complex landscapes of transcription initiation. The TSS is important for the annotation of cis-regulatory sequences, because it provides a link between the mRNA transcript and the promoter. The patterns of TSS distributions observed within mRNA 5' end profiling studies prevent straightforward annotation of putative promoters. To address this challenge, we developed a method to identify- on a genome-wide basis- the putative promoter, which we define by TSS distributions and designate the transcription start region (TSR). We applied a clustering method to identify and annotate TSRs within the budding yeast Saccharomyces cerevisiae using a full-length cDNA dataset (Miura et al., 2006). To validate these TSR annotations, we performed an integrative genomic analysis using multiple datasets. Our method identified TSRs at positions consistent with bona fide promoters in S. cerevisiae. In addition, using 5'RACE, we find overall agreement between computationally-defined TSRs and TSSs identified experimentally. From this analysis, we find that a significant proportion of genes exhibiting alternative promoter usage within sporulation are associated with respiration, suggesting that this is regulated on a condition-specific basis in budding yeast. We further developed our TSS clustering method into a bioinformatics tool called TSRchitect, which identifies and annotates TSRs from large-scale TSS profiling information. TSRchitect is capable of handling both tag and sequence-based TSS information and efficiently computes TSRs from global TSS datasets on a desktop computer. We find support for TSRchitect's annotations in human from a CAGE experiment from the ENCODE (Encyclopedia of DNA Elements) project. Finally, we use TSRchitect to identify TSRs from the transcriptomes of diverse eukaryotes. We investigated the conservation of TSRs among orthologous genes. We frequently identify multiple TSRs for a given gene, suggesting that alternative promoter usage is widespread. Overall, using TSS profiling data derived from separate tissues within mouse and human, we find that the positions of TSRs are relatively stable across tissues surveyed; however, a small fraction of genes exhibit tissue-specific differences in TSR use. As transcriptome profiling information continues to be generated at an rapid pace, computational approaches are increasingly important. It is anticipated that the method and approach we describe within this dissertation will contribute to an improved of gene regulation and promoter architecture in eukaryotes.

Understanding the composition, evolution, and function of the cotton (Gossypium) genome is complicated by the joint presence of two genomes in its nucleus (AT and DT genomes). Specifically, read-mapping (a fundamental part of next-generation sequence analysis) cannot adequately differentiate reads as belonging to one genome or the other. These two genomes were derived from progenitor A-genome and D-genome diploids involved in ancestral allopolyploidization. To better understand the allopolyploid genome, we developed PolyCat to categorize reads according to their genome of origin based on homoeo-SNPs that differentiate the two genomes. We re-sequenced the genomes of extant diploid relatives of tetraploid cotton that contain the A1 (Gossypium herbaceum), A2 (Gossypium arboreum), or D5 (Gossypium raimondii) genomes. We identified 24 million SNPs between the A-diploid and D-diploid genomes. These analyses facilitated the construction of a robust index of conserved SNPs between the A-genomes and D-genomes at all detected polymorphic loci. This index can be used by PolyCat to assign reads from an allotetraploid to its genome-of-origin. Continued characterization of the Gossypium genomes will further enhance our ability to manipulate fiber and agronomic production of cotton. We re-sequenced 34 allotetraploid cotton lines, representing all 7 tetraploid cotton species. The analysis of these genomes-using PolyCat and PolyDog-provides us with the beginnings of a HapMap-like resource for cotton species, including indices of both homoeo-SNPs and allele-SNPs. With this information, we explore the phylogenetic relationships among cotton species, including the newly characterized species G. ekmanianum and G. stephensii. We examine gene conversion both recent and ancient, discovering that recent gene conversion is extremely rare, and ancient gene conversion is far less extensive than previously believed, with many previously identified conversion events being more probably due to autapamorphic SNPs in the descent of diploid relatives. In order to carry out these experiments, many tools for next-generation sequence analysis were developed. These tools, along with PolyCat and PolyDog, comprise the tool suite BamBam.

Given the high salinity, prevailing annual high temperatures, and ultra-oligotrophic conditions in the Red Sea isolation and characterization of important microbial groups thriving in this environment is important in understanding the ecological significance and metabolic capabilities of these communities. By using a high-­throughput cultivation technique in natural seawater amended with minute amounts of nutrients, members of the rare biosphere (PS1), methylotrophic Betaproteobacteria (OM43), and the ubiquitous and abundant SAR11 group (Pelagibacterales), were isolated in pure culture. Phylogenetic analyses of Red Sea isolates along with comparative genomics with close representatives from disparate provinces revealed ecotypes and genomic differentiation among the groups. Firstly, the PS1 alphaproteobacterial clade was found to be present in very low abundance in several metagenomic datasets form divergent environments. While strain RS24 (Red Sea) harbored genomic islands involved in polymer degradation, IMCC14465 (East (Japan) Sea) contained unique genes for degradation of aromatic compounds. Secondly, methylotrophic OM43 bacteria from the Red Sea (F5, G12 and H7) showed higher similarities with KB13 isolate from Hawaii, forming a ‘H-­RS’ (Hawaii-­Red Sea) cluster separate from HTCC2181 (Oregon isolate). HTCC2181 members were shown to prevail in cold, productive coastal environments and had an nqrA-­F system for energy generation by sodium motive force. On the contrary, H-­RS cluster members may be better adapted to warm and oligotrophic environments, and seem to generate energy by using a proton-­translocating NADH:Quinone oxidoreductase (complex I; nuoA-­N subunits). Moreover, F5, G12, and H7 had unique proteins related to resistance to UV, temperature and salinity, in addition to a heavy metal ‘resistance island’ as adaptive traits to cope with the environmental conditions in the Red Sea. Finally, description of the Red Sea Pelagibacterales isolates from the Ia (RS39) and Ib (RS40) subgroups, principally revealed unique putative systems for iron uptake and myo-inositol utilization in RS39, and a potential phosphonates biosynthetic pathway present in RS40. The findings presented here reflect how environments influence the genomic repertoire of microbial communities and shows novel metabolisms and putative pathways as unique adaptive qualities in diverse microbes encompassing from rare to predominant bacterioplankton groups from the Red Sea.

Teleost fishes share a duplication of their entire genomes. We report here on a computational survey of structured non-coding RNAs (ncRNAs) in teleost genomes, focusing on the fate of fish-specific duplicates. As in other metazoan groups, we find evidence of a large number (11,543) of structured RNAs, most of which (~86%) are clade-specific or evolve so fast that their tetrapod homologs cannot be detected. In surprising contrast to protein-coding genes, the fish-specific genome duplication did not lead to a large number of paralogous ncRNAs: only 188 candidates, mostly microRNAs, appear in a larger copy number in teleosts than in tetrapods, suggesting that large-scale gene duplications do not play a major role in the expansion of the vertebrate ncRNA inventory.

Genome-wide multiple sequence alignments (MSAs) are a necessary prerequisite for an increasingly diverse collection of comparative genomic approaches. Here we present a versatile method that generates high-quality MSAs for non-protein-coding sequences. The NcDNAlign pipeline combines pairwise BLAST alignments to create initial MSAs, which are then locally improved and trimmed. The program is optimized for speed and hence is particulary well-suited to pilot studies. We demonstrate the practical use of NcDNAlign in three case studies: the search for ncRNAs in gammaproteobacteria and the analysis of conserved noncoding DNA in nematodes and teleost fish, in the latter case focusing on the fate of duplicated ultra-conserved regions. Compared to the currently widely used genome-wide alignment program TBA, our program results in a 20- to 30-fold reduction of CPU time necessary to generate gammaproteobacterial alignments. A showcase application of bacterial ncRNA prediction based on alignments of both algorithms results in similar sensitivity, false discovery rates, and up to 100 putatively novel ncRNA structures. Similar findings hold for our application of NcDNAlign to the identification of ultra-conserved regions in nematodes and teleosts. Both approaches yield conserved sequences of unknown function, result in novel evolutionary insights into conservation patterns among these genomes, and manifest the benefits of an efficient and reliable genome-wide alignment package. The software is available under the GNU Public License at http://www.bioinf.uni-leipzig.de/Software/NcDNAlign/.

No description available.

Chez les organismes sexués, des suppressions de recombinaison peuvent évoluer dans certaines régions génomiques pour conserver des combinaisons d’allèles bénéfiques, ce qui aboutit à la transmission de plusieurs gènes en un seul locus, alors appelé « supergène ». Les supergènes déterminent des phénotypes complexes, comme l’identité sexuelle chez les organismes qui ont des chromosomes sexuels. Sur certains chromosomes sexuels, la région sans recombinaison s’est étendue plusieurs fois successivement, produisant des « strates évolutives ». Il est communément admis que ces strates évolutives sont issues de liaisons successives de gènes sexuellement antagonistes (qui ont des allèles bénéfiques à un sexe mais délétère à l’autre) à la région qui détermine le sexe, mais peu de preuves empiriques soutiennent cette hypothèse. Les champignons constituent des modèles intéressants pour étudier les causes évolutives des suppressions de recombinaison parce qu’ils peuvent avoir des chromosomes de types sexuels non recombinants sans être associés à des fonctions mâles ou femelles. Dans cette thèse, nous avons étudié l’évolution de la suppression de recombinaison sur les chromosomes de type sexuel chez les champignons castrateurs de plantes du genre Microbotryum. Chez les champignons Microbotryum, les croisements ne sont possibles qu’entre des gamètes qui ont des allèles distincts aux deux locus de types sexuels. Nous avons montré que les suppressions de recombinaison ont évolué plusieurs fois indépendamment pour lier les deux locus de types sexuels, depuis l’état ancestral avec les locus de types sexuels situés sur deux chromosomes différents. La suppression de recombinaison a soit lié les locus de types sexuels à leur centromère respectif, ou a lié les locus de types sexuels entre eux après que des réarrangements chromosomiques, différents dans les différentes espèces, les aient amenés sur le même chromosome. Les deux sortes de suppression de recombinaison sont bénéfiques sous le mode de reproduction par auto-fécondation intra-tétrade de Microbotryum, parce qu’ils augmentent le taux de compatibilité entre gamètes. Les suppressions de recombinaison ont donc évolué plusieurs fois indépendamment via des chemins évolutifs et des changements génomiques différents, ce qui renseigne sur la répétabilité de l’évolution. De plus, nous avons révélé l’existence de strates évolutives sur les chromosomes de type sexuels de plusieurs espèces de Microbotryum, ce qui remet en cause le rôle de l’antagonisme sexuel dans la formation de strates évolutives, les types sexuels n’étant pas associés à des fonctions mâles / femelles. Des études précédentes ont rapporté peu de différences phénotypiques associées aux types sexuels, ce qui rend peu probable qu’une sélection antagoniste existe entre types sexuels sur de nombreux gènes (l’existence de gènes avec des allèles bénéfiques à un type sexuel mais délétère à l’autre). Certains gènes situés dans les régions non-recombinantes des chromosomes de types sexuels étaient différentiellement exprimés entre types sexuels, mais nos analyses suggèrent qu’un tel différentiel d’expression peut être dû à la dégénérescence. En effet, des mutations délétères s’accumulent dans les régions non-recombinantes, ce qui peut modifier l’expression des gènes ou les séquences protéiques. Nous avons donc conclu que la sélection antagoniste ne peut pas expliquer la formation des strates évolutives chez les champignons Microbotryum. Par conséquent, des mécanismes alternatifs doivent être considérés pour expliquer l’extension progressive des régions non-recombinantes, et ces mécanismes pourraient aussi générer des strates évolutives sur les chromosomes sexuels. Ces travaux incitent de futures études à d’une part identifier d’autres strates évolutives qui ne sont pas associées à des fonctions mâles/femelles, et d’autre part à identifier leurs causes évolutives et leurs conséquences en termes de dégénérescence. / In sexual organisms, recombination suppression can evolve in specific genomic regions to protect beneficial allelic combinations, resulting in the transmission of multiple genes as a single locus, which is called a supergene. Supergenes determine complex phenotypes, such as gender in organisms with sex chromosomes. Some sex chromosomes display successive steps of recombination suppression known as “evolutionary strata”, which are commonly thought to result from the successive linkage of sexually antagonistic genes (i.e. alleles beneficial to one sex but detrimental to the other) to the sex-determining region. There has however been little empirical evidence supporting this hypothesis. Fungi constitute interesting models for studying the evolutionary causes of recombination suppression in sex-related chromosomes, as they can display non-recombining mating-type chromosomes not associated with male/female functions. Here, we studied the evolution of recombination suppression on mating-type chromosomes in the Microbotryum plant-castrating fungi using comparative genomic approaches. In Microbotryum fungi, mating occurs between gametes with distinct alleles at the two mating-type loci, as is typical of basidiomycete fungi. We showed that recombination suppression evolved multiple times independently to link the two mating-type loci from an ancestral state with mating-type loci on two distinct chromosomes. Recombination suppression either linked the mating-type genes to their respective centromere or linked mating-type loci after they were brought onto the same chromosome through genomic rearrangements that differed between species. Both types of linkage are beneficial under the intra-tetrad mating system of Microbotryum fungi as they increase the odds of gamete compatibility. Recombination suppression thus evolved multiple times through distinct evolutionary pathways and distinct genomic changes, which give insights about the repeatability and predictability of evolution. We also reported the existence of independent evolutionary strata on the mating-type chromosomes of several Microbotryum species, which questions the role of sexual antagonism in the stepwise extension of non-recombining regions because mating-types are not associated with male/female functions. Previous studies reported little phenotypic differences associated to mating-types, rending unlikely any antagonistic selection between mating types (i.e. “mating-type antagonism”, with genes having alleles beneficial to one mating-type but detrimental to the other). The genes located in non-recombining regions on the mating-type chromosomes can be differentially expressed between mating types, but our analyses indicated that such differential expression was more likely to result from genomic degeneration than from mating-type antagonism. Deleterious mutations are indeed known to accumulate in non-recombining regions resulting in modifications of gene expression or of protein sequence. We concluded that antagonistic selection cannot explain the formation of evolutionary strata in Microbotryum fungi. Alternative mechanisms must be therefore be considered to explain the stepwise expansion of non-recombining regions, and they could also be important on sex chromosomes. This work thus prompts for future studies to identify further evolutionary strata not associated with male/female functions as well as to elucidate their evolutionary causes and consequences in terms of genomic degeneration.

The Central Arctic Ocean is a repository of rich and diverse biota, whose major portion is one of the most important drivers of global biogeochemical cycles, including carbon cycling. In this study, the functional potential of the microbiota to fix carbon with alternative carbon fixation pathways were investigated along with their chemolithotrophic characteristics. Samples from two expeditions MOSAiC & SAS-Oden (2019-2021) resulted in metagenomic data consisting of about 1200 mOTUs (metagenomic Operational Taxonomic Units). Kofamscan based annotation explained by KEGG pathways database was analysed to explore prevalence of the alternative Carbon Fixation pathways across different taxa. From the six carbon fixation pathways, three were consolidated for their presence (rTCA, DC-HP, HP-HP). In order to explain the other metabolic processes that these organisms employ to survive, a functional annotation tool for metabolic pathways was used. that Reductive Tricarboxylic acid cycle pathway was found to be most present and observed in 5 out of 6 mOTUs selected from filtering the dataset. The taxa include bacterial phyla Proteobacteria, Actinobacteria, Chloroflexota and Marinisomatota and archaeal phyla Thermoplasmota. However, for the other pathways and less studied organisms less resolution were observed across the dataset for the presence of other pathways. These CFPs found were also supported by oxidation of inorganic compounds with high redox potentials. This study provides a glimpse of the metabolic potential of the Central Arctic Ocean microbiota, shines light on the importance of understanding and unravelling the intricacies of this rich and diverse environment.

Spiders are one of the most diverse clades of multicellular life on earth, and their success has been attributed to several key genomic features, such as weaving silk, deploying various forms of venom, and being able to metabolize many types of prey. We have created a database of whole genome information from ten spider species from across major branches of the spider tree of life to help contextualize the genomic events occurring along the evolutionary history of the spider clade. Genes from these ten spiders were clustered into gene families which were used to detect duplications, losses, and evidence of selection (dN/dS) along the branches of these spiders' phylogeny. These results also allow us to weigh in on a contentious taxonomic placement for one of these spiders, the Uloborus diversus, which we found to be paraphyletic with other orb-web weaving spiders. One future applications of this database will involve phenotype reconstruction, with the goal of forging a genotype to phenotype map. / Biology

The U.S. aquaculture industry has become well established in the last three decades, and channel catfish aquaculture is the most significant component of this industry. Virulent Aeromonas hydrophila has been a serious disease problem since 2009 in the U.S. catfish aquaculture, and Aeromonas veronii and Edwardsiella piscicida are emerging pathogens of catfish. Therefore, this study aims to address fundamental questions on virulence mechanisms of these three fish pathogens, which I expect to support the development of control measures for preventing these diseases. In this study, E. piscicida and virulent Aeromonas hydrophila (vAh) genomes were sequenced, and comparative analyses were conducted using the genome sequences. Average nucleotide identity (ANI) calculations showed that E. piscicida strains share high sequence identity, yet they are from diverse host species and geographic regions. vAh isolates share very high sequence identity, while the other A. hydrophila genomes are more distantly related to this clonal group. We applied several comparative genomics approaches to evaluate E. piscicida genomes and E. ictaluri genomes, providing valuable information about unique and shared features of these two important pathogens in the Edwardsiella genus. Comprehensive secretion system analysis of 55 A. hydrophila genomes and deletion of tssD and tssI core elements of T6SS from vAh isolate ML09-119 has provided new knowledge. We sequenced the genome of virulent Aeromonas veronii strain ML09-123 from catfish indicated that it was highly similar to an A. veronii strain from China. Evaluation of all 41 A. veronii genomes available in the National Center for Biotechnology Information (NCBI) provides a base platform to investigate in detail the molecular mechanism of A. veronii biology and virulence. Lastly, we constructed deletion mutants vAhΔsia, vAhΔent, vAhΔcol, vAhΔhfq1, vAhΔhfq2, and vAhΔhfq1Δhfq2 to determine roles of A. hydrophila secreted proteins and regulatory proteins on virulence in catfish. Results showed that sialidase (vAhΔsia) and enterotoxin (vAhΔent) mutants were significantly attenuated.