京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22354号 / 医博第4595号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 武藤 学, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Comparing genomes in a computationally efficient manner is a difficult problem. Methods that provide the highest resolution are too inefficient and methods that are efficient are too low resolution. In this thesis, we show that the Colored de Bruijn Graph (CdBG) is a suitable method for comparing genomes because it is efficient while maintaining a useful amount of resolution. To illustrate the usefulness of the CdBG, the phylogenetic tree for 12 species in the Drosophila genus is reconstructed using pseudo-homologous regions of the genome contained in the CdBG.

Many angiosperms have undergone some series of polyploidization events over the course of their evolutionary history. In these genomes, especially those resulting from multiple autopolyploidization, it may be relatively easy to recognize all the sets of n homeologous chromosomes, but it is much harder, if not impossible, to partition these chromosomes into n subgenomes, each representing one distinct genomic component of chromosomes making up the original polyploid. Thus, if we wish to infer the polyploidization history of the genome, we could make use of all the gene trees inferred from the genes in one set of homeologous chromosomes to construct a consensus tree, but there is no evident way of combining the trees from the different sets because we have no labelling of the chromosomes that is known to be consistent across these sets. We suggest here that lacking a consistent leaf-labelling, the topological structure of the trees may display sufficient resemblance so that a higher level consensus could be revealing of evolutionary history. This would be especially true of the peripheral structures of the tree, likely representing events that occurred more recently and have thus been less obscured by subsequent evolutionary processes. Here, we present a statistical test to assess whether the subgenomes in a polyploid genome could have been added one at a time. The null hypothesis is that the accumulation of chromosomes follows a stochastic process in which transition from one generation to the next is through randomly choosing an edge, and then subdividing this edge in order to link the new internal vertex to a new external vertex. We analyze the probability distributions of a number of peripheral tree substructures, namely leaf- or terminal-pairs, triples and quadruples, arising from this stochastic process, in terms of some exact recurrences. We propose some conjectures regarding the asymptotic behaviours of these distributions. Applying our analysis to a sugarcane genome, we demonstrate that it is unlikely that the accumulation of subgenomes has occurred one at a time in this genome.

Aspergillus fumigatus is a filamentous saprophytic mold that is found abundantly in the biosphere. A. fumigatus is also an airborne human pathogen and is considered the major cause of aspergillosis, infections caused by inhalation of conidia. In immunocompetent individuals, the spores rarely cause any harm as they are cleared by innate pulmonary defences; however, in immunocompromised patients, the host immune system can fail to clear the inhaled conidia and aspergillosis may develop. Indeed, aspergillosis represents a major cause of morbidity and mortality in these populations. Aspergillosis is commonly treated using triazole and amphotericin B (AMB) antifungal agents. However, the increasing prevalence of triazole resistant strains and emergence of AMB resistance has become a challenge in treatment. To further expand our knowledge on the mechanisms of antifungal resistance in the species, we tested previously known or associated genes for antifungal resistance as well as investigated novel mechanisms via multiple genome-wide association studies (GWAS), which used a total of 211 genomes from A. fumigatus strains in 12 countries. Our results identified many novel mutations related to triazole and AMB resistance. Specifically, using stepwise GWAS analyses, we identified 6 and 18 missense variants to be significantly associated with itraconazole and voriconazole resistance, respectively. A linkage disequilibrium analysis identified six additional missense variants associated with triazole resistance, with two of these six being consistently associated with pan-azole resistance across subsets of samples. Furthermore, examination of known mutation sites and genes overexpressed with triazole exposure found a total of 65 SNPs implicated in triazole resistance. For the AMB study, we identified a total of 34 mutations associated with AMB tolerance using a GWAS. Subsequent analysis with 143 progeny strains, generated from a laboratory cross and genotyped with PCR-RFLP, identified epistatic interactions between five of these SNP sites that impacted growth in different concentrations of AMB. With the expanding immunocompromised population and increasing frequency of antifungal resistance, our results will help in investigating novel resistance mechanisms in A. fumigatus and in expanding the molecular diagnostic toolset in resistance screening, to enable rapid and accurate diagnosis and treatment decision-making. / Thesis / Master of Science (MSc)

Chytridiomycosis is a fungal skin disease in amphibians that is primarily caused by Batrachochytrium dendrobatidis (Bd). We analyzed whole genome sequences of 40 bacterial isolates that had been previously cultured from the skin of four amphibian species from Virginia, USA, and tested for their ability to inhibit Bd growth via an in vitro challenge assay. These 40 isolates spanned 11 families and 13 genera. The aim of this study was to identify genomic differences among the amphibian skin bacterial isolates and generate hypotheses about possible differences that could contribute to variation in their ability to inhibit the growth of Bd. We identified sixty-five gene families that were present in all 40 isolates. We also looked for the presence of biosynthetic gene clusters. While this set of isolates contained a wide variety of biosynthetic gene clusters, the two most abundant clusters with potential anti-fungal activity were siderophores (N=17) and Type III polyketide synthases (N=20). We then analyzed the isolates belonging to the phylum Proteobacteria in more detail. We identified 197 gene families that were present in all 22 Proteobacteria. We examined various subsets of the Proteobacteria for genes for specific compounds with known activity against fungi, including chitinase and violacein. We identified a difference in the number, as well as amino acid sequences, of predicted chitinases found in two isolates belonging to the genus Agrobacterium that varied in their inhibition of Bd. After examining the annotated genomes, we identified a predicted chitinase in a Sphingomonas isolate that inhibited the growth of Bd that was absent from the five Sphingomonas isolates that did not inhibit Bd growth. The genes vioA, vioB, vioC, vioD and vioE are necessary to produce violacein, a compound which inhibits the growth of Bd. Differences in these genes were identified in three out of the four Janthinobacterium isolates. Of these three isolates, two showed strong inhibition of Bd growth, while the third inhibited Bd growth to a lesser extent. Using comparative genomics, we generated several testable hypotheses about differences among bacterial isolates that could contribute to variation in ability to inhibit Bd growth. Further work is necessary to test the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd. / Master of Science / Many amphibian population declines around the world have been caused by chytridiomycosis, a skin disease. This disease is caused by the fungus Batrachochytrium dendrobatidis (Bd). The skin of amphibians is also home to many bacteria that can provide important functions for the amphibian host, like preventing infection by Bd. To understand how these bacteria might provide protection, we examined the entire genomes of 40 bacterial isolates that reside on the skin of four amphibian species from Virginia, USA. These bacteria were previously tested for their ability to prevent Bd growth and 40 of them were chosen for sequencing based on selecting closely related isolates that varied in their ability to inhibit Bd growth. This allowed us to compare their genomes and generate hypotheses about possible genomic differences that could contribute to the variation in Bd growth inhibition. We identified sixty-five gene families that were present in all 40 bacteria. We also looked for sets of genes (biosynthetic gene clusters) that are known to produce secondary metabolites, which are compounds that can include antifungals. The two most abundant clusters we identified that had the potential to produce compounds that inhibit fungal growth were siderophores and Type III polyketide synthases. We then looked for genes that were not part of biosynthetic gene clusters that could produce specific compounds that can inhibit Bd growth, such as chitinase and violacein. We found variation in chitinase genes in several isolates that seemed to be associated with the ability to inhibit Bd growth. In addition, there were some differences in violacein genes that should be examined more in future studies. Overall, we suggest that using comparative genomic approaches can be valuable for identifying key bacterial functions in the microbiome.

Tandemly arrayed genes (TAG) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. We identified all the TAGs in the genomes of human, chimp, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG gene orientations and intergenic distances, and TAG gene functions. TAGs account for about 14-17% of all the genomic genes and nearly one third of all the duplicated genes in the four genomes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests while others are enriched with TAG deserts. The majority of TAGs are of size two for all genomes, similar to the previous findings in C. elegans, A. thaliana, and O. sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes with parallel orientation in the genome. The analysis of GO function indicate that genes with receptor or binding activities are significantly over-represented by TAGs. Simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. It is noteworthy to mention that gene family sizes are significantly correlated with the extent of tandem duplication, suggesting that tandem duplication is a preferred form of duplication, especially in large families. There has not been any systematic study of TAG genes' expression patterns in the genome. Taking advantage of recent large-scale microarray data, we were able to study expression divergence of some of the TAGs of size two in human and mouse for which the expression data is available and examine the effect of sequence divergence, gene orientation, and physical proximity on the divergence of gene expression patterns. Our results show that there is a weak negative correlation between sequence divergence and expression similarity between the two members of a TAG, and also a weak negative correlation between physical proximity of two genes and their expression similarity. No significant relationship was detected between gene orientation and expression similarity. Moreover, we compared the expression breadth of upstream and downstream duplicate copies and found that downstream duplicate does not show significantly narrower expression breadth. We also compared TAG gene pairs with their neighboring non-TAG pairs for both physical proximity and expression similarity. Our results show that TAG gene pairs do not show any distinct differences in the two aspects from their neighboring gene pairs, suggesting that sufficient divergence has occurred to these duplicated genes during evolution and their original similarity conferred by duplication has decayed to a level that is comparable to their surrounding regions. / Master of Science

The formation and persistence of species are the subject of much debate among biologists. Many species of Drosophila are behaviourally isolated, meaning that heterospecific individuals are not attracted to one another and do not interbreed. Often, this behavioural isolation is at least in part due to differences in pheromonal preference. Drosophila pheromones are long-chain cuticular hydrocarbons (CHCs). Desaturases are enzymes that are important for the production of CHCs. This thesis investigates the evolution of the gene family across 12 species of Drosophila. Desaturase genes were located in all species. Some genes, those that have previously been shown to have important roles in pheromonal communication, have experienced duplication and loss in several species. Two previously undiscovered duplicates were identified. Generally the desaturase gene family is governed by purifying selection, although following duplication these constraints are relaxed and in some cases duplicated genes show compelling evidence of positive selection. One of the loci under positive selection, the novel duplicate desat1b of the obscura group, was found to have a sex-biased expression pattern and alternative splicing in its 5′ UTR. In RNAi knock-down experiments of desaturase gene function in D. melanogaster, several desaturases were shown to affect CHC profiles of males and females, including some that were previously unlinked to CHC production.

BACKGROUND: Alternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins. DESCRIPTION: We have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform. CONCLUSION: Recent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu.

Background: Mutator-like transposable elements, a class of DNA transposons, exist pervasively in both prokaryotic and eukaryotic genomes, with more than 10,000 copies identified in the rice genome. These elements can capture ectopic genomic sequences that lead to the formation of new gene structures. Here, based on whole-genome comparative analyses, we comprehensively investigated processes and mechanisms of the evolution of putative genes derived from Mutator-like transposable elements in ten Oryza species and the outgroup Leersia perieri, bridging similar to 20 million years of evolutionary history. Results: Our analysis identified thousands of putative genes in each of the Oryza species, a large proportion of which have evidence of expression and contain chimeric structures. Consistent with previous reports, we observe that the putative Mutator-like transposable element-derived genes are generally GC-rich and mainly derive from GC-rich parental sequences. Furthermore, we determine that Mutator-like transposable elements capture parental sequences preferentially from genomic regions with low methylation levels and high recombination rates. We explicitly show that methylation levels in the internal and terminated inverted repeat regions of these elements, which might be directed by the 24-nucleotide small RNA-mediated pathway, are different and change dynamically over evolutionary time. Lastly, we demonstrate that putative genes derived from Mutator-like transposable elements tend to be expressed in mature pollen, which have undergone de-methylation programming, thereby providing a permissive expression environment for newly formed/transposable element-derived genes. Conclusions: Our results suggest that DNA methylation may be a primary mechanism to facilitate the origination, survival, and regulation of genes derived from Mutator-like transposable elements, thus contributing to the evolution of gene innovation and novelty in plant genomes.

Microsatellites are strings of short DNA motifs (≤6 bp) repeated in tandem across genomes of both prokaryotes and eukaryotes. In 20 years, they became popular genetic markers, successfully employed in the field of genetic mapping and gene hunting, as well as to address various biological questions at the individual, family, population and species level. However, evolutionary and demographic inferences from microsatellite polymorphism are hampered by controversy and ambiguity in the mutational processes of microsatellite sequences. Drawing on new data from genome projects, I review in Chapter 1 the concept of a microsatellite life cycle, which hypothesizes that microsatellites follow a life cycle from birth, through expansion, contraction, death and potentially resurrection. To document and understand this integrative concept of evolution, which could help improve current models of microsatellite evolution, there is an implicit need to study the evolution of microsatellites above the species level. A prerequisite of such comparative studies is therefore to find microsatellite loci that are conserved between different species. The near or full completion of many vertebrate genomes and their alignment against one another offer the ultimate approach to find genomic elements conserved over a large evolutionary scale. In Chapter 2, I present a new comprehensive method to find conserved microsatellites in whole genomes. Using the multiple-alignment of the human genome against those of 11 mammalian and five non-mammalian vertebrates, I examine the genomewide conservation of microsatellites, and challenge the general assumption that microsatellites are too labile to be maintained in distant species. In Chapter 3, I present similar results using the alignment of the newly sequenced platypus genome against those of three mammals, the chicken and the lizard, and incorporate these data into the framework created by the 17-genome analysis. This enlarged dataset was ground for attempting to reconstruct a vertebrate phylogeny from the presence/absence of microsatellites in the different genomes. Maximum parsimony analyses resulted in a tree much similar to that of the current view of the vertebrate phylogeny, while Bayesian analyses showed some discrepancies. This work opens a way for novel theoretical developments regarding the inference of ancestral states of microsatellites. In Chapter 4, I show how knowledge on conserved microsatellite sites can help for the development of a set of comparative primers useful across the Mammalia; implementing a similar protocol, nine conserved dinucleotide repeats were genotyped in 20 unrelated individuals of 18 species (nine sister species) encompassing the mammalian phylogeny, including marsupials and monotremes, and four microsatellites were sequenced in 4 individuals per species. My results emphasize conserved microsatellites as a new resource for genetic mapping and population studies. Finally, in Chapter 5, I recount the unexpected extent of structural change among mammalian orthologous microsatellites, including change of complexity, motif replacement and overall length variability. Altogether, these findings provide a comprehensive framework that may help in many areas of research, including molecular ecology, genome mapping, population genetics, and genome and microsatellite evolution.