xiv, 196 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Establishing homologous (evolutionary) relationships among a set of genes allows us to hypothesize about their histories: how are they related, how have they changed over time, and are those changes the source of novel features? Likewise, aggregating related genes into larger, structurally conserved regions of the genome allows us to infer the evolutionary history of the genome itself: how have the chromosomes changed in number, gene content, and gene order over time? Establishing homology between genes is important for the construction of human disease models in other organisms, such as the zebrafish, by identifying and manipulating the zebrafish copies of genes involved in the human disease. To make such inferences, researchers compare the genomes of extant species. However, the dynamic nature of genomes, in gene content and chromosomal architecture, presents a major technical challenge to correctly identify homologous genes. This thesis presents a system to infer ancient homology between genes that takes into account a major but previously overlooked source of architectural change in genomes: whole-genome duplication. Additionally, the system integrates genomic conservation of synteny (gene order on chromosomes), providing a new source of evidence in homology assignment that complements existing methods. The work applied these algorithms to several genomes to infer the evolutionary history of genes, gene families, and chromosomes in several case studies and to study several unique architectural features of post-duplication genomes, such as Ohnologs gone missing. / Committee in charge: John Conery, Chairperson, Computer & Information Science; Virginia Lo, Member, Computer & Information Science; Arthur Farley, Member, Computer & Information Science; John Postlethwait, Member, Biology; William Cresko, Outside Member, Biology

Tineola bisselliella, the webbing clothes moth, is an economically important, globally distributed, synanthropic pest species. Tineola bisselliella is a member of Tineidae, the fungus moth family, which is a basal moth lineage in Lepidoptera. They are facultatively keratinophagous and can digest both keratin and detritus. The mechanism of keratin digestion has mostly been investigated from a symbiotic lens and is specifically thought to be performed by Bacillus bacterium that have been found in the gut of T. bisselliella larvae. However, expression of candidate digestion genes postulated to be in the genome have also been hypothesized as a probable mechanism. Here, we present the first draft de novo reference genome assembly and annotation for this species to investigate of the presence of keratin digestion genes, chromosomal synteny, and the evolution of the ancestral karyotype. Our final pseudochromosome-level assembly, which was assembled using syntenic comparisons with the closely related species Tinea pellionella, is 243.630 Mb and has an N50 length of 8.708 Mb. To facilitate quality genome annotation, we sequenced, assembled, and annotated a transcriptome. The annotated transcriptome had 13,615 protein-coding genes, while the final annotated genome contains 11,267 genes; 10,769 genes were functionally identified. We also performed systematic synteny comparisons of our T. bisselliella genome assembly and other basal moths and butterflies to investigate chromosome evolution. Comparison of synteny conservation between Melitea cinxia, which possesses the ancestral lepidopteran karyotype, and Tineola bisselliella suggests small fragmentation and fusion events were the mechanism by which Tineola bisselliella’s karyotype was reduced. This contrasts observations in other Lepidoptera with reduced karyotypes (e.g., Heliconius melpomene), which have undergone whole chromosomal fusion events. Finally, we performed preliminary differential gene expression and gene ontology enrichment analyses to investigate the adaptation of the ability to digest keratin in this organism. Tineola bisselliella adults do not ingest or digest food; therefore, we hypothesized genes related to keratin digestion would be enriched in larvae when compared to adults. Differential expression analysis of RNA sequencing data revealed 5,066 genes significantly differentially expressed between larval and adult stages out of 39,404 genes in the assembled and annotated transcriptome. GO enrichment analysis of differentially expressed genes revealed significant enrichment of GO terms associated with DNA replication in adults and enrichment of GO terms associated with serine proteases, oxidative-reduction reaction enzymes, and other proteases in larvae. Depletion of GO terms related to DNA replication in larvae suggest they were experiencing a nutrient poor environment when reared in the lab and entered larval diapause to slow their development and conserve energy. Our observations are consistent with previous research that found DNA replication was slowed in other Lepidoptera during diapause. Our data also illustrates in larvae digesting keratin, GO terms associated with serine proteases and oxidative-reduction reaction enzymes as well as other proteases are enriched. This suggests they are involved in the digestion process of keratin. However, this does not rule out that symbiotic bacteria that express keratinases are also part of this process. Future experiments should include differential expression and GO enrichment analyses with a larger sample size to confirm the results we obtained. Additionally functional experiments should be performed to investigate the expression of serine proteases, oxidative-reduction reaction enzymes, and other proteases in larvae in the absence of symbionts to determine if these ancestral digestion genes were able to facilitate the transition to a facultative keratin diet.

Researchers interested in examining a given species of interest (or target species) that lacks complete sequence data can infer some knowledge of that species from one or more related species that has a complete set of data. To infer knowledge, it is desired to compare the available sequence data between the two species to find orthologs. However, without complete data sets, one cannot be certain of the validity of the detected orthologs. Using ortholog detection systems in concert with species’ mapping data, researchers can find regions of shared synteny, allowing for more certainty of the detected orthologs as well as allowing inference of some genetic information based on these regions of shared synteny. A pipeline software solution, Detection of Orthologs via Genetic Mapping Augmentation (DOGMA), was developed for this purpose. DOGMA’s functionality was tested using a target species, Phaseolus vulgaris, which only had partial sequence data available, and a closely related species, Glycine max, which has a fully se- quenced genome. On sequence similarity alone, which is the standard technique for detecting or- thologs, 205 potential orthologs were detected. DOGMA then filtered these results using mapping data from each species to determine that 121 of the 205 were quite likely true orthologs, referred to as putative orthologs, and the remaining 84 were categorized as reduced orthologs as there was either insufficient information present or were clearly outside a noted region of shared synteny. This provides evidence that DOGMA is capable of reducing false positives versus traditional techniques, such as applications based on Reciprocal Best BLAST Hits. If we interpret the output of the Or- tholuge program as the correct answer, DOGMA achieves 95% sensitivity. However, it is possible that some of the reduced orthologs classified by DOGMA are actually Ortholuge’s false positives, since DOGMA is using mapping data. To support this idea, we show DOGMA’s ability to detect false positives in the results of Ortholuge by artificially creating a paralog and removing the real ortholog. DOGMA properly classifies this data as opposed to Ortholuge.

The mop2 gene is required for epigenetic silencing; it was originally defined as a mutation, Mop2-1, which when dominant prevented paramutation at b1. Paramutation is an allele communication that causes a mitotically and meiotically heritable change in gene expression. Mop2-1 was subsequently shown to be involved in maintaining the silenced paramutant state and to prevent dsRNA-mediated transcriptional gene silencing (activities revealed only when the mutation is homozygous). Understanding the product encoded by mop2 will help dissect the underlying mechanisms involved in paramutation and dsRNA-mediated transcriptional silencing. This dissertation describes map-based cloning and candidate gene approaches directed toward the eventual goal of identification of mop2.Initial mapping of mop2 placed it within a region delineated by the markers umc1823 and eks1. On the maize physical map this region contains 21 BAC (Bacteria Artificial Chromosome) clones, representing 2.9 Mb. Skim sequencing identified additional markers for mapping and revealed the gene content. Extensive candidate gene examinations, including gene sequencing, expression profiling with microarrays and RT-PCR, and complementation tests with mutant alleles did not identify any of the four chromatin and RNAi-related genes as mop2.The new markers developed from the skim sequence enabled further mapping and molecular genotyping, which revealed that the Mop2-1 mutation was unstable. Approxi¬mately 10% of phenotypic heterozygous plants were actually genotypic homozygous. Further mapping using only Mop2-1 homozygous plants reduced the mop2 interval to a region of nine BACs, containing 57 genes.The mop2 region is highly syntenic to a rice region of 1.25 Mb on chromosome 4. The gene alignment and repetitive sequence analyses between the syntenic regions in these two species revealed both syntenic and non-syntenic blocks of sequences. Analyses suggested several potential mechanisms for the collinearity breakage, including, but not limited to, tandem duplications of genes in one species but not the other and the presence of gene fragments in maize, but not in rice.The research described herein provides the basis for continued efforts to clone mop2. Fine-structure mapping with new markers and a larger population, as well as candidate gene sequencing in the Mop2-1 BAC library, should be pursued to clone mop2.

Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.

As two species diverge, much of their genomes begin to differentiate. In many lineages, however, the genomic structure remains remarkably intact, with orthologous gene content maintained across millions of years and significant changes to their biological characteristics. The maintenance of gene content is defined as conserved synteny while the preservation of gene order is defined as conserved linkage; the conservation of both can be incredibly informative when interrogating and comparing two genomes. In non-model organisms, linkage conservation to a well-developed model allows informed, cost-effective and rapid answers to fundamental biological questions without generation of equivalent resources. With the development of new model organisms, we can begin to discuss more fundamental evolutionary concepts, such as the maintenance of chromosomal gene content across larger evolutionary time-scales, or the reorganization that occurs in chromosomes following major genomic events such as whole-genome duplications. In this work, I utilized the rapid development of primary genomic resources in the non-model teleost sablefish (Anoplopoma fimbria) to demonstrate that conserved linkage to a model genomic reference can identify the gene most likely responsible for genetic sex-control. I then assembled the first genome for a non-duplicated member of the teleost lineage Protacanthopterygii, the northern pike (Esox lucius), and demonstrated the conservation of synteny between three major lineages of teleosts, the Protacanthopterygii, the Acanthopterygii and the Ostariophysi. I further showed that the genome of northern pike retains an ancestral teleost organization and pre-duplicated genome in comparison to the economically important Salmoniformes. Finally, with continued improvements of the genome to the chromosome level, I demonstrated the degree of conserved linkage maintained between Atlantic salmon and northern pike and explained how conserved linkage through both genomes could be used to improve the genome assembly of the other, even with over 125 million years of separation. As genomic technology continues to advance and new genomic resources become available, the continued refinement of genome re-organization post duplication will be revealed, and this pre-duplication outgroup will continue to push our understanding of the effects of genome duplication, as we transition from genome organization to functional modifications of gene duplicates following duplication. / Graduate / 2018-12-01

Small fibrils termed actinotrichia are involved with the growth and structure of the fin fold during fin development in fish. The actinodin (and) genes are required for actinotrichia formation, and the loss of these genes from the genomes of tetrapods has been implicated in the tetrapod-specific loss of actinotrichia, loss of a fin fold and the concurrent evolution of paired fins into limbs. This study focuses on the function of the and genes and their role in actinotrichia formation. The results reveal cis-acting regulatory elements required for and1 expression in the fin epithelium. Furthermore, it is shown that the And proteins display similarities to the secreted signaling molecule, Ecrg4, implying a possible role in cell differentiation during fin fold development. In the final section of this report, I use a genomic analysis to show that the and genes were lost from otherwise well-conserved syntenic loci in fish and tetrapod genomes. These results suggest possible causes for the evolutionary loss of and genes and the associated developmental changes that may have permitted fin to limb evolution.

Background: Transfer RNAs (tRNAs) are ubiquitous in all living organism. They implement the genetic code so that most genomes contain distinct tRNAs for almost all 61 codons. They behave similar to mobile elements and proliferate in genomes spawning both local and non-local copies. Most tRNA families are therefore typically present as multicopy genes. The members of the individual tRNA families evolve under concerted or rapid birth-death evolution, so that paralogous copies maintain almost identical sequences over long evolutionary time-scales. To a good approximation these are functionally equivalent. Individual tRNA copies thus are evolutionary unstable and easily turn into pseudogenes and disappear. This leads to a rapid turnover of tRNAs and often large differences in the tRNA complements of closely related species. Since tRNA paralogs are not distinguished by sequence, common methods cannot not be used to establish orthology between tRNA genes. Results: In this contribution we introduce a general framework to distinguish orthologs and paralogs in gene families that are subject to concerted evolution. It is based on the use of uniquely aligned adjacent sequence elements as anchors to establish syntenic conservation of sequence intervals. In practice, anchors and intervals can be extracted from genome-wide multiple sequence alignments. Syntenic clusters of concertedly evolving genes of different families can then be subdivided by list alignments, leading to usually small clusters of candidate co-orthologs. On the basis of recent advances in phylogenetic combinatorics, these candidate clusters can be further processed by cograph editing to recover their duplication histories. We developed a workflow that can be conceptualized as stepwise refinement of a graph of homologous genes. We apply this analysis strategy with different types of synteny anchors to investigate the evolution of tRNAs in primates and fruit flies. We identified a large number of tRNA remolding events concentrated at the tips of the phylogeny. With one notable exception all phylogenetically old tRNA remoldings do not change the isoacceptor class. Conclusions: Gene families evolving under concerted evolution are not amenable to classical phylogenetic analyses since paralogs maintain identical, species-specific sequences, precluding the estimation of correct gene trees from sequence differences. This leaves conservation of syntenic arrangements with respect to "anchor elements" that are not subject to concerted evolution as the only viable source of phylogenetic information. We have demonstrated here that a purely synteny-based analysis of tRNA gene histories is indeed feasible. Although the choice of synteny anchors influences the resolution in particular when tight gene clusters are present, and the quality of sequence alignments, genome assemblies, and genome rearrangements limits the scope of the analysis, largely coherent results can be obtained for tRNAs. In particular, we conclude that a large fraction of the tRNAs are recent copies. This proliferation is compensated by rapid pseudogenization as exemplified by many very recent alloacceptor remoldings.

Self-incompatibility (SI) is an important genetic mechanism to prevent the inbreeding of flowering plants and also an excellent system for studying cell-cell recognition and signal transduction. During evolution, several SI systems have been evolved. A unique SI system widely spreads in the grasses. In the grasses, two unlinked, multi-allelic loci (S and Z) determine SI specificity. A putative self-incompatibility gene (Bm2) was previously cloned. In this study, the role of Bm2 in self-incompatibility was investigated first. The cDNA homologues of Bm2 were sequenced from two pollen-only mutants. The results indicated that Bm2 is not the one of SI genes in Phalaris, but represents a subclass of thioredoxin h. Thus a map-based cloning strategy was then adopted to clone the SI genes from Phalaris. Fine linkage maps of the S and Z regions were constructed. RFLP probes from wheat, barley, oat and rye were screened and the S locus was delimited to 0.26 cM and the Z locus to 1.0 cM from one side using specially designed segregating populations. The S locus was located to the sub-centromere region of triticeae chromosome group 1 and the Z locus to the middle of the long arm of group 2. Finally, barley and rice bacterial artificial chromosome (BAC) clones corresponding to the S and Z region were identified to analyse the chromosome structures and to seek candidate SI genes. The abundant repetitive sequences in the identified barley BAC clones limit their usefulness. Identification of Rice BAC clones orthologous to the S and Z regions open the gate to use rice genome information to clone SI genes from the grasses. A positive rice clone (139.9 kb) orthologous to the S region contained 19 predicted genes. Several of these genes might be involved in pollen tube germination and pollen-stigma interaction, which are the major parts of SI reaction. A positive clone (118.9 kb) orthologous to the Z region gave 16 predicted genes. The predicted genes on the outmost ends of these clones could be used to construct contigs to cover the S and Z regions and delimit the S and Z loci in the grasses. / Thesis (Ph.D.)--Department of Plant Science, 2001.

The differentiation of plant vascular tissue is regulated by plant hormones and transcription factors. One of the key plant hormones involved in this process is auxin. Auxin signals are mediated by auxin response factor transcription factors (ARFs). These transcription factors are involved in the perception of auxin signals and the subsequent activation or deactivation of suites of downstream genes. Based on its mutant phenotype, one of the most interesting members of this family is the ARF MONOPTEROS (MP). This thesis investigates the role played by MP in secondary vascular differentiation, as well as taking a look at other molecular aspects of secondary vascular differentiation, with a focus on the model plants Arabidopsis thaliana and poplar (Populus trichocarpa and hybrid poplar). A dexamethasone inducible RNAi silencing strategy was developed, and transgenic Arabidopsis lines produced. When silencing was induced in these lines from germination, a phenotype closely resembling the mp mutant was observed. When MP silencing was induced in bolting stems, early senescence, as well as a dramatic reduction in interfascicular fibre production was observed, and these stems were thinner and less rigid than empty vector controls. RNA from these stems was isolated and used in a global transcript profiling microarray experiment. This experiment showed that several auxin-related genes, as well as several transcription factors, were differentially regulated in response to MP silencing. Because Arabidopsis is not a typical woody plant, further investigation into the role played by MP in wood formation was done using the model tree poplar. A BLAST search of a poplar xylem EST database identified a single promising partial sequence. Based on this sequence information, a poplar MP homolog was isolated and named PopMP1. The full-length sequence of this gene demonstrated remarkable structural conservation when compared with that of Arabidopsis. Subsequent complete sequencing of the poplar genome revealed a second copy of the MP gene in poplar and named PopMP2. Expression profiling across a range of tissues suggests that subfunctionalization has occurred between the two copies. Overexpression transgenic lines for PoptrMP1 were developed. AtHB8 is known to be regulated by MP in Arabidopsis, and a poplar HB8 homolog was upregulated in the transgenic lines. However, no obvious physical phenotype in these lines was apparent. To investigate the transcriptome-wide changes associated with initiation of cambium formation in poplar stems, a global transcript profiling experiment was performed. Out of 15400 genes tested, 2320 met an arbitrary cutoff of >1.3 fold and p-value <0.05 and were labeled differentially expressed (DE). These included several transcription factors and showed remarkable similarity to analogous data from Arabidopsis. The conclusions drawn from this thesis support the hypothesis that MP plays roles in later development, and do not rule out the possibility that MP is directly involved in wood development. The data reported also offer a large number of candidate for further investigation into the genetic control of wood development.