Traditionally, conservation genetics has examined neutral-marker (e.g microsatellite) surveys to inform the conservation and management of species. The field expanded together with the expansion of molecular biology, primarily enabled by polymerase chain reaction (PCR) and DNA sequencing technologies. Recently, advances in genomics and bioinformatics, notably next-generation sequencing (NGS), have demonstrated the ability to further enhance conservation genetic assessments. As a result, conservation genetics is rapidly transforming into a field of conservation genomics. Although complete genome sequencing and analysis is still beyond the reach of many conservation genetic projects, researchers can benefit by producing large amounts of genetic data quickly for their species of interest, or by exploiting existing genomic data for a closely related species. The research presented below serves as an example of these two different approaches. First, I review the current state of conservation genomics, utilizing examples when appropriate to illustrate different techniques and approaches. Next, I describe the development of a tool using NGS that is useful for the rapid genetic analysis of pumas (Puma concolor) called PumaPlex. This work details the methods involved and will be useful for anyone interested in working with a species where little genomic data is available. The last three chapters focus on using an existing genomic tool for the domestic dog to analyze admixture, quantify inbreeding, and identify potential adaptive variation in the endangered Mexican wolf (Canis lupus baileyi). The results demonstrated the Mexican wolf has no significant recent ancestry from domestic dogs, and that several loci may potentially be effective in increasing fitness in the reintroduced population.

Comparative sequence analyses were performed with members of the Solanaceae and the Brassicaceae. These studies investigated genomic organization, determined levels of microcolinearity, identified orthologous genes and investigated the molecular basis of trait differences. The first analysis was performed by comparison of tomato (Solanum lycopersicum) genomic sequence (119 kb) containing the JOINTLESS1 (J1) locus with orthologous sequences from two potato species, a diploid, Solanum bulbocastanum (800-900 Mb, 2N=2X=24), and a hexaploid, Solanum demissum (2,700 Mb, 2N=6X=72). Gene colinearity was well maintained across all three regions. Twelve orthologous open reading frames were identified in identical order and orientation and included three putative J1 orthologs with 93-96% amino acid sequence identity in both potato species. Although these regions were highly conserved, several local disruptions were detected and included small-scale expansion/contraction regions with intergenic sequences, non-colinear genes and transposable elements. Three putative Solanaceous-specific genes were also identified in this analysis. The second analysis was performed by comparison of a Thellungiella halophila (T. halophila) genomic sequence (193 kb) containing the SALT OVERLY SENSITIVE1 (SOS1) locus with the orthologous sequence (146 kb) in Arabidopsis thaliana (Arabidopsis). T. halophila is a halophytic relative of Arabidopsis thaliana that exhibits extreme salt tolerance. Twenty-five genes, including the putative T. halophila SOS1 (ThSOS1), showed a high degree of colinearity with Arabidopsis genes in the corresponding region. Although the two sequences were significantly colinear, several local rearrangements were detected which were caused by tandem duplications and inversions. Three major expansion/contraction regions in T. halophila contained five LTR retrotransposons which contributed to genomic size variation in this region. ThSOS1 shares similar gene structure and sequence with Arabidopsis SOS1 (AtSOS1), including 11 transmembrane domains and a cyclic nucleotide-binding domain. Three Simple Sequence Repeats (SSRs) were detected within a 540 bp region upstream of the putative translational start site in ThSOS1. The (CTT)n repeat is present in different copy numbers in ThSOS1 (18 repeats) and AtSOS1 (3 repeats). When present in the 5' UTRs of some Arabidopsis genes, (CTT)n serves as a putative salicylic acid responsive element. These SSRs may serve as cis-acting elements affecting differential mRNA accumulation of SOS1 in the two species.

The goal of this dissertation was to evaluate genomic regulation during bovine mammary epithelial cell (BMEC) growth and differentiation. To accomplish this goal, a collagen gel cell culture system was developed that was capable of mimicking the prepartum stages of epithelial development and differentiation. In addition, a 4,600-cDNA bovine microarray was developed in order to profile gene expression. Analysis of BMEC in collagen cultures using various lactogenic conditions highlighted the critical importance of both hormonal and structural signals. The objective of the first study utilizing the microarray was to evaluate the contribution of the two prominent lactogenic factors in vitro, 1) prolactin and 2) gel release. Collectively, lactogenic stimulation appears to turn off genes associated with structural progression and morphogenesis, and turn on genes involved in alveolar MEC differentiation such as cell polarization, milk protein synthesis and ER/Golgi transport. The objective of the second study utilizing these resources was to evaluate the direct effects of thermal stress on BMEC growth and development. The structural response to thermal stress was characterized by morphogenic inhibition and dramatic regression of the ductal branches. Microarray analysis revealed an overall up-regulation of genes associated with stress response, DNA repair, protein degradation and cell death. In contrast, genes associated with cellular and MEC-specific biosynthesis, metabolism, and morphogenesis, were generally down-regulated. Subsequent to the analysis of BMEC differentiation was a targeted effort focusing on two small molecules hypothesized to be involved in regulating the BMEC secretory response: serotonin and prostaglandin E2. A pilot study suggested that serotonin is produced by bovine MEC and a model was proposed that describes serotonin's role as a feedback inhibitor during milk synthesis and secretion. A second pilot study demonstrated that PGE2 had a consistently positive influence on lumen diameter of alveolar structures in vitro. Overall, this dissertation provides new resources for studying bovine functional genomics, particularly within the mammary gland, and it provides a strong foundation for understanding genomic regulation of mammary epithelial structure and function. Furthermore, it establishes potential roles for local regulation of milk production by serotonin and PGE2.

The domestic dog, Canis lupus familiaris, plays many roles in the lives of humans. Additionally, the dog is recognized for its potential as a model for many human hereditary diseases. Thus, the genetics and genomics of the dog are being studied extensively in order to facilitate its use as a model, as well as to help the dog for its own sake. As part of this research effort, our laboratory has added type I markers (i.e., the acidic and basic keratins, c-kit, type I and IV collagens, and the gene encoding uromodulin) to the emerging map of the canine genome. The mapping of genes, particularly those in large gene families such as the collagens, is valuable because it rapidly increases the density of gene loci on the map and provides insight regarding conservation of synteny between the dog and other mammals. The major focus of work reported here is the genetics of X-linked Alport syndrome (XLAS), a terminal renal disease that affects the human and the dog. The disease results from mutations in COL4A5, a type IV collagen gene. Reported here are the 1) sequencing and mapping of the canine cDNA encoding uromodulin, 2) mapping of the type I and type IV collagen genes, 3) sequencing of the full-length cDNA of canine COL4A5, 4) identification of a 10 bp deletion in COL4A5, causative for XLAS in our colony of mixed breed dogs, 5) development of a genetic test for identification of affected and carrier dogs in the colony and 6) assessment of gene expression in the kidneys of normal and XLAS-dogs. This assessment was performed using a canine-specific oligonucleotide microarray. XLAS dogs demonstrated up-regulation of many genes involved in extracellular matrix reorganization, cell structure, and immune response, as expected in a glomerulopathy with tubulointerstitial nephritis. Trends were verified by quantitative RT-PCR. A review of the current status of canine genetics research, and current understanding of hereditary diseases in the dog, concludes this dissertation.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

<p>In an ever-changing world, evolution is an essential process that may allow organisms to adapt to their environment through natural selection. All evolutionary processes act through a single fundamental medium: genetic variation. Polyploidy, or whole genome duplication, is a major mechanism for evolutionary change because it is both widespread across taxa and results in a proliferation of genetic material that evolution can act upon. The key questions addressed here are: (1) How does chromosome pairing during meiosis in allopolyploids affect the magnitude of genetic variation?, (2) How does the genome of polyploids evolve following formation, and what genetic mechanisms govern this evolution?, and (3) How does genetic and genomic evolution in polyploids affect phenotypic evolution? I use the shy monkeyflower, <italic>Mimulus sookensis</italic>, a tetraploid of hybrid origin between <italic>Mimulus guttatus</italic> and <italic>Mimulus nasutus</italic>, to address these focal questions. In order to develop a foundation to aid in interpretation of my findings, I first investigate the evolutionary history of <italic>M. sookensis</italic>. Chromosome counts establish that <italic>M. sookensis</italic> is indeed an allotetraploid, and a review of taxonomic literature reveals that this species is heretofore undescribed. By analysing the patterns of genetic variation at chloroplast and nuclear loci in <italic>M. guttatus</italic>, <italic>M. nasutus</italic>, and <italic>M. sookensis</italic>, I show that <italic>M. sookensis</italic> has recurrently formed from <italic>M. guttatus</italic> and <italic>M. nasutus</italic>. Crossing experiments within <italic>M. sookensis</italic> indicate that recurrent origins can contribute to genetic diversity without contributing to reproductive isolation among independently arisen polyploid lineages.</p><p>To address my focal questions, I take advantage of an intriguing and striking difference in flower size among <italic>M. sookensis</italic>, <italic>M. guttatus</italic>, and <italic>M. nasutus</italic>. The flowers of <italic>M. sookensis</italic> and <italic>M. nasutus</italic> are small and remarkably similar to one another, while the flowers of <italic>M. guttatus</italic> and diploid and tetraploid F1 hybrids between <italic>M. guttatus</italic> and <italic>M. nasutus</italic> are large and showy. This phenotypic divergence in flower size between <italic>M. sookensis</italic> and <italic>M. guttatus</italic>-like hybrids indicates that small flower size has evolved in <italic>M. sookensis</italic>. Using genetic marker data and phenotypic measurements from synthetic neoallotetraploid <italic>Mimulus</italic>, I demonstrate that there are low levels of fragment loss and phenotypic variation in neoallotetraploids; this suggests that homeologous pairing and recombination following polyploidization is not a major source of genetic variation or phenotypic evolution in <italic>M. sookensis</italic>. Analysis of the whole genome sequence of two <italic>M. sookensis</italic> lines reveals that <italic>M. sookensis</italic> is a fixed heterozygote throughout its entire genome, in that it has retained both a <italic>M. guttatus</italic>-like and <italic>M. nasutus</italic>-like subgenome, neither of which have been removed through homeologous recombination. These subgenomes have been homogenized by widespread gene conversion, and do not appear to have been differentially affected by deletions or deleterious mutations. Finally, to directly characterize the genetic architecture of flower size in <italic>M. sookensis</italic>, I cross a large-flowered synthetic neoallotetraploid <italic>Mimulus</italic> to small-flowered <italic>M. sookensis</italic>. I then employ a novel genotyping-by-sequencing approach to identify quantitative trait loci (QTL) associated with flower size. I find that there is one locus that accounts for a large proportion of phenotypic variation, and four other loci also contribute to flower size variation between the parental lines. Some of these loci co-localize with previously identified loci for flower size in diploid <italic>Mimulus</italic>, while others do not. Altogether, genetic marker data, phenotypic analysis of neoallotetraploids, whole genome sequence data, and QTL mapping data suggest that the genetic variation necessary for flower size evolution was likely caused by both gene conversion and new mutations, but not homeologous recombination. These results suggest that trait evolution in polyploids may be affected by the unique attributes of polyploids, but that new mutations are always an important source of genetic variation, regardless of ploidy level.</p> / Dissertation

The genus Vitis (the grapevine) is a group of highly diverse, diploid woody perennial vines consisting of approximately 60 species from across the northern hemisphere. To gain insights into the use of wild Vitis species during the past century of interspecific grape breeding and to provide a foundation for marker- assisted breeding programmes, we present a principal components analysis based ancestry estimation method to calculate admixture proportions of hybrid grapes in the United States Department of Agriculture grape germplasm collection using genome-wide polymorphism data. We find that grape breeders have backcrossed to both the domesticated V. vinifera and wild Vitis species and that reasonably accurate genome-wide ancestry estimation can be performed on interspecific Vitis hybrids using a panel of fewer than 50 ancestry informative genetic markers.

The integrity of the genome is critical to organisms and it is affected by many factors. Radiation, for example, poses a serious threat to genome stability of human beings. While physical monitors for radiation hazard are present, the biological consequences of long term exposure to radiation are not well understood. With the opportunity as part of the International Caenorhabditis elegans Experiment-1 flight project, several approaches using C. elegans were taken to measure mutational changes that occurred during the spaceflight. Among these methods, the eT1 balancer system was demonstrated to be well-suited as an integrating biological dosimeter for spaceflight. The dog-1 gene in C. elegans is required to prevent mutations at poly-G/poly-C tracts, and previous work has described that in the absence of DOG-1, small deletions initiate within these tracts, most likely as a consequence of improperly repaired replication blocks. The eT1 balancer system was adapted to investigate the broad mutational spectrum of dog-1 mutants. Using this system, I was able to determine a forward mutation rate of approximately 1 x 10-3, 10 fold higher than spontaneous. Both small deletions as reported previously and unreported large chromosome rearrangements were observed, and most of mutations analyzed are associated with G/C tracts. Thus, I propose that following dog-1-induced replication blocks, repair leads to a wide range of mutational events and chromosomal instabilities, similar to those seen in human cancers. The existence of the G/C tracts in C. elegans creates a fortuitous but perplexing problem. They are hotspots for genome instability and need enzymatic protection. In the genome of C. elegans, approximately 400 G/C tracts exist and are distributed along every chromosome in a non-random pattern. G/C tracts are also over-represented in another Caenorhabditis species, C. briggsae. However, the positions and distribution differ from those in C. elegans. Furthermore, in C. elegans, analysis of SAGE data showed that the position of the G/C tracts correlated with the level of gene expression. Although being a threat to genome stability, the genomic distribution of G/C tracts in C. elegans and their effect on regional transcription levels suggest a role for G/C tracts in chromatin structure.

The highly developed and specialized anatomical and physiological characteristics observed for eukaryotes in general and mammals in particular are underwritten by an elaborate and intricate process of genome regulation. This precise control of the location, timing and amplitude of gene expression is achieved by a variety of genetic and epigenetic tools and mechanisms. While several of these regulatory mechanisms have been extensively studied, our understanding of the complex and diverse associations between various epigenetic marks and genetic elements with genome regulatory systems has remained incomplete. However, the recent profound improvements in sequencing technologies have significantly improved the depth and breadth to which their functions and relationships can be understood. The objective of this thesis has been to apply bioinformatics, computational and statistical tools to analyze and interpret various recent high throughput datasets from a combination of Next generation sequencing and Chromatin immune precipitation (ChIP-seq) experiments. These datasets have been analyzed to further our understanding of the dynamics of gene regulation in humans, particularly as it relates to repetitive DNA, cis-regulation and DNA methylation. The thesis thus resides at the intersection of three major areas; transposable elements, cis-regulatory elements and epigenetics. It explores how those three aspects of regulation relate with gene expression and the functional implications of those interactions. From this analysis, the thesis provides new insights into; 1) the relationship between the transposable element environment of human genes and their expression, 2) the role of mammalian-wide interspersed repeats (MIRs) in the function of human enhancers and enhancement of tissue-specic functions, 3) the existence and function of composite cis-regulatory elements and 4) the dynamics and relationship between human gene-body DNA methylation and gene expression.