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Title: Leveraging Angiosperm Pangenomics to Understand Genome Evolution
Creator: Yocca, Alan E.
Date: 2022
Collection: Electronic Theses & Dissertations
Description: My dissertation work focused on species-level comparative genomics and pangenomics to describe patterns of genetic variation. I studied multiple systems and unsurprisingly discovered different patterns of variation. Within a species, individuals are genetically diverse. There are some DNA regions present in every individual (core), while others may be specific to a single individual or lineage (variable). The sum of the genetic sequences found across an entire taxonomic group is called the...
Show moreMy dissertation work focused on species-level comparative genomics and pangenomics to describe patterns of genetic variation. I studied multiple systems and unsurprisingly discovered different patterns of variation. Within a species, individuals are genetically diverse. There are some DNA regions present in every individual (core), while others may be specific to a single individual or lineage (variable). The sum of the genetic sequences found across an entire taxonomic group is called the pangenome. This DNA variation greatly contributes to observed phenotypic differences between individuals. Therefore, to understand genome evolution and the link between genotype and phenotype, we must understand the pangenome. In this work, I compare the core and variable genetic regions both coding and noncoding across different flowering plant lineages. I note many consistent features across lineages as well as ways in which each pangenomic pattern is unique. These consistencies and differences can be leveraged in the future to better understand genome evolution as well as how genotype relates to phenotype. Specifically, my dissertation includes four chapters; (1) Evolution of Conserved Noncoding Sequences in Arabidopsis thaliana, (2) Machine learning identifies differences between core and variable genes in Brachypodium distachyon and Oryza sativa, (3) Current status and future perspectives on the evolution of cis-regulatory elements in plants, and (4) A pangenome for Vaccinium.
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Title: A nitrogen-responsive small peptide signaling mechanism modulates plant root system architecture
Creator: Lay-Pruitt, Katerina Sibala
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The plant root system changes dynamically in response to environmental cues. Plants utilize their root system for uptake of essential mineral nutrients that are heterogeneously distributed in the soil environment. Nutrient-dependent modulation of root system architecture (RSA) traits such as primary root growth, lateral root emergence, and the angles at which these roots grow allows for optimization of nutrient acquisition. Among signaling pathways by which plants may sense the availability...
Show moreThe plant root system changes dynamically in response to environmental cues. Plants utilize their root system for uptake of essential mineral nutrients that are heterogeneously distributed in the soil environment. Nutrient-dependent modulation of root system architecture (RSA) traits such as primary root growth, lateral root emergence, and the angles at which these roots grow allows for optimization of nutrient acquisition. Among signaling pathways by which plants may sense the availability of nutrients from the environment, small signaling peptide (SSP) pathways play important roles in optimizing root functions. These SSP pathways may further regulate molecular processes underlying RSA, such as the biosynthesis and transport of the major plant growth hormone, auxin. Characterization of these nutrient-responsive SSP pathways is thus of great importance and critical for understanding plant development in nutrient-poor environments. For my dissertation, I have identified and characterized a nitrogen (N)-responsive SSP pathway modulating root gravitropic response and lateral root development. Co-regulation of these RSA components by this module is proposed to prevent root outgrowth into N-poor regions and drive deeper root growth towards mobile nitrate (NO3-) resources stratified deeper in the soil profile. First, I show that a signaling pathway involving the CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) family of peptides and the CLAVATA1 (CLV1) receptor kinase, which is involved in N-dependent repression of lateral root emergence, also enhances root gravitropic response under N-limited conditions. Transcriptomic profiling of a clv1 mutant and CLE3 overexpressing lines identified Arabidopsis thaliana CENTRORADIALIS (ATC), a mobile protein previously characterized for its role in flowering regulation, as a downstream target of CLE-CLV1 signaling. Loss of ATC function significantly weakens root gravitropic response and has a moderate impact on lateral root emergence under low NO3- availability. ATC promoter activity and protein localization are also detected throughout the phloem and in the root columella cells, which are major centers for gravity sensing. Second, I demonstrate the relevance of ATC function on the molecular processes underlying root gravitropic response. While mutation in ATC does not impact gravity sensing via amyloplast sedimentation, it does inhibit the asymmetric transport of auxin needed for gravitropic bending. I determine that this occurs via the significant reduction of the PIN3 auxin efflux transporter in the vasculature and root tip of atc mutant lines. Lastly, I examine how the known roles of ATC in floral development could be implicated in root developmental processes. ATC binds to phosphatidic acid and phosphatidylserine, which is contrary to the binding capacity of its homolog FLOWERING LOCUS T (FT) to phosphatidylcholine and may contribute to its activity in N-limited environments. I also investigate the interaction of ATC and the transcription factor FD in the transcriptional regulation of PIN3. Although FD appears to have an impact on root gravitropic response, FD inhibits the expression of PIN3, suggesting potentially complex control of this gene via floral regulatory components. Taken together, the results presented in this dissertation contribute greatly to our understanding of how plant root architecture alters in response to N. These results can be further utilized in plant engineering strategies to regulate root growth in nutrient-limited soils.
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Title: USING FRAGARIA AS A MODEL SYSTEM FOR THE STUDY OF SUBGENOME DOMINANCE AND ADAPTATION IN CROPS
Creator: Alger, Elizabeth
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Polyploidy, or the presence of three or more complete genomes in a single organism, has occurred frequently in plants, especially in the angiosperm lineage. Allopolyploids, or polyploids resulting from the merging of different genomes in an interspecific hybrid, have often been shown to experience subgenome dominance. Subgenome dominance is the phenomenon where there is bias in the gene loss and expression between the different genomes in a polyploid, known as subgenomes. Despite the...
Show morePolyploidy, or the presence of three or more complete genomes in a single organism, has occurred frequently in plants, especially in the angiosperm lineage. Allopolyploids, or polyploids resulting from the merging of different genomes in an interspecific hybrid, have often been shown to experience subgenome dominance. Subgenome dominance is the phenomenon where there is bias in the gene loss and expression between the different genomes in a polyploid, known as subgenomes. Despite the prevalence of polyploids and subgenome dominance, little is known about the factors and mechanisms that influence this process. Strawberry (Fragaria sp.) is emerging as a powerful model system to investigate polyploid subgenome dominance evolution due to the recent identification of the four extant diploid progenitor species of the cultivated octoploid strawberry (Fragaria x ananassa). Having the diploid progenitors in hand allows us to identify differences between the dominant subgenome, F. vesca, and the other three progenitors that may have an impact of subgenome dominance. One possible factor is transposable element (TE) abundance, as low TE density has been consistently associated with the dominant subgenome in allopolyploids. Epigenetic silencing of TEs by DNA methylation to suppress TE activity has been shown to result in decreased expression of neighboring genes and this lowered gene expression may affect the establishment of subgenome dominance. F. vesca will be used as a diploid model for the study of subgenome dominance in strawberry where I can examine how TE abundance and other factors influence gene expression in a single accession and in hybrid crosses between different accessions. Tracking changes in gene expression in the hybrids will allow us to examine how genomes with difference sizes and genomic factors interact. The results and insights observed from this study can then be applied to subgenome dominance research in octoploid strawberry. In addition to the germplasm and genomic resources, strawberries are also a high value crop and the loss of their production due to (a)biotic stressors results in the loss of millions of United States dollars annually. Using a population of octoploid strawberries segregating for salt tolerance, I will identify candidate genes related to salt tolerance. Together this work will identify factors and mechanisms related to subgenome dominance and use genotypic data in a practical breeding context.
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Title: GENERATION OF HT-B AND HT-B PLUS S-RNASE KNOCKOUT LINES TO UNDERSTAND SELF-COMPATIBILITY IN DIPLOID POTATO
Creator: Lee, Sarah
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Domesticated potato (Solanum tuberosum L.) is the world’s third most important food crop and is a food security crop according to the Food and Agriculture Organization of the United Nations. Currently, commercial potatoes are autotetraploid and mainly produced via asexual clonal propagation. The autotetraploid nature of most cultivated potatoes in combination with acute inbreeding depression when self-fertilized over multiple generations cause challenges in making advances with traditional...
Show moreDomesticated potato (Solanum tuberosum L.) is the world’s third most important food crop and is a food security crop according to the Food and Agriculture Organization of the United Nations. Currently, commercial potatoes are autotetraploid and mainly produced via asexual clonal propagation. The autotetraploid nature of most cultivated potatoes in combination with acute inbreeding depression when self-fertilized over multiple generations cause challenges in making advances with traditional breeding schemes. The benefits of moving potato to a diploid breeding model include a simplified breeding scheme, easier fixation of desirable alleles, and generation of inbred lines that may be used to generate F1 hybrids with heterotic potential. A major hinderance to self-compatibility originates from the gametophytic self-incompatibility (GSI) system in which the S-RNase and HT-B genes play a critical role. Utilizing CRISPR-Cas9 gene editing, HT-B and HT-B + S-RNase knockout (KO) lines were produced. HT-B KOs produced parthenocarpic fruit but remained self-incompatible. However, the S-RNase and HT-B double KOs were self-compatible. Self-compatibility was measured quantitatively using fruit set, fruit weight, and seed count. Fruit set varied across both self-incompatible and self-compatible lines, with no clear trend in statistical significance. Double KO lines consistently displayed higher fruit weight than incompatible lines. Seed count served as the best measure of self-compatibility, with S-RNase and HT-B double KO lines producing up to three times mean seed per fruit when compared to S-RNase only KOs from prior studies. The lines with the highest levels of self-compatibility will serve as useful additions in advancing potato breeding to the diploid level.
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Title: DECIPHERING THE GENETIC BASIS OF SOLANUM CHACOENSE MEDIATED COLORADO POTATO BEETLE (LEPTINOTARSA DECEMLINEATA) RESISTANCE AND SELF- FERTILITY IN A DIPLOID SOLANUM CHACOENSE RECOMBINANT INBRED LINE POPULATION
Creator: Kaiser, Natalie
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The Colorado potato beetle (Leptinotarsa decemlineata) is the most widespread and destructive insect defoliator pest of potato and its control has historically been achieved through the use of insecticide. The diploid potato species Solanum chacoense has been utilized for over four decades in an attempt to introgress glycoalkaloid-based insect resistance into cultivated tetraploid potato. Despite these efforts, insect resistant cultivars have not been achieved, due in part to the complex...
Show moreThe Colorado potato beetle (Leptinotarsa decemlineata) is the most widespread and destructive insect defoliator pest of potato and its control has historically been achieved through the use of insecticide. The diploid potato species Solanum chacoense has been utilized for over four decades in an attempt to introgress glycoalkaloid-based insect resistance into cultivated tetraploid potato. Despite these efforts, insect resistant cultivars have not been achieved, due in part to the complex genetics underlying the trait. The creation of inbred diploid lines would allow more efficient examination and deployment of this economically important trait. We introduced self-compatibility into diploid insect resistant S. chacoense germplasm and developed the first potato recombinant inbred line (RIL) population to study, understand and deploy this mechanism of host-plant insect resistance in cultivated, diploid breeding lines.We first examined the genetic features underlying leptine glycoalkaloid mediated Colorado potato beetle host plant resistance in the F2 generation derived from a cross between S. chacoense lines USDA8380-1 (80-1) and M6. Using biparental linkage mapping, a major overlapping QTL region with dominant effects was identified on chromosome 2 explaining 49.3% and 34.1% of the variance in Colorado potato beetle field resistance and leptine accumulation, respectively. Bulk segregant whole genome sequencing of the same F2 population detected QTL associated with Colorado potato beetle resistance on chromosomes 2, 4, 6, 7, and 12. Candidate genes within these QTL regions were identified by weighted gene co-expression network analysis of parental lines and resistant and susceptible F2 individuals. Second, we exploited M6-mediated self-compatibility and established vigorous, F5 inbred diploid lines to further examine loci associated with Colorado potato beetle resistance and explore the practicality of inbreeding in diploid potato. F5 inbred lines carrying Colorado potato beetle resistance equivalent to the resistant donor parent were created without field selection during the inbreeding process. We report that the ratio of acetylated to non-acetylated glycoalkaloids measured under greenhouse conditions is a powerful metabolite marker to predict field performance without incurring the costs of conducting a Colorado potato beetle field trial. Leptine production was successfully introduced into diploid breeding germplasm. Single nucleotide polymorphism (SNP) genotyping coupled with stylar analysis of pollen tube growth and self-fertility phenotyping of the F4 and F5 generations revealed that multiple factors mediate the self-compatible response in this RIL population. Third, we assessed the initial transcriptional and metabolite response to Colorado potato beetle herbivory in beetle resistant and beetle susceptible S. chacoense lines over a 48-hour time course. To facilitate genome editing modification of the leptine biosynthesis pathway, we characterized the allelic variation between S. chacoense 80-1 and M6 in a candidate leptine biosynthesis gene identified by transcriptional profiling. This work highlights the challenges of establishing inbred germplasm, reinforces the complexity of selecting for self-fertility in diploid potato, and lays the foundation for optimization of potato RIL development. The availability of highly homozygous Colorado potato beetle resistant lines will enable further genomic inquiry of the loci contributing to this trait and will facilitate rapid deployment of beetle resistant diploid potato varieties.
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Title: NOVEL IMPACTS OF HOST-ENVIRONMENT INTERACTIONS IN ENTERIC GLIA THROUGH SEQUENCING AND IN-SITU EXPRESSION
Creator: Ponnampalam, Christine Dharshika
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The enteric nervous system (ENS) is comprised of enteric neurons and glia that facilitate essential gastrointestinal (GI) function including motility, visceral sensation, absorption, and gut permeability. Enteric neurons and glia are responsive to environmental cues and stressors ranging from the local gut microenvironment to the host’s psychosocial state and understanding how the ENS integrates these cues to modulate local and systemic function is critical. Novel roles for enteric neurons in...
Show moreThe enteric nervous system (ENS) is comprised of enteric neurons and glia that facilitate essential gastrointestinal (GI) function including motility, visceral sensation, absorption, and gut permeability. Enteric neurons and glia are responsive to environmental cues and stressors ranging from the local gut microenvironment to the host’s psychosocial state and understanding how the ENS integrates these cues to modulate local and systemic function is critical. Novel roles for enteric neurons in host-environmental interactions have been discovered using specialized sequencing technologies but these tools have not yet readily investigated enteric glia. The goal of this dissertation was to develop and utilize genetic technologies to characterize enteric glial responses to environmental mediators. First we adapted existing genetic tools to study molecular changes in the ENS and specifically enteric glia. We developed effective means of characterizing enteric glial expression within complex in vivo models using the RiboTag model with RNA-sequencing and subsequently visualized changes in gene expression within enteric ganglia in situ. We then utilized these techniques to investigate sex-specific responses to early life stress in enteric glia. Enteric glia from male and female mice have contrasting expression profiles including differences in GPCR signaling that could contribute to sex-specific ENS signaling mechanisms and ultimately GI disease outcomes. This supports recent findings of sexual dimorphism in glial functional connectivity and may highlight a critical difference in the way enteric glia communicate with other cell types between males and females. Additionally enteric glia from male mice ‘feminize’ following iiiearly life stress through altered expression of GI and neurological disease genes including mechanisms of glial-immune communication like type I interferon signaling. Together these data highlight striking differences in the physiologic molecular patterns and nature of stress response in enteric glia between males and females that likely contribute to sexually dimorphic GI disease patterns and symptom presentation. Next we investigated ENS type I interferon responses through the stimulator of interferon genes (STING) pathway. STING responds to both microbial and host mediators to contribute to GI inflammation. However the role of STING signaling in the gut is complex and can either exacerbate or ameliorate inflammation likely dependent on complex microenvironmental factors. We provide the first known investigation of STING expression and signaling within the ENS. STING is expressed in both enteric neurons and glia but IFNB is only expressed in enteric neurons. ENS STING is activated by its canonical ligands toproduce type I interferons. However this is likely primarily mediated through canonical activation of enteric neuronal STING and the contribution of enteric glial STING to type I IFN response is minor. Additionally enteric glial STING does not alter gastrointestinal outcomes during acute colitis within the DSS colitis model. Taken together these findings suggest enteric glia do not utilize STING for canonical type I IFN signaling or contribute to disease pathology in acute DSS colitis. Enteric glial STING may instead utilize primordial and specialized signaling pathways that more selectively alter local function. Together our data provide novel genetic tools and data to further uncover molecular functions in enteric glia and their role in GI and systemic health. Using these we discovered entirely novel molecular interaction effects between sex and early life stress that shift the framework of these risk factors in GI disease. Furthermore we highlight a novel potential mediator of ENS-microbe communication with STING. Our findings further characterize the molecular patterns used by glia in response to complex environmental factors and highlight unique heterogeneity in glial intercellular communication.
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Title: A FRAMEWORK FOR BIOLOGICAL DATA INTEGRATION AND FEATURE SELECTION IN LARGE DATA SETS
Creator: Gonzalez-Reymundez, Agustin
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The increasing volume of high-dimensional biological data (omics) has intensified the discovery of thousands of biomarkers across the different fundamental components of the cell (e.g., genome, transcriptome, proteome, epigenome) and allowed the characterization of complex phenotypes (e.g., metabolome, imaginome, phenome). However, the ability to integrate omics into informative results is constantly challenged by a seemingly ever-increasing volume of data. Furthermore, huge data sizes impose...
Show moreThe increasing volume of high-dimensional biological data (omics) has intensified the discovery of thousands of biomarkers across the different fundamental components of the cell (e.g., genome, transcriptome, proteome, epigenome) and allowed the characterization of complex phenotypes (e.g., metabolome, imaginome, phenome). However, the ability to integrate omics into informative results is constantly challenged by a seemingly ever-increasing volume of data. Furthermore, huge data sizes impose a tradeoff between how complex an omic integration algorithm can be and how much data it can handle (e.g., how fast can the algorithm be scaled to integrate large data sizes). In this dissertation, we explore statistical frameworks to face the challenges of modern omic data, including the integration of high-dimensional data of large sample sizes. We have developed a novel framework of competitive analytical performance compared with existing methods but suitable for omic data reaching biobank scales (i.e., hundreds of thousands of samples and variables). We implemented this method as an R package and showed its application on two traits of a complex molecular basis: cancer and regulation of energy intake and expenditure. In chapter one, we review the technologies and methods used to generate and integrate omic data. Chapter two describes our novel method and software of omic integration, shows examples in synthetic data, and evaluates its computational and analytical performance. Chapter three presents an application of our method to reveal a novel pan-cancer classification of tumors beyond the tissue of origin, regulated by distinct sets of molecular signatures. In chapter four, we present an application of our method to integrate phenomics data and identify patterns of energy balance regulated by genomic variation. Finally, in chapter five, we offer general conclusions to the entire thesis.
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Title: Genomic basis of electric signal variation in African weakly electric fish
Creator: Losilla-Lacayo, Mauricio
Date: 2021
Collection: Electronic Theses & Dissertations
Description: A repeated theme in speciation is reproductive isolation centered around divergence in few, highly variable traits, specially in cases without strong geographic isolation and high speciation rates. Understanding the genomic basis of highly variable traits that are key to speciation is a major goal of evolutionary biology, because they can characterize crucial drivers and foundations of the speciation process. African weakly electric fish (Mormyridae) are a decidedly speciose clade of teleost...
Show moreA repeated theme in speciation is reproductive isolation centered around divergence in few, highly variable traits, specially in cases without strong geographic isolation and high speciation rates. Understanding the genomic basis of highly variable traits that are key to speciation is a major goal of evolutionary biology, because they can characterize crucial drivers and foundations of the speciation process. African weakly electric fish (Mormyridae) are a decidedly speciose clade of teleost fish, and their electric organ discharges (EODs) are highly variable traits central to species divergence. However, little is known about the genes and celullar processes that underscore EOD variation. In this dissertation, I employ RNAseq and Nanopore sequencing to study the genomic basis of electric signal variation in mormyrids. In Chapter 1, I take a transcriptome-wide approach to describe the molecular basis of electric signal diversity in species of the mormyrid genus Paramormyrops, divergent for EOD complexity, duration and polarity. My results emphasize genes that influence the shape and structure of the electrocyte cytoskeleton, membrane, and extracellular matrix, and the membrane’s physiological properties. In Chapter 2, I compare gene expression patterns between electric organs that produce long vs short EODs. The results strongly support known aspects of morphological and physiological bases of EOD duration, and for the first time I identified specific genes and broad cellular processes expected to that alter morphological and physiological properties of electrocytes, most striking among these is the differential expression of multiple potassium voltage-gated channels. These two chapters independently identified the gene epdl2 as of interest for EOD divergence. In Chapter 3, I study the molecular evolutionary history of epdl2 in Mormyridae, with emphasis on Paramormyrops. My results suggest that three rounds of gene duplication produced four epdl2 paralogs in a Paramormyrops ancestor. In addition, I identify ten sites in epdl2 expected to have experienced strong positive selection in paralogs and implicate them in key functional domains. Overall, the results of this dissertation greatly solidify and expand our understanding of how the genome underpins changes to electrocytes, and in turn, divergence in their electric signals, a highly variable trait that may facilitate speciation in African weakly electric fish. This work provides an evidence-grounded list of candidate genes for functional analyses aimed to corroborate their contribution to the EOD phenotype.
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Title: Developing Reverse Genetic Tools in Weakly Electric Fish : Investigating Electric Organ in vivo scn4aa Function Through CRISPR Knockouts and Morpholino Knockdowns
Creator: Constantinou, Savvas James
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The ability to determine gene function allows research to progress at one of the finest scales in biology and is a goal in electric fish research. Reverse genetics allows researchers to determine gene function and would aid the electric fish community in beginning to answer some of the broadest and most complicated questions in biology such as linking genotype to phenotype and understanding the processes that lead to biological diversity. In this dissertation, I describe the development of...
Show moreThe ability to determine gene function allows research to progress at one of the finest scales in biology and is a goal in electric fish research. Reverse genetics allows researchers to determine gene function and would aid the electric fish community in beginning to answer some of the broadest and most complicated questions in biology such as linking genotype to phenotype and understanding the processes that lead to biological diversity. In this dissertation, I describe the development of two major reverse genetic tools for use in the electric fish system: CRISPR/Cas9 genome editing and morpholinos. To develop these tools, I also produced protocols for in-vitro breeding, husbandry, and single-cell embryo microinjections. In the first chapter, I describe in-vitro breeding, husbandry, and single-cell embryo microinjections and demonstrate that CRISPR/Cas9 is a promising tool for future electric fish research by targeting nonsense mutations to scn4aa in the mormyrid Brienomyrus brachyistius and gymnotiform Brachyhypopomus gauderio, two independently evolved lineages of weakly electric fish, resulting in a reduction in the electric organ discharge amplitude. In the second chapter, I provide electric fish researchers with a detailed analysis of our many successes and failures applying CRISPR/Cas9 methods to this system and discuss future suggestions on how to best apply them to novel electric fish research. In the third chapter, I describe my efforts to utilize vivo-morpholinos in mormyrid electric fish. While a single early pilot study I performed demonstrated vivo-morpholinos can reduce target gene mRNA levels and cause a phenotypic effect, my efforts to replicate these findings demonstrate inconsistent performance: control vivo-morpholino and scn4aa targeting vivo-morpholino injected fish had indistinguishable effects on electric organ discharge amplitude. Due to additional concerns of toxicity, I suggest morpholinos are not an ideal reverse genetic tool in Brienomyrus brachyistius and should only be utilized for future research with caution.
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Title: Genetic And Genetic By Environment Effects On Tar Spot Resistance And Hybrid Yield In Maize
Creator: Trygestad, Blake
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The phenotype of any plant can be broken down into the three primary sources of variation, genetic (G), environment (E), and genetic by environmental interaction (GxE). Producers and researchers alike will harness repeatable G and GxE effects to maximize their resource efficiency. This study studied the G and GxE effects in the biotic stress of the fungi Phyllachora maydis and the environment patterns in advanced yield trial data. In rating 800 genotypes over two seasons, we genetically...
Show moreThe phenotype of any plant can be broken down into the three primary sources of variation, genetic (G), environment (E), and genetic by environmental interaction (GxE). Producers and researchers alike will harness repeatable G and GxE effects to maximize their resource efficiency. This study studied the G and GxE effects in the biotic stress of the fungi Phyllachora maydis and the environment patterns in advanced yield trial data. In rating 800 genotypes over two seasons, we genetically mapped and identified over 100 significant Single Nucleotide Polymorphisms (SNPs) associated with tar spot resistance using a genome-wide association study. We then conducted genomic prediction, which was 81.5% accurate for predicting tar spot severity within the location and 48% accurate in predicting disease resistance in a new environment. Also, using Genetic and Genotype x Environment (GGE) biplots, we investigated environmental patterns of nine locations in three maturity Zones in the advanced yield trials in the Michigan Yield Performance Trials. First, we identified two locations, one in the late and one in the mid maturity zone, with equal G and GxE effects and should be removed. Then, using a sliding window of year combinations, we analyzed the optimal number of replications needed across the three maturity zones. We determined that an average of three replications are needed to achieve 75% of the maximum repeatability across the zones.
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Title: INTEGRATED ANALYSIS OF GENETIC MARKER, MIRNA, AND MRNA DATA TO UNRAVEL MECHANISMS CONTROLLING GROWTH AND MEAT QUALITY TRAITS IN PIGS
Creator: Daza, Kaitlyn R.
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Determining mechanisms regulating complex traits in pigs is essential to improve the production efficiency of this globally important protein source. MicroRNAs (miRNAs) are a class of non-coding RNAs known to post-transcriptionally regulate gene expression affecting numerous tissues and phenotypes, including those important to the pig industry. However, further research is needed to characterize the miRNAs expressed in pig skeletal muscle and assess their impact on the regulation of growth,...
Show moreDetermining mechanisms regulating complex traits in pigs is essential to improve the production efficiency of this globally important protein source. MicroRNAs (miRNAs) are a class of non-coding RNAs known to post-transcriptionally regulate gene expression affecting numerous tissues and phenotypes, including those important to the pig industry. However, further research is needed to characterize the miRNAs expressed in pig skeletal muscle and assess their impact on the regulation of growth, carcass composition, and meat quality traits. Additionally, little is known about the genetic architecture controlling miRNA expression in pigs, which can be elucidated by combining high-density genotypic data with miRNA expression profiles from the same animals in the mapping of miRNA expression quantitative trait loci (miR-eQTL). This analysis reveals associations between genomic regions harboring single nucleotide polymorphisms (SNPs) and variation in the expression of miRNAs. By integrating mRNA expression profiles and phenotypic data from the same individuals, putative regulatory relationships can be revealed underlying variation in phenotypes relevant to pig production. In this study, our objectives were to profile and characterize the small RNA population present in longissimus dorsi (LD) skeletal muscle samples from a F2 Duroc x Pietrain resource population, and to conduct an integrated miR-eQTL analysis to identify regulators of miRNA expression and candidate genes regulating phenotypic traits in adult pig skeletal muscle.MicroRNA expression profiling was performed on total RNA extracted from (LD) muscle samples from 174 F2 pigs. The composition of small RNA classes present in this dataset was characterized through a series of homology searches against human, mouse, and pig databases. MicroRNA quantification and novel miRNA prediction was conducted, profiling the abundance of 295 known mature pig miRNAs and producing 27 unique candidate novel miRNA precursors. The 295 miRNA expression profiles were subsequently used as response variables in a GBLUP-based GWA analysis. Results for associating these miRNAs with 36,292 SNPs identified 315 significant miRNA-SNP associations (FDR < 0.05), comprising 23 significant eQTL peaks associated with 17 unique miRNAs. Five of the 23 miR-eQTL peaks were defined as local-acting, meaning the genomic positions of the significantly associated SNPs comprising the miR-eQTL peak overlapped that of the miRNA precursor transcript. We then investigated the potential effects of these miRNAs through miRNA target prediction, correlation, and colocalization analyses. Notably, one miR-eQTL miRNA exhibiting a strong local¬-acting miR-eQTL, miR-874, had predicted target genes colocalizing with previously identified phenotypic QTL for 12 production traits including backfat thickness, dressing percentage, muscle pH at 24 h post-mortem, and cook yield. The results of this study revealed putative pig-novel miRNAs for further study and validation, contributing to our understanding of the miRNA landscape present in adult pig skeletal muscle. Additionally, we identified genomic regions underlying variation in miRNA expression, and candidate miRNAs and genes for future investigation of their regulatory effects on growth, carcass composition, and meat quality traits of importance to the global pig industry.
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Title: Germplasm Evaluation of Common Beans for End-Use Quality : Seed Coat Color and Cooking Time of Yellow Beans and Paste Qualities of Michigan Bean Varieties
Creator: Sadohara, Rie
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Consumption of dry beans is low in the U.S. and the genetic improvement of end-use quality traits of dry beans and the development of new uses for beans offer potential opportunities to increase consumption. This research aimed to 1) characterize a yellow bean diversity panel for seed coat color and cooking time, 2) investigate the G×E and inheritance of seed coat color and cooking time of the diversity panel via genome-wide association (GWA), 3) evaluate the suitability of MI-bred bean...
Show moreConsumption of dry beans is low in the U.S. and the genetic improvement of end-use quality traits of dry beans and the development of new uses for beans offer potential opportunities to increase consumption. This research aimed to 1) characterize a yellow bean diversity panel for seed coat color and cooking time, 2) investigate the G×E and inheritance of seed coat color and cooking time of the diversity panel via genome-wide association (GWA), 3) evaluate the suitability of MI-bred bean varieties for sweet paste qualities, and 4) inform bean breeders of the food industry’s needs for bean flour. A Yellow Bean Collection with 295 lines grown in MI and NE were evaluated for seed coat color, post-harvest darkening, and cooking time. Machine-learning was used to develop a procedure to automatically exclude hilum and corona areas in bean images and extract L*a*b* color values from seed coat. Color values, post-harvest darkening, hilum, and corona colors mapped to the ground factor P gene. Cooking time evaluation identified 20 lines that cooked within 20 min in MI and five lines that cooked within 31 min in higher-altitude NE. GWA identified a polygalacturonase gene as a candidate gene for cooking time. G×E was significant for seed coat color and cooking time, and MI-grown seeds were generally darker and faster-cooking than NE-grown. Six white bean varieties and one cranberry genotype were evaluated for sweet bean paste qualities: paste yield, color, stickiness, and flavor. Large-seeded white varieties performed similar or higher for many of the quality traits than Hime, an industry standard, showing their potential as a novel food application in the U.S. An electronic survey was conducted targeting U.S. food industry professionals who use pulse and/or wheat flour. Seventy-five valid responses were collected, and flavor and functionality rather than lectins or gluten contamination were the most important challenges in utilizing bean flour. These studies will be useful in selecting germplasm for favorable qualities to consumers such as bright yellow color, non-darkening after storage, fast cooking time, bean paste qualities, and bean flour attributes.
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Title: Assessment of state-specific DNA methylation patterns to improve functional annotation of farm animal genomes
Creator: Corbett, Ryan James
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Over the past several decades, genetic advancements in the domestic pig (Sus scrofa) and other farm animal species have resulted in increased economic output and expanded use of these organisms as biomedical models to study human disease. However, limited functional annotation of the porcine genome—particularly in non-coding regulatory regions—hinders both identification of causal genes for complex traits and translational research capabilities. The Functional Annotation of Animal Genomes...
Show moreOver the past several decades, genetic advancements in the domestic pig (Sus scrofa) and other farm animal species have resulted in increased economic output and expanded use of these organisms as biomedical models to study human disease. However, limited functional annotation of the porcine genome—particularly in non-coding regulatory regions—hinders both identification of causal genes for complex traits and translational research capabilities. The Functional Annotation of Animal Genomes consortium seeks to map functional elements in domesticated animal genomes in part by performing sequencing assays to characterize the animal epigenome, as specific chromatin modifications have been shown to be predictive of regulatory regions. DNA methylation is the most ubiquitous epigenetic modification made to the DNA molecule, and in mammals occurs almost exclusively at cytosines in CpG dinucleotides. DNA methylation exerts regulatory effects through numerous mechanisms, including the occlusion of transcription factors at activating regulatory regions, and as such has been shown to play important roles in establishing spatiotemporal gene expression. Furthermore, differential methylation has been associated with genomic imprinting and stress-induced physiological changes in mammals. Assessment of DNA methylation in the pig and other farm animal species has thus far been limited in scope. In this dissertation, I have characterized state-specific DNA methylation patterns in farm animal genomes across a diverse collection of cell types, developmental stages, and environmental conditions, to enhance understanding of epigenetic gene regulation in livestock and poultry. First, I demonstrate that sorted porcine immune cells exhibit unique DNA methylation landscapes that are strongly correlated with local and distal gene expression as well as binding sites for transcription factors regulating immune cell-specific functions. The co-localization of immune cell differentially methylated regions with GWAS SNPs for immune-related traits supports the use of epigenomics assays to increase functional annotation of economically relevant genomic regions. Second, I show that development of four porcine fetal tissues (whole brain, liver, loin muscle, and placenta) is associated with increased differentiation of DNA methylation profiles that likely contributes to tissue-specific transcriptomes and transcription factor regulatory potential. I also report widespread allele-biased methylation in fetal tissues associated with breed-specific gene regulation as well as putative regions of genomic imprinting events. Third, I characterize associations between environmental stimuli and DNA methylation patterns in two studies. I show that piglet weaning correlates with changes in peripheral blood mononuclear cell DNA methylation, and that increased weaning stress is associated with increased methylation and decreased expression of T cell-enriched genes, suggesting a diminished adaptive immune response. Lastly, I assess the impact of broiler chick incubation parameters on cardiac DNA methylation and observe significant temperature-associated differential methylation of genes involved in heart morphogenesis. I identified differentially methylated and expressed genes between temperature treatments that may influence environment-driven differences in cardiovascular development. In conclusion, I have performed the most expansive survey of whole-genome DNA methylation in farm animal species to date and have identified thousands of putative regulatory elements influencing state-specific gene and phenotype expression. These data will be a valuable resource for future functional annotation efforts seeking to identify mechanistic links between genetic and phenotypic variation in animal species.
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Title: EVALUATING GENOMIC ESTIMATES AND RECONSTRUCTED PEDIGREES AS ASSESSMENT TECHNIQUES FOR SEA LAMPREY POPULATIONS
Creator: Weise, Ellen M.
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Sea lamprey (Petromyzon marinus) are a harmful invasive species in the Great Lakes. Their invasion resulted in the decimation of native fish populations. A large annual control program has been dedicating to reducing lamprey population abundance. Control measures are mainly based on construction of barriers at the mouths of Great Lakes rivers and through the use of lampricides such as 3-trifluormethlyl-4-nitrophenol (TFM). Current population assessment techniques in Great Lakes tributaries...
Show moreSea lamprey (Petromyzon marinus) are a harmful invasive species in the Great Lakes. Their invasion resulted in the decimation of native fish populations. A large annual control program has been dedicating to reducing lamprey population abundance. Control measures are mainly based on construction of barriers at the mouths of Great Lakes rivers and through the use of lampricides such as 3-trifluormethlyl-4-nitrophenol (TFM). Current population assessment techniques in Great Lakes tributaries include mark-recapture to quantify census size of sea lamprey spawning populations. Larval assessments characterize stream population abundance and size/age composition to prioritize streams for TFM treatment. We expanded traditional assessment methodologies by generating reconstructed pedigrees and estimates of effective breeding size (Nb) and minimum spawning size (Ns) of sea lamprey populations using single nucleotide polymorphism (SNP) genotypes developed from RAD-capture of larval sea lamprey collected during annual river assessments. In Chapter 1, we evaluated efficacy of barriers to adult upstream passage in three streams by assigning larvae to reconstructed pedigrees to estimate the number and effective number of spawning adults. In Chapter 2, we elucidated the effects of several sampling, biotic, and environmental factors on Nb and Ns using multi-locus SNP genotypes from larval populations from 18 streams across the Great Lakes. Correlations between mark-recapture estimates of adult abundance and Nb and Ns were also tested, and no significant correlation was found. Additional analyses were conducted to examine the effects of sample size, number of SNP loci, and true Nb on estimated Nb and Ns using simulated sea lamprey populations. As true Nb increased, different methods of estimating Nb and Ns showed different types and levels of bias, highlighting the need for multiple methods of estimating these parameters. Additionally, bias decreased across all estimates when sample size and SNP size were large. Overall, the analyses conducted provided unique insight into sea lamprey spawning populations and have potential as an annual assessment technique for evaluating both current control and future alternative controls.
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Title: ARID1A MUTANT PATHOGENESIS OF THE ENDOMETRIAL EPITHELIUM
Creator: Reske, Jake Jordan
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Women’s health diseases represent an understudied, widespread medical concern with historically limited treatment options. Diseases of the endometrium, the innermost lining of the uterus, are a highly prevalent public burden. Endometriosis occurs in 1 in 10 women, and endometrial cancer is the most common gynecologic malignancy in the United States. Recent advances have revealed recurrent genetic causes of endometrial diseases, including gene mutations known to play a role in cancer...
Show moreWomen’s health diseases represent an understudied, widespread medical concern with historically limited treatment options. Diseases of the endometrium, the innermost lining of the uterus, are a highly prevalent public burden. Endometriosis occurs in 1 in 10 women, and endometrial cancer is the most common gynecologic malignancy in the United States. Recent advances have revealed recurrent genetic causes of endometrial diseases, including gene mutations known to play a role in cancer development. ARID1A is one such gene that is commonly mutated in endometrial diseases, and it encodes a protein involved in regulating DNA packaging and activity in the cell nucleus within a large complex known as SWI/SNF. The focus of this dissertation is to improve our understanding of how ARID1A mutations promote endometrial diseases at multiple biological levels, with a particular focus on how disrupted chromatin regulation affects physiologically relevant gene expression. In these works, genetic engineering techniques are leveraged in mice and human cell-based models supported by public and clinical data to establish the consequences of ARID1A mutations in the endometrium and how they relate to other common genetic alterations in these diseases. These studies have revealed that ARID1A is a tumor suppressor in the endometrial epithelium, such that ARID1A loss drives cellular invasion into nearby tissue. ARID1A mutations also promote invasive metastasis and squamous metaplasia in the context of aggressive TP53 mutations. At the level of chromatin, ARID1A and SWI/SNF directly regulate endometrial epithelial identity genes through both promoter and distal enhancer chromatin interactions. Mechanistically, ARID1A mutant invasion is driven by cell identity control regions known as super-enhancers that become hyperactivated, which can be reversed pharmacologically. Moreover, ARID1A physically and genomically interacts with other nuclear chromatin regulators to govern gene activation states through variant histone regulation. These works have contributed in multiple aspects toward deciphering how ARID1A mutations promote disease in the endometrium, including pre-clinical support for using epigenetic therapies to treat invasive ARID1A mutant endometrial conditions. Future efforts will aim to further identify and understand molecular and biochemical mechanisms linking ARID1A and SWI/SNF chromatin remodeling activity to regulation of gene expression. Ongoing work seeks to explain roles of ARID1A and SWI/SNF epigenetic regulation in normal physiological processes of the endometrium, such as hormone signaling across the menstrual cycle.
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Title: THE GENOMIC BASIS FOR FITNESS AND ECOMORPHOLOGICAL VARIATION IN RECOVERING POPULATIONS OF LAKE TROUT (SALVELINUS NAMAYCUSH) IN THE GREAT LAKES
Creator: Smith, Seth Robert
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Here I describe the development of novel genomic resources that will be fundamental for advancing a new generation of genomic research on Lake Trout (Salvelinus namaycush) including a high-density linkage map, an annotated chromosome-anchored genome assembly, and three high-throughput genotyping panels. We used these resources to identify genomic regions exhibiting signals of adaptive divergence between Lake Trout hatchery strains, some of which were found to underlie differences in fitness ...
Show moreHere I describe the development of novel genomic resources that will be fundamental for advancing a new generation of genomic research on Lake Trout (Salvelinus namaycush) including a high-density linkage map, an annotated chromosome-anchored genome assembly, and three high-throughput genotyping panels. We used these resources to identify genomic regions exhibiting signals of adaptive divergence between Lake Trout hatchery strains, some of which were found to underlie differences in fitness (survival and reproduction) between strains in the contemporary Lake Huron environment. Loci associated with differences in fitness between Seneca and Great Lakes origin strains were localized using local ancestry inference and local ancestry outlier tests. By evaluating locus specific allelic contributions of the ancestral Seneca Lake and Great Lakes-derived hatchery strains to naturally-produced wild Lake Huron populations across the genomes of F2 wild born individuals, we were able to determine that a subset of 7 genomic regions contributed to differences in fitness between Seneca Lake and Great Lakes origin individuals during the re-emergence of wild populations in Lake Huron. We also identified 2 genomic regions where Great Lakes origin alleles were favored by selection, 4 regions associated with hybrid vigor, and 2 regions potentially associated with hybrid inferiority in recovering wild Lake Trout populations. We also found that some Seneca origin alleles are only favored by selection on certain genetic backgrounds. For instance, F2 hybrids between the Seneca strain and Lake Michigan origin strains have an excess of Seneca origin haplotypes on chromosome Sna11, while this is not the case in hybrids between Lake Superior and Seneca origin individuals. Collectively, these results indicate that elevated performance of the Seneca strain can at least be partially attributed to a number of adaptive alleles at a small subset of genes. Additionally, the fact that Great Lakes origin alleles were favored by selection at two loci indicates that native strains contain some variation that provides a fitness advantage in Lake Huron. These admixture outlier regions contained a significant excess of genes related to swimming behavior and negative regulation of vascular wound healing, which strongly suggests that differences in fitness between strains are due to behavioral and physiological factors associated with the ability to avoid and survive predation by Sea Lamprey. Additionally, we carried out two studies seeking to identify genetic variation associated with habitat occupancy and phenotypic variation in Lake Trout. First, we carried out a quantitative trait locus (QTL) mapping study in which we identified loci associated with length and condition related traits, skin pigmentation, and body shape. We produced a linkage map for Lake Trout as a prerequisite for this work. The information on locus order obtained from the linkage map was also critical to the assembly of the Lake Trout genome and recombination rate estimates obtained from the linkage map were critical to the goal of assigning haplotype segments to their hatchery strain of origin for wild-born Lake Huron individuals. This study also allowed us to determine the location of the Lake Trout sex determination locus, determine centromere locations, and characterize structural differences (i.e., chromosomal inversions and translocations) between Lake Trout and other salmonid species. Second, we performed a genome-wide scan for loci associated with ecomorphological divergence in Lake Superior Lake Trout (specifically between lean, siscowet, and humper forms), and identified numerous regions with abnormally high levels of divergence between forms. These loci likely underlie variation in traits that differentiate forms, as well as traits that contributed to reproductive isolation historically. For example, the genomic region most strongly associated with length and condition (from our QTL mapping study) was also associated with ecomorphological divergence in Lake Superior and this region also contains a putative chromosomal inversion. Interestingly, we find that hybridization primarily occurred between humpers and siscowets and humpers and leans immediately preceding a genetic homogenization event that occurred in the late 1990s or early 2000s. Using a collection of samples over a multi-decade time series collected from the Apostle Islands, we show that levels of hybridization with humpers increased substantially starting in the 1980s.
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Title: DEVELOPMENT OF FINE-MAPPING RESOURCES FOR INTERROGATION OF A YIELD IMPACTING QTL ON THE 2D CHROMOSOME IN A BREAD WHEAT (TRITICUM AESTIVUM) AND AEGILOPS TAUSCHII NESTED ASSOCIATION MAPPING POPULATION
Creator: Turkus, Jonathan Dubau
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Bread wheat (Triticum aestivum) is one of the dominant grain crops across the world. As a mainstay of diets across the world, there is a constant pressure by the breeders and geneticists to identify yield promoting loci. In previous work, significant SNP associations were found on the 2D chromosome (approximately at 23.5Mb and 25.2Mb) for yield variation in a genome-wide association study (GWAS) of the D-genome Nested Association Mapping population (DNAM), an advanced-backcross nested...
Show moreBread wheat (Triticum aestivum) is one of the dominant grain crops across the world. As a mainstay of diets across the world, there is a constant pressure by the breeders and geneticists to identify yield promoting loci. In previous work, significant SNP associations were found on the 2D chromosome (approximately at 23.5Mb and 25.2Mb) for yield variation in a genome-wide association study (GWAS) of the D-genome Nested Association Mapping population (DNAM), an advanced-backcross nested association mapping population of five Ae. tauschii lines into an elite bread wheat background. To identify the signal source more precisely, fine-mapping tools were created in this work. Seven Kompetitive Allele Specific PCR (KASP) primer sets were designed for SNP markers between 22Mb and 30Mb, six of which displayed codominant behavior and one dominant. The KASP primer sets were identified the U6718 BC1F1 subpopulation within the DNAM as segregating for bread wheat and Ae. tauschii alleles between 22Mb and 30Mb on 2D chromosome. Linkage maps were created for the seven D-genome chromosomes for the U6718 subpopulation. QTL analysis defined the QTL as being between 21.3cM to 28.3cM on the 2D linkage map and 23.3Mb to 30.3Mb on the physical map. Using the defined 2DS QTL and the KASP primer sets, a fine-mapping population was created consisting of five heterozygous inbred families fixed 16 unique recombination events across the 2DS QTL. These developed tools will prove instrumental tools towards refining the understanding of causal loci of this yield QTL.
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Title: USING THE REGULATION OF GA2-OXIDASE AS A MODEL TO UNDERSTAND GIBBERELLIN’S ROLES IN APPLE AND ARABIDOPSIS THALIANA
Creator: Zhang, Songwen
Date: 2021
Collection: Electronic Theses & Dissertations
Description: In the model plant Arabidopsis thaliana (Arabidopsis), where the gibberellin (GA) signaling pathway has been best studied, GAs promote flowering. However, in many woody perennial plants such as apple (Malus x domestica Borkh.), GAs generally have a repressive role in flowering. The genetic and molecular mechanisms of GA-mediated flowering repression in apple and other plants remain unknown. In this study, we carried out transcriptional profiling of apple shoot apices during the anticipated...
Show moreIn the model plant Arabidopsis thaliana (Arabidopsis), where the gibberellin (GA) signaling pathway has been best studied, GAs promote flowering. However, in many woody perennial plants such as apple (Malus x domestica Borkh.), GAs generally have a repressive role in flowering. The genetic and molecular mechanisms of GA-mediated flowering repression in apple and other plants remain unknown. In this study, we carried out transcriptional profiling of apple shoot apices during the anticipated period of floral induction and examined how gene regulation is influenced by exogenous GAs. GA treatment resulted in downregulation of a diversity of genes participating in GA biosynthesis, transport and reception, and strong upregulation of the GA catabolic GA2-ox (GA2-oxidase) genes, suggesting GA homeostasis is mediated at multiple levels in these tissues. We also observed rapid and strong upregulation of both of two copies of a gene previously observed to inhibit flowering in apple, MdTFL1 (TERMINAL FLOWER 1), offering a potential explanation for the flowering-inhibitory effects of GA in apple. These results provide a context for investigating factors that may transduce the GA signal in apple and contribute to a preliminary genetic framework for the repression of flowering by GAs in a woody perennial plant. GA2ox expression and the enzyme activity determines concentrations of specific GAs in the right structure at the right time and contributes to enabling GAs to execute their function precisely. In return, their expression is under tight regulation by GA. Taking advantage of this tight link between GA2ox and GA activity, we aimed to develop regulation of GA2ox as a model to evaluate GA activity and function in both apple and Arabidopsis with a focus on floral induction. Through analysis of high-quality apple genomes and our transcriptional datasets, we identified 16 canonical GA2ox-like genes in the apple genome, representing eight pairs of homoeologous genes. By analyzing their spatial and developmental expression, we found that at least half of the genes were expressed in all the structures studied. In addition, individual members of homoeologous pairs displayed both overlapping and distinct expression patterns, suggesting function redundance and divergency. Finally, we found that two pairs of homoeologous genes (MdGA2ox1A, -1B, -2A and -2B) may be responsible for regulating GA activity in the shoot apex during floral induction. These findings provide biological and evolutionary insights into GA biology in apple, particularly in floral induction. In Arabidopsis, where GA2ox has been relatively well studied, we developed a genetic tool kit comprising both CRISPR/Cas-induced, transgene-free single knockout mutants and transcriptional and translational GUS fusion lines for the seven canonical Arabidopsis GA2ox genes (AtGA2ox1-AtGA2ox8). Mutations in the loss-of-function mutants have presumably disrupted function of both two functional domains of the GA2ox proteins. By comparing GUS activity in the GUS fusion lines, we found that the transcribed region is required for appropriate expression for several genes. This tool kit can be used for a wide spectrum of GA biology.
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Title: Systematic analysis of the signal responsive gene regulatory network governing Myxococcus xanthus development
Creator: Saha, Shreya
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Studies of signal-induced gene expression in bacteria have contributed to understanding of how bacteria cope with environmental stress. As an extensively studied model, Myxococcus xanthus provides fascinating insights into how changes at the level of gene expression enable which bacteria to survive environmental insults such as nutrient limitation. Upon starvation M. xanthus cells glide into aggregates and form mounds that mature into fruiting bodies as some cells form spores. Previously, our...
Show moreStudies of signal-induced gene expression in bacteria have contributed to understanding of how bacteria cope with environmental stress. As an extensively studied model, Myxococcus xanthus provides fascinating insights into how changes at the level of gene expression enable which bacteria to survive environmental insults such as nutrient limitation. Upon starvation M. xanthus cells glide into aggregates and form mounds that mature into fruiting bodies as some cells form spores. Previously, our group defined 24-30 h poststarvation as the critical period for commitment to spore formation, when cells commit to form spores despite perturbation of the starvation signal by nutrient addition. The process of multicellular development that culminates in sporulation is governed by a network of signal-responsive transcription factors that integrate signals for starvation and cellular alignment. In this dissertation I present the first systematic approach to elucidate the network dynamics during the commitment period.In the network, MrpC is a starvation-responsive transcription factor, whereas FruA is a transcription factor that responds to cellular alignment conveyed by C-signaling. Transcription of fruA is dependent on MrpC binding, and FruA activity is proposed to be posttranslationally regulated by C-signaling, although the mechanism is unknown. FruA and MrpC cooperatively regulate transcription of the dev operon. My systematic analysis of the network dynamics supported a model in which posttranslational activation of FruA by C-signaling is critical for dev transcription and for commitment to spore formation. Similar to dev, MrpC and C-signal-activated FruA combinatorially controlled transcription of the late-acting fadIJ operon involved in spore metabolism. Regulation of late-acting operons implicated in spore coat biogenesis (exoA-I, nfsA-H, MXAN_3259-MXAN_3263) was discovered to be under complex control by MrpC and FruA. My evidence suggests that transcription of these operons depends at least in part on a C-signal-dependent switch from negative regulation by unactivated FruA to positive regulation by activated FruA during the period leading up to and including commitment to sporulation. MrpC negatively regulated exo and MXAN_3259 during mound formation, but positively regulated nfs. During commitment to sporulation, MrpC continued to positively regulate nfs, switched to positive regulation of MXAN_3259, and continued to negatively regulate exo. A third transcription factor, Nla6, appeared to be a positive regulator of all the late genes. We propose that in combination with regulation by Nla6, differential regulation by FruA in response to C-signaling and by MrpC controls late gene expression to ensure that spore resistance and surface characteristics meet environmental demands.
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Title: Characterization of cyclic-GMP-AMP signaling and novel gene networks in the Vibrio Seventh Pandemic Islands of El Tor Vibrio cholerae
Creator: Severin, Geoffrey Blaine-Graessley
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which there have been seven pandemics. The first six pandemics (1817-1923) have been attributed to strains of the classical biotype while the seventh pandemic (1961- current) is being perpetuated by circulating strains of the El Tor biotype. It is hypothesized that El Tor's acquisition of two unique genomic islands of unknown origins, Vibrio Seventh Pandemic Island 1 & 2 (VSP-1 & 2), potentiated its displacement of...
Show moreVibrio cholerae is the causative agent of the diarrheal disease cholera, for which there have been seven pandemics. The first six pandemics (1817-1923) have been attributed to strains of the classical biotype while the seventh pandemic (1961- current) is being perpetuated by circulating strains of the El Tor biotype. It is hypothesized that El Tor's acquisition of two unique genomic islands of unknown origins, Vibrio Seventh Pandemic Island 1 & 2 (VSP-1 & 2), potentiated its displacement of classical strains in both environmental and clinical reservoirs prior to the seventh pandemic. Despite their connection to the pandemic evolution of the El Tor biotype and the likelihood they encode a wealth of novel biological functions the majority of the 303036 ORFs in the VSP islands have remained uncharacterized. The works presented in the thesis represent examples of our collective efforts to understand the unique traits afforded to the El Tor biotype by the VSP islands. .In 2012, ten years after the initial discovery of the VSP islands, the cyclic di-nucleotide synthase DncV was identified in VSP-1 and shown to synthesize the novel second messenger cyclic-GMP-AMP (cGAMP). Despite this significant discovery, cGAMP remained an orphan second messenger in bacteria for six years. In Chapter 2, I present our identification of the first cGAMP signaling network in bacteria by connecting the synthesis of cGAMP to the allosteric activation of the VSP-1 encoded patatin-like phospholipase vc0178, now named CapV. This finding helped catalyze the revelation that homologous cyclic-dinucleotide signaling networks contribute to bacteriophage-immunity and are distributed widely across the bacterial phyla.dncV and capV represent just two of the 103031 ORFs encoded in VSP-1, therefore we hypothesized that cGAMP signaling may extend to include additional genes within the island. Bioinformatic analysis of VSP island genes performed in collaboration with the laboratory of Eva Top at the University of Idaho predicted that dncV was likely to share a biological pathway with the VSP-1 encoded putative deoxycytidylate deaminase vc0175, we have now named DcdV. In Chapter 3, I describe our ongoing efforts to understand the biological function of DcdV and the discovery of its novel negative regulator DifV.The work presented in this thesis has made fundamental contribution to understanding the pandemic evolution of the El Tor biotype and the utility homologous genes afford to their bacterial host while providing a roadmap for further exploration of the biological role of the VSP-1 and VSP-2 islands in pandemic V. cholerae.
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