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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
Genome-wide association study reveals genes associated with mite recruitment phenotypes in the domesticated grapevine (vitis vinifera
Creator
LaPlante, Erika R.
Date
2020
Collection
Electronic Theses & Dissertations
Description
Plants in the grape genus Vitis have varying densities of trichomes and mite-domatia on their leaves, which facilitate the recruitment, retention, and reproduction of beneficial mites. By increasing the abundance of mites on grape leaves, these phenotypes promote a defense mutualism contributing to the control of grape pests and pathogens. Identification of the genes controlling these phenotypes would inform our understanding of the genetics underlying mite-plant mutualistic interactions and...
Show morePlants in the grape genus Vitis have varying densities of trichomes and mite-domatia on their leaves, which facilitate the recruitment, retention, and reproduction of beneficial mites. By increasing the abundance of mites on grape leaves, these phenotypes promote a defense mutualism contributing to the control of grape pests and pathogens. Identification of the genes controlling these phenotypes would inform our understanding of the genetics underlying mite-plant mutualistic interactions and could lead to breeding domesticated Vitis vinifera L. varieties that are naturally defended against pathogens. Little is known about the genetics underlying mite recruitment phenotypes in Vitis. We conducted a GWAS to determine the genetic architecture of mite recruitment traits in V. vinifera using 399 cultivars from a common garden diversity panel. We investigated eight traits previously established in the literature associated with an increase in beneficial mite abundance. We found single nucleotide polymorphisms (SNPs) significantly associated with each mite recruitment trait investigated. Corresponding gene annotations of SNP genetic coordinates revealed notable gene associations, including a trichome development gene, and a physiological defense response gene, suggesting these genetic regions may have a large impact on mediating mite-plant interactions in this species. Our findings are among the first to investigate which genes underly ecologically important mutualisms between plants and beneficial mites and suggest promising candidate genes for breeding and genetic editing to increase naturally occurring predator-based defenses in grapes.
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Title
Local adaptation and fitness trade-offs
Creator
Dittmar, Emily Loring
Date
2017
Collection
Electronic Theses & Dissertations
Description
Adaptation generates and maintains genetic and phenotypic diversity. This is thought to occur due to trade-offs, where adaptation to one environment comes at a cost in another. Although trade-offs are believed to play a prominent role in the generation and maintenance of genetic and phenotypic diversity, the mechanisms by which adaptation leads to trade-offs are not well understood.My research explores the forces that lead to adaptive trade-offs in two systems. First, using a RIL mapping...
Show moreAdaptation generates and maintains genetic and phenotypic diversity. This is thought to occur due to trade-offs, where adaptation to one environment comes at a cost in another. Although trade-offs are believed to play a prominent role in the generation and maintenance of genetic and phenotypic diversity, the mechanisms by which adaptation leads to trade-offs are not well understood.My research explores the forces that lead to adaptive trade-offs in two systems. First, using a RIL mapping population created from natural populations of Arabidopsis thaliana, I studied the genetic basis of flowering time, a putatively adaptive trait and one that differs between the parental populations. I identified flowering time QTL in growth chambers that mimicked the natural temperature and photoperiod variation across the growing season in each native environment and compared the genomic locations of flowering time QTL to those of fitness (total fruit number) QTL from a previous three-year field study.In addition, I studied two populations of Leptosiphon parviflorus, an annual wildflower native to California. At Jasper Ridge biological preserve, populations of L. parviflorus grow on and off serpentine soil in close proximity. Due to its harsh growing conditions, serpentine soil exerts strong selective pressures on plants. Despite the close proximity of study populations (<100 m) and ongoing gene flow, reciprocal transplant studies demonstrate that these populations are locally adapted to their native soil types. To determine the selective agents operating in both habitats and the forces underlying fitness trade-offs, I performed manipulative experiments in the field and greenhouse. Results from these studies show that both soil moisture and competitive interactions are important for mediating fitness differences among the populations, and adaptation to serpentine soil might result in a cost to competitive ability.I also addressed the causes of flowering-time differences in these populations. Field reciprocal-transplant studies and watering manipulations in the greenhouse demonstrate the contribution of both the genotype and the environment to observed flowering-time differences. The plasticity of flowering time in response to soil type appears to be driven by differences in soil moisture. In addition, selection on flowering time was measured in both soil types across four years of study using a set of F5 advanced generation hybrids and found to differ among the habitats. Therefore, both selection and plasticity contribute to flowering-time differences between these populations and thus have likely played an important role in the initiation and/or maintenance of adaptive divergence in this system.Finally, the two populations differ in their flower color, a Mendelian trait. Pollinators do not discriminate among flower colors and are unlikely to exert selection on this trait. Instead, flower color may be related to stress tolerance if the causal gene has pleiotropic effects on other traits. Using a set of Near Isogenic Lines (NILs), I found that the flower color locus has an effect on survival in field soil and fecundity in benign conditions. Ongoing work is aimed at addressing the mechanisms underlying the relationship between flower color and soil adaptation.
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Title
Genome evolution of Campylobacter jejuni during experimental adaptation
Creator
Jerome, John Paul
Date
2012
Collection
Electronic Theses & Dissertations
Description
Campylobacter jejuni is a leading cause of foodborne bacterial enteritis in humans. An important reservoir for C. jejuni is in chickens, but it has been shown to colonize a large host range. Passage through a mouse model of campylobacteriosis resulted in a hypervirulent phenotype in mice for C. jejuni strain NCTC11168. After analyzing the wild-type and mouse-adapted variants by phenotype assays, expression microarray, pulse-field gel...
Show moreCampylobacter jejuni is a leading cause of foodborne bacterial enteritis in humans. An important reservoir for C. jejuni is in chickens, but it has been shown to colonize a large host range. Passage through a mouse model of campylobacteriosis resulted in a hypervirulent phenotype in mice for C. jejuni strain NCTC11168. After analyzing the wild-type and mouse-adapted variants by phenotype assays, expression microarray, pulse-field gel electrophoresis and whole genome sequencing we discovered that the genetic changes in the mouse-adapted variant were confined to thirteen hypermutable regions of DNA in contingency loci. We also show that specific contingency loci changes occurred in parallel during mouse infection when reisolates from multiple mice were analyzed. Furthermore, a mathematical model that considers contingency loci mutation rates and patterns does not explain the observed changes. Taken together, this is the first experimental evidence that contingency loci play a role in the rapid genetic adaptation of C. jejuni to a host, which results in increased virulence. In contrast to the observed virulence increase by serial host passage, we showed that C. jejuni rapidly loses an essential host colonization determinant during adaptive laboratory evolution. Passage in broth culture selected for flagellar motility deficient C. jejuni cells in parallel for five independently evolved lines. Moreover, the loss of motility occurred by two genetic mechanisms: reversible and irreversible. Reversible loss of motility occurred early during broth adaptation, followed by irreversible motility loss in the majority of cells by the end of the experiment. Whole genome sequencing implicated diverse mutation events that resulted in the loss of gene expression necessary for flagellar biosynthesis. Furthermore, reversible mutations in homopolymeric DNA tracts of adenine/thymine residues, and irreversible types of mutation such as gene deletion, were discovered in the broth-evolved populations. In all evolved lines, an alternative sigma factor necessary for flagellar structural gene expression was removed from the genome. Overall, this dissertation contains the first accounts of C. jejuni experimental evolution. The results provide insight into the biological importance of reversible mutations in homopolymeric DNA tracts, and provide a basis for future studies of C. jejuni evolvability.
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