Browsing by Author "Sloan, Daniel, committee member"
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Item Open Access aPPRove: an HMM-based method for accurate prediction of RNA-pentatricopeptide repeat protein binding events(Colorado State University. Libraries, 2015) Harrison, Thomas, author; Boucher, Christina, advisor; Ben-Hur, Asa, committee member; Sloan, Daniel, committee memberPentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The P class contains tandem P-type motif sequences, and the PLS class contains alternating P, L and S type motif sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a PLS-class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. [1]. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the PLS class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for PLS-class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular we use our method to make a conjecture regarding a novel binding event between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at www.cs.colostate.edu/~aPPRove and the soft- ware is available at that website for stand alone usage.Item Open Access Decreased dicamba transport due to increased flavonoid biosynthesis: a candidate dicamba resistance mechanism(Colorado State University. Libraries, 2016) Pettinga, Dean J., author; Gaines, Todd A., advisor; Ward, Sarah, committee member; Sloan, Daniel, committee memberResistance to dicamba (a synthetic auxin herbicide) has been documented in Kochia scoparia (L.) Schrad. populations since 1994, but the molecular mechanisms of observed resistance cases remain elusive. An RNA-Seq approach was used to identify transcripts with significantly differential transcription responses between inbred lines of dicamba-resistant (9425R) and dicamba-susceptible (7710S) K. scoparia in response to dicamba application. Among the significantly differentially expressed transcripts was both Chalcone Synthase (CHS), the first enzyme and rate-limiting step in the flavonoid biosynthesis pathway, and Flavono 3'-Hydroxylase (F3'H), which catalyzes the conversion of quercetin into kaempferol, known inhibitors of auxin transport. In silico expression patterns of both transcripts were confirmed with qRT-PCR. An F2 population derived from a cross of 9425R x 7710S segregating for the resistance phenotype was assayed for CHS and F3'H expression using qRT-PCR. Dicamba-resistant F2 individuals displayed significantly higher CHS transcript abundance compared to dicamba-susceptible F2 individuals, associating the resistance phenotype of 9425R with a greater overall flux through the flavonoid biosynthesis pathway. Increased production of the auxin transport inhibitors quercetin and kaempferol could reduce intercellular transport and vascular loading of dicamba, causing a substantial reduction in dicamba efficacy by reducing its translocation to sensitive meristematic tissue, thereby conferring the observed resistance phenotype.Item Embargo DNA replication in the environmental extremes(Colorado State University. Libraries, 2024) Liman, Geraldy Lie Stefanus, author; Santangelo, Thomas J., advisor; Markus, Steven, committee member; Schauer, Grant, committee member; Sloan, Daniel, committee memberDNA replication is an essential biological process across all life on Earth. For the prokaryotic Archaea domain, which contains organisms that can thrive in inhospitable environments like hydrothermal vents or salt deposits in the Dead Sea, the cell machinery for these conserved processes have acclimated over the course of evolution to encourage survival. While the origin of replication (ori), a predetermined position within the genome where DNA replication starts, is conserved in all Domains, its significance is not equal between them. Surprisingly, the model hyperthermophilic archaeon, T. kodakarensis, replicates its genome without relying on origin-dependent replication (ODR), and instead, relies mostly on recombination-dependent replication (RDR). In fact, the ori in T. kodakarensis is dispensable from the organism without much phenotypic consequence. Although dispensable, ori persists after millions of years of evolution in this organism, suggesting some functional significance under certain conditions. Not to mention, archaeal replisomes are comprised of unique components that are distinct from the other two domains of life, though surprisingly more similar to those found in Eukarya. Central to all replisomes is the activity of the DNA polymerase (DNAP). Most archaeal organisms, except for the Creanarchaea, encode two main replicative DNAPs, the eukaryotic-like B-family DNAP (PolB) and the archaeal-specific D-family DNAP (PolD). In T. kodakarensis, PolD is the essential replicative DNAP while PolB is dispensable. This thesis aims to (1) characterize the activity and regulation of RadA, the main recombinase in Archaea, (2) characterize the exaptation of inteins to regulate DNA replication, (3) delineate the in vivo function(s) of PolB. Furthermore, I hope to further characterize DNA replication in the context of evolutionary biology and how it relates to the three Domains of life.Item Open Access Evaluation of wastewater as a nutrient source for the cultivation of the model cyanobacterium Synechocystis sp. PCC6803(Colorado State University. Libraries, 2017) Hughes, Alexander, author; Peers, Graham, advisor; Sloan, Daniel, committee member; Reardon, Kenneth, committee memberThe rising demand for more sustainable and renewable energy sources has led to the development of using photosynthetic microalgae and cyanobacteria for biofuel feedstocks. Microalgae and cyanobacteria offer an attractive solution over the cellulosic and lignocellulosic feedstocks of first and second generation biofuels that compete for arable land, nutrients and water necessary for sufficient food crop production. It has been proposed for several decades that wastewater could be a sustainable and affordable source of water and nutrients. Phycoremediation of wastewater by microalgae as biofuel feedstocks could provide beneficial environmental health impacts by preventing eutrophication of fresh water supplies. Many eukaryotic microalgae have been grown in diluted and/or modified wastewaters. The growth of cyanobacteria on wastewaters has not been nearly as well characterized. Cyanobacteria grown on wastewaters could be an ideal feedstock for advanced biofuels, since cyanobacteria have a more extensively established molecular toolbox for genetic engineering. The first aim of this thesis was to evaluate wastewater centrate as a growth medium for the cultivation of the cyanobacterium Synechocystis sp. strain PCC6803 (Chapter 1). Centrate was collected from the Drake Water Reclamation Facility (Fort Collins, CO) and filter sterilized to allow axenic culturing of PCC6803 under controlled laboratory conditions. PCC6803 was grown in up to 21% filtered centrate diluted in sterile water; while higher concentrations completely inhibited growth. Nitrogen drawdown from centrate by PCC6803 was then analyzed. Surprisingly, the drawdown of nitrogen from the centrate media correlated poorly with the amount of cyanobacterial biomass. The cell densities of cultures grown in centrate were all significantly lower than that of PCC6803 grown in BG-11 indicating that diluted centrate does not provide adequate nutrients to support optimal growth. Abiotic precipitation of nitrogen was then determined to dominate the removal of nitrogen from the cultivation media. Furthermore, it suggested that centrate lacks a critical nutrient to support robust growth of PCC6803. The second aim of this thesis was to augment the nutrient composition of wastewater in order to optimize PCC6803 growth and nutrient removal (Chapter 2). A series of bioassays were performed to elucidate the limiting macronutrient in centrate. Adding 304 μM Na2SO4 – equivalent to the concentration of SO42in BG-11 media – yielded final cell densities that were only 4% lower than those observed in cultures grown in the synthetic, standard media (BG-11). Exogenous supplementation of Na2SO4 also improved total dissolved nitrogen (TN) drawdown for centrate grown PCC6803 cultures. In Na2SO4 amended centrate, PCC6803 was able to grow to significantly higher cell densities, permitting the removal of 69% of the TN in diluted centrate. Transcript abundance of the sulfate transporters encoded by the spbA-cysTWA operon were found to be upregulated when grown in centrate, confirming that PCC6803 experienced S-limitation during growth on this media. Hydrogen sulfide gas (H2S) is an undesirable product of the biological nutrient removal process due to its pungent odor. Currently, H2S produced at the DWRF is vented to biofilters consisting of wood chips and compost where sulfide oxidizing microbes convert sulfide into elemental sulfur. Therefore, endogenously sourced sulfur supplementation from H2S into centrate could provide a viable sulfur source to support the cultivation of PCC6803. We have shown that sulfur supplementation improves the phycoremediation of nutrients in centrate. Cultivation of PCC6803 on centrate supplemented with endogenously sourced sulfur provides an industrially feasible method for combining wastewater treatment with advanced biofuel production.Item Open Access Explorations in West Nile virus ecology and evolution(Colorado State University. Libraries, 2021) Byas, Alexandria D., author; Ebel, Gregory D., advisor; Bowen, Richard, committee member; Kading, Rebekah, committee member; Sloan, Daniel, committee memberWest Nile virus (WNV) continues to be a major cause of human arboviral neuroinvasive disease. Susceptible non-human vertebrates are particularly diverse, ranging from commonly affected birds and horses to less commonly affected species such as alligators. The literature review in Chapter 1 summarizes the pathology caused by West Nile virus during natural infections of humans and non-human animals. While the most well-known findings in human infection involve the central nervous system, WNV can also cause significant lesions in the heart, kidneys and eyes. Time has also revealed chronic neurologic sequelae related to prior human WNV infection. Similarly, neurologic disease is a prominent manifestation of WNV infection in most non-human non-host animals. However, in some avian species, which serve as the vertebrate host for WNV maintenance in nature, severe systemic disease can occur, with neurologic, cardiac, intestinal and renal injury leading to death. The pathology seen in experimental animal models of WNV infection and knowledge gains on viral pathogenesis derived from these animal models are also briefly discussed. A gap in the current literature exists regarding the relationship between the neurotropic nature of WNV in vertebrates, virus propagation and transmission in nature. This and other knowledge gaps, and future directions for research into WNV pathology, are addressed. In Chapter 2, experimental evolution work is described. For arboviruses, the vertebrate and invertebrate hosts in which they circulate shape viral evolution and can lead to the emergence of new genotypes. Previous work in mosquitoes and birds has identified species-specific effects on viral populations when species were assessed in isolation. We united mosquito and bird species to perform experimental evolution studies which paired Culex (Cx.) pipiens with American crows, Cx. quinquefasciatus with American crows and Cx. quinquefasciatus with American robins. Crow and Cx. pipiens transmission cycles were the most successful and robin and Cx. quinquefasciatus transmission cycles were the least successful at reaching three complete rounds of bird-to-mosquito transmission. These findings suggest that crows may be more important to WNV maintenance in nature over robins. The greater success of crow cycles when paired with Cx. pipiens in comparison to crows paired with Cx. quinquefasciatus may also suggest fitness losses associated with Cx. quinquefasciatus. In multiple rounds of transmission, infection rates (WNV-positive mosquito midgut) and transmission-capability (WNV-positive mosquito saliva) decreased with each subsequent round of transmission, suggesting that pairings in isolation experience fitness losses. Competitive fitness assays of transmission cycles exhibited cyclical increases and decreases in fitness as virus moved through crows and mosquitoes, respectively. That the stronger competitive fitness tended to occur with samples from the avian host while virus from mosquitoes tended to have decreased fitness may be consistent with genetic restriction and strong purifying selection in birds and genetic expansion and weak purifying selection in mosquitoes. Sequencing is needed to assess whether differences in transmission cycle success and competitive fitness can be attributed to genetic changes. In Chapter 3, the avian single cell viral environment is assessed. Error-prone replication of RNA viruses generates the viral diversity required for adaptation to rapidly changing environments. This is crucial for arboviruses whose viral populations exist as mutant swarms maintained between both mosquito and vertebrate hosts. By infecting cells and birds with barcoded WNV stock and sequencing single cells, we demonstrated that the richness and frequency of rare variants in crows far exceeded that found in robins. Moreover, those rare occurring variants were maintained by crows more than they were by robins. We further demonstrated that bird viremia functions as a determinant of multiplicity of infection in peripheral blood mononuclear cells (PBMCs), a significant site of viral replication. We found that increased viremia leads to increased polyinfections of individual PBMCs with maintenance of defective genomes and less prevalent variants, specifically in crows, presumably through complementation. When two pairings of variably-fit viruses were used to co-infect American robins and American crows, we observed increases in replication for one of the less fit viruses when viremia was higher. The ability of the low fitness virus to better replicate at higher viremia is likely a result of polyinfections and complementation at the cellular level. Our findings suggest that weak purifying selection in highly susceptible crows is attributable to higher viremia, polyinfections and complementation while viral divergence and fewer variants rising to fixation in robins is a result of overall lower levels of viremia and fewer polyinfections. In Chapter 4, the potential contributions of American alligators to natural WNV ecology are examined. West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle. We performed a diverse array of experiments which utilize novel techniques and technologies to characterize the mechanisms of WNV evolution. We also identified a potential non-avian WNV amplifier host in alligators. This work represents a significant contribution to the West Nile virus literature by working with the unique species which contribute to virus propagation and assessing their effects on viral evolution and ecology.Item Open Access Floristic inventory of private and public lands in southwestern Gunnison County, Colorado, A; and a software tool to assist in the generation of herbarium specimen labels(Colorado State University. Libraries, 2018) Maher, Madeline A., author; Simmons, Mark, advisor; Maurer, Ruth, committee member; Sloan, Daniel, committee member; Osborne Nishimura, Erin, committee memberI conducted an inventory of vascular flora on public and private property in southwestern Gunnison County, Colorado. The study area consisted of 3,004 acres of private land and 1,850 acres of adjacent public land managed by the Bureau of Land Management (BLM). While a small part of the BLM area was surveyed in the late 1990s, the majority of the study area represented a gap in the existing floristic research to date in the Rocky Mountains. Fieldwork was conducted in the growing seasons of 2016 and 2017. Six hundred five herbarium specimens were generated, representing 315 species and infraspecific taxa. Combining my findings with reliable observations and existing collections from the area, a checklist of 330 species and infraspecific taxa was compiled, representing ten percent of the known Colorado flora. The variable landscape of the area includes submontane to subalpine forests, wetlands, grasslands, sagebrush shrublands, xeric mesa tops, and rocky cliffs. Taken together these areas provide habitat for two species previously unknown in the county (Trifolium kingii, Pyrola picta), two further species that are considered vulnerable or imperiled in Colorado (Iliamna rivularis, Draba rectifructa), and 30 vulnerable or imperiled plant community types. The presence of species and communities of conservation concern not previously documented in the region emphasizes the need for continued floristic study of private lands and other undersampled areas. In order to efficiently make labels for the aforementioned herbarium specimens, I developed a Python program to generate formatted, print-ready labels from a comma-separated value (CSV) file. Efficiently generating specimen labels can be challenging for newcomers to the field, or for those unable to use existing tools. Several software tools exist for automatic specimen label generation, but these either require a significant learning curve to use, are not consistently maintained, are not compatible with multiple operating systems, or rely on proprietary software. My program generates labels in HTML, and these are formatted for print using Cascading Style Sheets (CSS) and the Mustache template system. The program does not rely on any proprietary software, is compatible with any operating system on which Python 3.0 or later can be installed, and is easily customizable to suit the user's needs. The minimal nature of the tool makes it easy and efficient to use. The tool will be especially useful to students learning to manage their collection data and produce their own specimen labels. As such, it could be a convenient resource for plant-taxonomy and related courses in which students make collections.Item Open Access Myocardial afterload regulates atrioventricular valve development in zebrafish(Colorado State University. Libraries, 2020) Ahuja, Neha, author; Garrity, Deborah, advisor; Sloan, Daniel, committee member; Mykles, Don, committee member; Tjalkens, Ron, committee memberThe incidence of congenital heart disease (CHD) is estimated to be 1% of all human births. CHD of the heart valves occurs in over 50% of CHD cases. Despite significant clinical interest, the molecular mechanisms that govern valve development remain poorly elucidated. As the heart develops, blood flow and blood pressure increase rapidly to support the growing demands of the embryo. Our group has previously shown that pressure at the developing atrioventricular valve dramatically increases through development. Consequently, we hypothesized that afterload—defined as the pressure the ventricle must overcome in order to pump blood through the body—may be a cue that cardiac valve cells read and respond to build a valve leaflet. Here, we present a zebrafish model in which afterload has been increased through the use of vasopressin, a vasoconstrictive drug. We first show that application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. To evaluate cardiac function and valve leaflet dynamics, we took a quantitative live-imaging approach. Consistent with pathology seen in adult human patients with clinically high afterload, we see defects in both form and function of the valve leaflets. To identify the cause of this functional defect, we utilized in situ hybridization to evaluate makers of cell differentiation for both valve leaflet cells and the adjacent myocardial cells. Our results suggest that this valve defect is due to changes in atrioventricular myocyte differentiation and signaling, rather than pressure directly acting on the valve leaflet cells. We next took a transcriptomics approach to identify regulators of atrioventricular myocyte differentiation and identified a subset of differentially expressed transcription factors that are putatively responsible for sensing afterload. Together, our results show that afterload regulates the physiological and molecular state of the developing valve.Item Open Access RNA sequencing identifies genes putatively involved at the aphid-Buchnera symbiotic interface(Colorado State University. Libraries, 2020) Acharya, Shailesh Raj, author; Nalam, Vamsi J., advisor; Leach, Jan, committee member; Sloan, Daniel, committee memberIn aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium, Buchnera aphidicola. The endosymbiont converts abundant non-essential amino acids into essential amino acids that are supplied to the aphid. The long-term goal of the proposed work is to exploit the biochemical interdependence that exists between soybean aphid (Aphis glycines) and its primary endosymbiont to develop effective resistance in soybean (Glycine max). Little is known of the A. glycines and soybean amino acid transporters (AATs) that facilitate this exchange. The soybean aphid is the most important arthropod pest on soybean in North America and aphid outbreaks in major soybean growing regions of the country in the past has resulted in yield losses of up to 40%. In the current study, we used RNA-seq to identify amino acid transporters involved in the exchange of amino acids between the aphid and its endosymbiont. A total of 2121 genes were differentially expressed between the aphid and bacteriocytes with 516 genes showing up-regulation, while 1605 genes were down-regulated in the bacteriocytes. Analysis of GO terms revealed enrichment in membrane and transport associated processes. Our RNA-seq analysis of differentially expressed genes showed that one putative amino acid transporter: 72-RA, is up-regulated in the bacteriocytes. This work represents a first step towards understanding aphid dependency on its endosymbiotic bacteria and target them as a means of a novel aphid control strategy.Item Open Access The effects of genome expansion on transposable element diversity in salamanders(Colorado State University. Libraries, 2021) Haley, Ava, author; Mueller, Rachel, advisor; Sloan, Daniel, committee member; Stenglein, Mark, committee memberTransposable elements (TEs) are repetitive sequences of DNA that replicate and proliferate throughout genomes. Taken together, all the TEs in a genome form a diverse community of sequences, which can be studied to draw conclusions about genome evolution. TE diversity can be measured using ecological models for species distribution that consider richness and evenness of communities. It is currently not well studied how genome expansion impacts the diversity of transposable elements. However, there are a few models that predict TE diversity decreasing as genomes expand due to varying mechanisms such as selection against ectopic recombination and competition between TEs and silencing machinery. Salamanders are known to have some of the largest vertebrate genomes. Salamanders of the genus Plethodon in particular have very large genomes consisting of high levels of TEs, with sizes ranging from 30 to 70 Gigabases (Gb). Here, I use Oxford Nanopore sequencing to generate low-coverage genomic sequences for four species of Plethodon that encompass two independent genome expansion events, one in the eastern clade and one in the western clade: Plethodon glutinosus (41.4 Gb), P. cinereus (30.5 Gb), P. idahoensis (71.7 Gb), and P. vehiculum (50.5 Gb). I classified the TEs in these datasets using RepeatMasker and DnaPipeTE and found ~51 superfamilies which accounted for 27-32% of the genomes. For each genome I calculated the Simpson's and Shannon's diversity indices to quantify diversity, taking into account both TE richness and evenness. In all cases, the values for Simpson's index were within 0.75 and 0.79, and for Shannon's index all species were within 1.88 and 1.99. We conclude that once genomes reach large sizes, they maintain high levels of TE diversity at the superfamily level, in contrast to observations made by previous studies done on smaller genomes.Item Embargo The epitranscriptome in heat-loving Archaea enhances thermophily(Colorado State University. Libraries, 2023) Fluke, Kristin Alison, author; Santangelo, Thomas J., advisor; Wilusz, Carol, committee member; Sloan, Daniel, committee member; Abdo, Zaid, committee member>170 RNA modifications are known to decorate the transcriptome across all three Domains of life. The totality of RNA modifications in a cell is called the epitranscriptome. Modifications expand the form and function of RNA, often invoking new structures, activities, and interactions. The molecular consequences, fitness impacts, transcriptome-wide distribution, and genesis of the vast majority of modifications are largely unknown, but more > 100 human diseases are linked to mutations in the genes that encode RNA modifying enzymes. It is therefore critical to elucidate the generation and impact of RNA modifications on fitness and function. 5-methylcytidine (m5C) is one of the most abundant and conserved modifications across Domains and is generated through the post-transcriptional activities of several RNA m5C methyltransferases (R5CMTs). RNA modifications, especially m5C, have largely been studied in the context of abundant rRNA and tRNAs while research into the impact of mRNA modifications is lacking due to their low abundance in the cell. Archaeal model organisms have been shown to incorporate a higher abundance of select modifications compared to Eukarya, proving a new avenue to resolve fundamental questions regarding the phenotypic consequences of epitranscriptomic changes. In the model hyperthermophilic archaeon, Thermococcus kodakarensis, I comprehensively mapped m5C to the transcriptome. I identified at least five R5CMTs that site-specifically generate m5C and showed an unprecedented level of m5C incorporation that includes 10% of unique transcripts, mainly in mRNA. I demonstrated that R5CMTs target mRNAs for modification with both sequence and structural specificity. Cells lacking m5C exhibit a severe temperature dependent growth defect, indicating the m5C epitranscriptome is critical for cellular fitness under heat stress. The extensive m5C epitranscriptome coupled with the large collection of R5CMTs indicate that T. kodakarensis is the ideal model system to pursue fundamental questions regarding the epitranscriptome. Efforts to identify RNA methyltransferases that install m5C led to the discovery of a novel modification, N4,N4-dimethylcytidine (m42C) and the enzyme responsible for its in vivo and in vitro installation. I showed that m42C is robustly resistant to bisulfite-driven deamination, potentially indicating that all bisulfite-sequencing datasets may be falsely reporting m5C sites that are instead occupied by m42C. I mapped a single m42C residue to the ribosomal decoding center in the 16S rRNA and showed that cells lacking m42C exhibit a severe growth defect at higher temperatures. Structural studies of the enzyme that generates m42C, tentatively named m42C synthase, demonstrate it adopts a canonical class I Rossman fold at the C-terminal lobe and a unique N-terminal lobe. I showed that m42C synthase methylates assembled ribosomes and defined the catalytic amino acid residue. Taken together, I report a novel writer enzyme and show that both m5C and m42C promote hyperthermophilic growth. The dense and chemically diverse epitranscriptome argues that Thermococcus provides an excellent model system for further epitranscriptomic studies that probe the impact of both ubiquitous and rare modifications on core biological processes.Item Open Access The genetics and genomics of herbicide resistant Kochia scoparia L.(Colorado State University. Libraries, 2018) Patterson, Eric L., author; Gaines, Todd, advisor; Saski, Chris, committee member; Sloan, Daniel, committee member; Pearce, Stephen, committee memberWeed genomics resources lag behind other plant biology disciplines despite larger annual crop losses occurring due to weeds than to plant pathogens or invertebrate pests. To date only a handful of weed genomes are assembled, and what is available is generally incomplete, poorly annotated, or only useful to a small group of researchers. Recent advancements in sequencing and an increased interest in the genetic foundations of weedy traits have contributed to driving de novo genome assemblies for key weed species. The introduced weed species Kochia scoparia (kochia) is the most important weed species in Colorado and severely impacts yield in various crop systems including sugar beet, wheat, and corn. Additionally, kochia rapidly invades disturbed land including roadsides, drainage areas, rangelands, and pastures. Kochia spans a massive geographic distribution, from as far south as Mexico, as far north as Saskatoon, Canada, as far east as the Mississippi river, and as far west as Oregon. Locally, kochia populations are well adapted to various abiotic stresses including drought, cold, high salinity, and high wind. Recently, and most importantly, kochia has evolved resistance to several modes of herbicide action. Currently kochia populations exist that are resistant to acetolactate synthase (ALS) inhibitors, photosystem II (PSII) inhibitors, several synthetic auxin compounds, and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor, glyphosate. Individuals have even been identified that are resistant to all four modes of action (MOA) simultaneously. Each herbicide mode of action (MOA) resistance case is caused by different mutations or even different mutation types (target site SNPs, copy number variation, translocation changes, etc.). Selection pressure from herbicides is intense as not having the proper allele is lethal; therefore, resistance alleles are selected and go to fixation quickly. Kochia populations may be especially prone to herbicide resistance for a variety of physiological reasons, as kochia plants can produce thousands of seeds, are wind pollinated, are primarily outcrossing, and have tumbleweed seed dispersal in the windier environments like eastern Colorado and Kansas. Additionally, there may be genetic and genomic explanations for rapid herbicide resistance evolution such as rapid mutation rates or dynamic responses to environmental stress. Glyphosate resistance, in particular, has driven a significant amount of herbicide resistance research in this species. In this case, resistance is caused by copy number variation of the target gene, EPSPS. Over production of the EPSPS enzyme makes normally lethal doses of glyphosate inadequate for control. Many of the details underlying gene amplification are missing, such as what are its origins and what genes are included in the duplication event. Understanding mechanisms of gene duplication is fundamental to understanding the evolution of resistance, predicting future gene duplication events, and understanding the significance of fitness and inheritance studies.Item Open Access The use of metagenomic sequencing as a tool for pathogen discovery with further investigation of novel reptilian serpentoviruses(Colorado State University. Libraries, 2019) Hoon-Hanks, Laura L., author; Stenglein, Mark, advisor; Ebel, Gregory, committee member; Sloan, Daniel, committee member; Han, Sushan, committee memberTo view the abstract, please see the full text of the document.Item Open Access Transcriptome and elemental analysis of the selenium hyperaccumulator Stanleya pinnata and non-accumulator Stanleya elata(Colorado State University. Libraries, 2015) Wang, Jiameng, author; Pilon-Smits, Elizabeth, advisor; Sloan, Daniel, committee member; Stargell, Laurie, committee memberTo view the abstract, please see the full text of the document.Item Open Access Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and development of molecular tools for early detection and monitoring(Colorado State University. Libraries, 2019) Dobbs, John, author; Stewart, Jane, advisor; Kim, Mee-Sook, committee member; Sloan, Daniel, committee memberPathogenic and non-pathogenic Fusarium oxysporum are morphologically indistinguishable from each other. Pathogenic F. oxysporum f. sp. koae (Fo koae) is a limiting factor for low to mid elevation, below 610m (2000 ft), Acacia koa forests and timber stands. These warmer, lower elevation sites are best suited for optimal growth of Acacia koa, but Fo koae hinders efforts to establish stands at these elevations. Detection of pathogenic isolates is necessary for informing land managers and disease resistance breeding programs. Current methods to distinguish pathogenic isolates are conducted through costly, extensive greenhouse virulence assays. Genomic comparisons of pathogens and non-pathogens such as amplified fragment length polymorphisms (AFLPs), single nucleotide polymorphisms (SNPs), Random Amplification of Polymorphic DNA (RAPDs), pathogenicity-related genes, and microsatellites have been effective for detecting pathogens, and these genomic comparisons are more time and cost effective than traditional greenhouse virulence assays. Chapter two of this thesis examined whole genomic comparisons of a pathogenic F. oxysporum f. sp. koae (Fo koae 44) and a F. oxysporum (Fo 170) isolate found to be non-pathogenic to Acacia koa, for the identification of genomic features that could be used to distinguish pathogenicity. Genome sizes were comparable at 48Mb and 50Mb, respectfully. Fo koae 44 and Fo 170 shared an average nucleotide identity of 96%. Eleven syntenic putative core chromosomes and one unique putative lineage-specific chromosome were identified when compared to a reference strain of F. oxysporum f. sp. lycopersici. Pathogenicity-related genes, including the secreted in xylem (SIX) genes, Fusarium transcription factors, and Fusarium transporters, and unique sequences were identified as exclusive to Fo koae 44 when compared to Fo 170. These variants were used to develop pathogen-specific primers. When tested on previously characterized pathogenic (highly and moderate virulence) Fo koae and low virulent or non-pathogenic Fo isolates, six primers only amplified moderate and highly virulent isolates of Fo koae. Haplotype networks were constructed based on sequencing data of previously characterized Fo koae and Fo isolates and field collected isolates with no pathogenicity data at the translation elongation factor 1-α and RNA polymerase II second largest subunit to determine the genetic relationships of these two groups. Some field collected isolates grouped with highly virulent Fo koae isolates. These results suggested that these field collected isolates might be highly virulent and contain the putative lineage-specific chromosome.