Browsing by Author "Pearce, Stephen, committee member"

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Open Access
Field population genetics of global Puccinia striiformis f. sp. tritici populations in wheat reveal a dynamic molecular battlefield
(Colorado State University. Libraries, 2021) Lyon, Rebecca, author; Trivedi, Pankaj, advisor; Broders, Kirk, committee member; Pearce, Stephen, committee member; Hufbauer, Ruth, committee member
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Open Access
From fields to genomes: towards a comprehensive understanding of the lifestyle and evolution of Claviceps purpurea the ergot fungus
(Colorado State University. Libraries, 2020) Wyka, Stephen Andrew, author; Nalam, Vamsi, advisor; Broders, Kirk, advisor; Charkowski, Amy, committee member; Pearce, Stephen, committee member; Jahn, Courtney, committee member
Claviceps purpurea (ergot), an ascomycete and member of the family Clavicipitacea, is considered a pathogen of all grass species (family Poaecea) including economically important cereal crops which infects ovaries resulting in the development of a fungal sclerotium rather than a plant seed. Ergot infections poses significant impacts to agriculture and livestock due to various toxic alkaloids present in the sclerotia. Severe ergot poisoning in humans and livestock, ergotism, can cause corrosion/loss of extremities from gangrene, internal bleeding, diarrhea, and reduced pregnancy and abortion. Due to these serious health concerns, strict restrictions are placed on the amount of ergot contaminated grain that can be accepted for food and livestock feed. However, these toxic alkaloids are also heavily researched in the field of pharmacology and have been shown to provide some beneficial aspects in human medicine. Despite the abundance of pharmacological and agricultural research on C. purpurea researchers have been unsuccessful in identifying crop or wild grass varieties that have resistance to ergot infection, leading to critical challenges in the control of ergot disease outbreaks. Recent studies have also suggested that C. purpurea is more of a conditional defensive mutualist as opposed to a plant pathogen. Taken together, these factors demonstrate that there are still gaps of knowledge surrounding the epidemiology, lifestyle, evolution, and adaptability of this species. We implemented a comprehensive analysis into the life history of C. purpurea through a combination of field surveys, greenhouse inoculations, and deep genomic data mining to help elucidate these gaps. Field surveys were conducted to investigate the role wild grass populations surrounding cereal crop fields play in epidemiology of ergot outbreaks. Results revealed that unmanaged grasses along ditch banks, even in drought years, represent significant inoculum reservoirs of ergot, particularly when Bromus spp. are present, and should be a focal point in future research for better disease control. Greenhouse inoculations were conducted to elucidate the effects of C. purpurea infections on hosts through inoculations of a single isolate on two commercial cereal crops in a controlled setting. Our results show that the effect of C. purpurea infections can range from negative to positive, depending on infection rate, plant species, and plant tissue, but overall showed a general trend of neutral effects. However, we did observe a potential for increased root growth as infection rates increased, which could signify an interesting plant-microbe interaction that imparts a benefit, of infection, on highly rhizomatous grass hosts such as Bromus spp.. Lastly, through a collaborative effort we sequenced, assembled, and annotated 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture, plasticity, and evolution within the genera. We also conducted a detailed analysis of C. purpurea through construction of a pangenome and investigations of the recombination and positive selection landscape across the genome. Our genus-wide comparison revealed that despite having nearly identical life-strategies, these closely related species have substantially altered genomic architectures and plasticity that are likely driving genome adaptation. One key difference we observed was a shift from characteristic one-speed genomes in narrow host-range Claviceps species of sections Citrinae and Paspalorum to two-speed genomes in broader host-range lineages of sections Pusillae and Claviceps. Claviceps purpurea was observed to have a large accessory genome that is likely influenced by a large effective population size, high recombination rates, and transposable element (TE) mediated gene duplication. Due to a lack of repeat-point induced (RIP) mutation, prolific TE expansion is likely controlled by high recombination rates, which subsequently may be influencing the overall trend of purifying selection observed within the species. However, secondary metabolites genes were found to have the highest rates of positive selection on codons within genes, indicating that these genes are a primary factor affecting the diversification of the species into new ecological niches and to potentially help maintain its global distribution and broad host range.
Embargo
Herbicide resistance in kochia and wheat: the loss and gain of weed control tools for wheat production
(Colorado State University. Libraries, 2024) Montgomery, Jacob S., author; Gaines, Todd, advisor; Dayan, Franck, committee member; Pearce, Stephen, committee member; Mason, Richard, committee member
Weeds are one of the main causes of yield loss across agricultural systems worldwide. Currently, weed control in crop production systems relies heavily on the use of chemical herbicides. While these herbicides are largely effective and very efficient, they are prone to herbicide resistance evolution in weeds. Herbicide resistance evolution is the most pressing issue facing weed scientists, currently. As researchers scramble to develop new weed control technologies, herbicide resistance traits enable the use of herbicides in new cropping systems. While this is not the long-term answer to issues facing weed managers, herbicide resistance traits offer a short-term solution to systems that may be desperate for answers. Kochia is a tumble weed that was introduced to North America in the 1800's. Since its introduction, kochia has invaded many settings of the American West. Its abiotic stress tolerance, tumble seed dispersion mechanism, and ability to outcross allow it to invade new areas and maintain genetic diversity for selection to act on. In a study reported here, I identify the genetic basis of resistance to the herbicide dicamba in a population of kochia collected from Colorado. I use linkage mapping to identify a region of the genome associated with resistance. Within this locus, I find a transposable element insertion within an exon of an AUXIN/INDOLE-3-ACETIC ACID gene. This insertion causes differential splicing that changes the amino acid sequence near the degron domain in resistant plants. I apply dicamba to Arabidopsis plants expressing wildtype ii and mutant alleles of this gene to demonstrate that this mutation is sufficient in causing dicamba resistance. Protein modeling suggests that while several amino acids are affected, a specific glycine-to-threonine substitution is likely the most important in causing resistance. Finally, the mutant allele of this gene segregates with reduced plant height and biomass, suggesting this resistance mechanism has a pleotropic effect of a fitness cost. This study demonstrates the diverse ways that adaptive alleles can be generated in weedy species and describes the genetic and physiological basis of dicamba resistance in kochia. These findings may be used to design new auxinic herbicides that are not affected by this resistance mechanism. With the loss of herbicide efficacy in the face of herbicide resistance evolution, as described above, wheat producers could benefit from the availability of more weed control options. In another study presented here, I utilize gene editing and traditional mutagenesis approaches to generate wheat plants that are deficient in sulfolipids. In rice, eliminating these sulfolipids clearly results in resistance to the herbicide oxyfluorfen. Liquid chromatography coupled with mass spectrometry (LC/MS) experiments confirmed that the edits I made to the gene UTP- GLUCOSE-1-PHOSPHATE URIDYLYLTRANSFERASE 3 indeed resulted in drastic reduction in sulfolipid content. Dose response experiments showed that mutant lines gained moderate resistance to oxyfluorfen, but not lactofen, a herbicide from the same chemical family. Finally, LC/MS experiments confirmed that mutant wheat plants accumulated less porphyrin following oxyfluorfen application. The wheat lines developed in this study provide germplasm for trait introgression or a framework to make similar edits in locally adapted varieties to reproduce this herbicide resistance trait. If stewarded correctly, the trait will extend the effective lifespan of currently used herbicides in wheat and improve weed control.
Open Access
Plasticity, allelic diversity, and genetic architecture of industrial hemp (Cannabis sativa L.)
(Colorado State University. Libraries, 2019) Campbell, Brian J., author; McKay, John, advisor; Jahn, Courtney, committee member; Pearce, Stephen, committee member; Brick, Mark, committee member
The first time in United States history that hemp was legally distinguished from high-THC Cannabis (marijuana) was in 2014 when the Farm Bill was passed. Although the two crops had been distinguished by their usage for thousands of years, their monospecific nature led to both psychoactive and non-psychoactive forms being legislated in tandem from the time that Americans began regulating Cannabis cultivation and usage. A simple statement in the 2014 Farm Bill distinguished hemp as Cannabis sativa L. with a tetrahydrocannabinol (THC) content of 0.3% or less. A second sentence enabled research into the crop and production within pilot programs in states where it is legal. This minor change in legality, followed by subsequent relaxation of laws surrounding hemp in the 2018 Farm Bill, has allowed a burgeoning hemp industry to form in the United States and enabled the return of a relict crop. Due to the long period of prohibition, hemp did not undergo the same type of crops research as other staple American crops. Consequently, little is known about the genetic mechanisms that control many of the key traits in hemp production. Understanding basic information about how traits are affected by environmental factors is highly important when regulation of the crop is based on a stringent and arbitrarily set threshold for chemical content. In 2016, we performed field trials of a diverse set of industrial hemp cultivars in multiple growing environments and assessed a wide range of traits. Expression of some traits, like days to maturity and THC content, were strongly influenced by genotype. Other traits, such as grain yield and plant height, exhibited large proportions of variance due to environmental factors and genotype-by-environment interactions. There were also varying ranges of plasticity exhibited between cultivars, underscoring the importance of selecting the right cultivar for target production environments. This highlights the importance of thoroughly characterizing genotype-by-environment interactions when breeding locally adapted hemp cultivars. Understanding genetic control of important traits and their range of plasticity enables the development of locally adapted cultivars for a wide range of end uses. Another aspect of Cannabis that is understudied is the genetic basis for differentiating hemp and high-THC Cannabis. Since the legal distinction is based on a strict threshold placed on a quantitative trait and not any known geographic or biological reproductive barriers, it is unclear whether or not there is genetic evidence to support the distinction or if the two groups are simply divergent phenotypes. A joint-site frequency and FST analysis show that individuals of the two groups mainly share common polymorphisms, with a small number of loci where differentiation occurs. These loci serve as the basis for distinguishing the two groups, but more study is needed to determine if alleles in these regions were driven to fixation via genetic drift and selection on unrelated traits, or if there is an evolutionary basis for the observed differences. When heterozygosity was assessed in these samples, the hemp group had higher overall heterozygosity levels, but the high-THC Cannabis group had more outliers which lead to a wider distribution with more extreme minimum and maximum values. Although it is clear that there are genetic differences distinguishing the two groups, extensive human vectoring and admixture between the groups, both historically and currently, makes it difficult to differentiate causes for the differences. A lack of centralized germplasm makes large-scale genomic studies of the species difficult, but, as more samples are surveyed over time, a more detailed picture of the genomic variation will emerge. These types of studies will be able to provide a more nuanced picture of the evolutionary history and current state of allelic variation within the species. In addition to plasticity and allelic diversity, genetic architecture of traits has also largely been ignored until recently. The first QTL study in Cannabis was performed in 2015 and was limited by legal restraints. Since understanding how economically relevant traits function is important to breeding improved hemp cultivars, we developed a genetic mapping population that captured variation for a wide range of traits. Utilizing whole-genome sequencing and phenotype data from a replicated field trial, we were able to detect 121 QTL associated with 38 agronomic and biochemical traits. Some traits, like days to maturity, had single loci of large effect accounting for the majority of trait variance, while other traits, like α-Pinene production, exhibited more complex polygenic architecture with epistatic interactions. Colocalization of QTL and significant trait correlations showed that there were positive relationships within both agronomic and biochemical trait groups. Although this study was limited by assessment of the population in a single environment, detecting these putative QTL serves as a substantial step forward in characterizing many relevant production traits.
Open Access
Quizalofop-resistant wheat: biochemical characterization of the AXigen™ trait and corresponding metabolism
(Colorado State University. Libraries, 2021) Bough, Raven A., author; Dayan, Franck E., advisor; Gaines, Todd A., committee member; Haley, Scott, committee member; Pearce, Stephen, committee member
A new weed management tool in wheat, the CoAXium™ Wheat Production System, incorporates quizalofop-resistant wheat, a specialized formulation of quizalofop (Aggressor™), and a stewardship management program for effective management of annual grasses with otherwise limited control options. The AXigen™ trait confers resistance primarily through a single-point mutation in ACC1. The mutation causes an alanine to valine substitution at position 2004 in wheat acetyl-CoA carboxylase (ACCase) relative to the Alopecurus myosuriodes reference. Through greenhouse and biochemical studies paired with protein homology modelling and simulations, the research presented herein provides strong evidence that a conformational change imparted by the amino acid substitution results in quizalofop-resistant ACCase. Conversely, the mutation conveys negative cross-resistance to haloxyfop, a similar herbicide to quizalofop with a smaller molecular volume. The remaining research objectives focus on quizalofop metabolism in CoAXium™ wheat. Liquid chromatography-mass spectrometry measurements of quizalofop content over time from liquid demonstrate cooler temperature conditions (4.5°C) delay quizalofop metabolism by 4 times compared to warmer temperature conditions (19°C). Reduced temperatures also delay quizalofop metabolism to the same extent in the following annual grass weed species: Aegilops cylindrica, Bromus tectorum, and Secale cereale. Further, additional studies suggest herbicide metabolism mechanisms enhance overall CoAXium™ wheat quizalofop resistance. Despite similar ACCase resistance, resistant winter and spring wheat varieties convey varying degrees of whole-plant resistance. In winter wheat but not spring wheat, increased resistance corresponds to a shorter quizalofop half-life, implying faster metabolism boosts overall resistance. Treatment of resistant spring wheat varieties with cloquintocet, a metabolism-boosting safener, increases overall resistance. Follow-up differential expression analysis of cloquintocet-treated plants may support differential metabolism findings and lead to identification of putative candidate genes associated with upregulated herbicide metabolism, such as cytochrome P450 monooxygenases, glutathione-S-transferases, and glycosyltransferases.
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 member
Weed 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.
Open Access
Understanding the molecular basis of insect pest resistance in Triticum aestivum using mass spectrometry
(Colorado State University. Libraries, 2018) Lavergne, Florent D., author; Heuberger, Adam, advisor; Broeckling, Corey, committee member; Pearce, Stephen, committee member; Jahn, Courtney, committee member
Bread wheat (Triticum aestivum L.) is a global staple crop and controlling for environmental stress that impacts grain yield is critical. Recently, Wheat Stem Sawfly (Cephus cinctus, hereafter WSS) has emerged as a new pest of wheat and is expanding across the Great Plains and southern United States. WSS is difficult to control using chemical, cultural or biological pest management methods. Currently, wheat breeders utilize a solid-stem trait to inhibit larval feeding and reduce lodging, however this trait only confers partial resistance and is thought to reduce grain yield. Models of metabolic-based resistance with demonstrated impact on reduction of insect pest fitness have been documented. Here, I investigate the broader hypothesis that wheat resistance to WSS is mediated by shifts in metabolism that promote avoidance and toxicity towards WSS. Four cultivars with contrasting phenotypes are used in our studies: Hatcher (resistant to WSS, hollow-stem, winter wheat); Conan (resistant, semi-solid-stem, spring); Denali (susceptible, hollow-stem, winter); and Reeder (susceptible, hollow-stem, spring). The first part of this work involved gas chromatography-mass spectrometry (GC-MS) metabolomics methods to provide a comprehensive characterization of the chemical composition of wheat cuticular waxes. A total of 263 putative compounds were detected among the four abovementioned wheat cultivars and comprised 58 wax compounds including alkanes and fatty acids. Many of the detected wax metabolites have known associations to important biological functions such as insect pest and drought resistance. Uni- and multivariate statistics were used to evaluate metabolite distribution between tissue types (leaf, stem) and cultivars. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were complemented using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan (resistant to WSS) was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. The second part of this study aimed at evaluating a selection of wheat cultivars in a WSS-infested field. Cultivars with increased yield and reduced WSS infestation values were found. The molecular basis of this resistance was evaluated in a greenhouse study that characterized proteomic and metabolomic signatures of wheat stems associated with WSS infestation. Stem proteins (1832) and metabolites (1823) were detected in the same four wheat cultivars using liquid chromatography-mass spectrometry. During infestation with WSS, 62 proteins and 29 metabolites were differentially regulated in the hollow-stem resistant cultivar Hatcher. Metabolic processes that were associated with resistance included enzymatic detoxification, proteinase inhibition, and anti-herbivory compound production, specifically the benzoxazinoids, neolignans, and phenolics. Compared to the semi-solid and resistant cultivar Conan, hollow-stem Hatcher had increased abundance of proteins and metabolites with known roles in plant defense against insects. These results will be invaluable to plant breeders as they contribute to the understanding of wax composition and metabolic regulation associated with important phenotypic traits in a major crop, including passive and active defense mechanisms to WSS.