Switchgrass is a C4 perennial grass that is currently being developed for use as a second generation lignocellulosic biofuel crop. For switchgrass to be fully utilized as a bioenergy crop, large-scale plantings of elite switchgrass germplasm, possibly in monoculture, are likely to occur. This practice may increase the selection pressure on plant pathogens, such as switchgrass rust, which could result in devastating disease epidemics. The identification and deployment of quantitative trait loci (QTLs) and major plant disease resistance genes (R) in switchgrass breeding programs could offer broad spectrum and durable disease resistance in commercial switchgrass cultivars. 'Alamo', a lowland cultivar, is generally resistant to switchgrass rust whereas 'Dacotah', an upland cultivar, is highly susceptible. I hypothesized that major R genes and/or QTLs were contributing to the differences in disease phenotypes of these two cultivars. In this dissertation, bioinformatics and molecular biology approaches were employed to dissect the genetic mechanisms underlying switchgrass rust disease resistance. Novel pseudo-F2 mapping populations were created from a cross derived from 'Alamo' and 'Dacotah'. RNA-sequencing of the pseudo-F2 progenies of 'Alamo' x 'Dacotah' was used to construct a genetic linkage map and to identify potential QTLs correlating with disease resistance. In addition, a homology-based computational method was used to identify 1,011 potential NB-LRR R genes in the switchgrass genome (v 1.1). These potential R genes were characterized for polymorphism and expression differences between 'Alamo' and 'Dacotah'. Moreover, I found that some NB-LRR genes are developmentally regulated in switchgrass. One of the major objectives of switchgrass breeding programs is to develop cultivars with improved feedstock quality; however, changes in the components of the plant cell wall may affect disease resistance. I hypothesized that genetically modified switchgrass plants with altered cell wall components will respond differently than the wild-type to switchgrass rust. Transgenic switchgrass plants overexpressing AtSHN3, a transcription factor with known functions in epicuticular wax accumulation and cell wall deposition, were created. I found that AtSHN3-overexpressing transgenic switchgrass lines were more susceptible than wild-type plants in their response to switchgrass rust. Overall, the results of this dissertation provide a platform for elucidating the molecular mechanisms underlying resistance of switchgrass to switchgrass rust. These findings will help breeders create switchgrass cultivars with improved disease resistance, and will ultimately allow switchgrass to be used for sustainable biomass production. / Ph. D.

There has been increased interest in utilizing switchgrass (Panicum virgatum) as biomass. There are several challenges to developing this industry, and these have led to the potential use of switchgrass as hay for feeding beef cattle in Kentucky. The effect of increasing maturity on crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and nutritive values of switchgrass hay has been well documented, but few in vivo intake and digestibility trials have been conducted to assess this effect on animal performance when feeding beef cattle. Two in vivo intake and digestibility trials were conducted in 2011 in which Angus x Hereford beef steers (200-265 kg) were fed Alamo and Cave-in-Rock switchgrass harvested as late vegetative, boot, and early flowering hay. The objectives of these trials was to evaluate the effect of increasing maturity on apparent dry matter intake (DMI), digestible dry matter intake (DDMI), and dry matter digestibility (DMD); and to discuss potential challenges that producers might face if incorporating switchgrass hay into their forage program for feeding beef cattle. Observed decreases in nutritive value, DMI, DDMI, and DMD indicate that producers should harvest Alamo and Cave-in-Rock switchgrass before it reaches the boot stage of maturity.

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As a warm season grass native to the North American continent, switchgrass is considered as one of the most promising biofuel crops in the USA. It is a C4 plant that makes it energy efficient. Switchgrass has a deep root system that allows it to grow on marginal land with low water and nutrient input. Switchgrass has been used as a forage crop and its use for biofuel will not affect food security. Biofuels are more environment-friendly than fossil fuels as they do not produce net greenhouse gases. However, the problem of high cost of production per unit for biofuel has to be overcome if we want to replace fossil fuels with biofuels. One of the major factors related to the high cost of biofuel are the expensive cellulase enzymes used in the pretreatment of feedstock. Endoglucanase is the key enzyme used for breaking down cellulose before fermentation. Currently, endoglucanase is produced from engineered E. coli or yeast strains, which is still expensive for enzyme production and purification of industrial scales. Expression of endoglucanase in plants has been previously reported. However, there are no reports of transgenic switchgrass producing cellulase enzyme. In this study, the catalytic domain of beta-endoglucanase gene was codon-optimized and synthesized based on the cDNA cloned from Hypocrea jecorina. Rice RuBisCO small subunit targeting signal peptide was fused to the N-terminus of the beta-endoglucanase gene, which was expected to target the fusion protein to chloroplast. This subcellular compartment targeting could minimize negative effects on cell function and plant development. The endoglucanase gene was cloned with maize ubiquitin promoter in a modified binary vector pCambia 1305-2 and transformed into switchgrass genotype HR8 by using Agrobacterium tumefaciens. In this study, I generated five independent transgenic switchgrass lines and they were confirmed by growing on the selection agent hygromycin, GUS assay, PCR amplification, southern blotting hybridization, for the presence of hygromycin and endoglucanase genes. However, based on RT-PCR analysis, only two transgenic lines were confirmed to produce mRNAs of the endoglucanase gene. These two transgenic lines were further characterized for their agronomic traits and the chlorophyll contents. Our results suggested that expression of endoglucanase in switchgrass could reduce chlorophyll content and affect plant development. Nevertheless, in this study, we demonstrated that a fungal endoglucanase gene could be expressed in switchgrass transgenic plants, though the gene expression level and the subcellular localization need to be carefully regulated in order to minimize the toxic effect of endoglucanase on plant cells. / Master of Science

In the United States, the Energy Independence and Security Act of 2007 mandates the annual production of 136 billion liters of renewable fuel in the US by 2022 (US Congress, 2007). As the nation moves towards energy independence, it is critical to address the current challenges associated with large-scale biofuel production. The biomass logistics network considered consists of three core operations: farmgate operations, highway-hauling operations, and receiving facility operations. To date, decision-making has been limited in post-production management (harvesting, in-field hauling, and storage) in farmgate operations. In this thesis, we study the impacts in the logistics network resulting from the selection of one of four harvest scenarios. A simulation model was developed, which simulated the harvest and filling of a Satellite Storage Location (SSL), using conventional hay harvest equipment, specifically, a round baler. The model evaluated the impacts of four harvest scenarios (ranging from short, October-December, to extended, July-March), on baler equipment requirements, baler utilization, and the storage capacity requirements of round bales, across a harvest production region. The production region selected for this study encompassed a 32-km radius surrounding a hypothetical bio-crude plant in Gretna, VA, and considered 141 optimally selected SSLs. The production region was divided into 6 sub-regions (i.e. tours). The total production region consisted of 15,438 ha and 682 fields. The fields ranged in size from 6 to 156 ha. Of the four scenarios examined in the analysis, each displayed similar trends across the six tours. Variations in the baler requirements that were observed among the tours resulted from variability in field size distribution, field to baler allocations, and total production area. The available work hours were found to have a significant impact on the resource requirements to fulfill harvest operations and resource requirements were greatly reduced when harvest operations were extended throughout the 9-month harvest season. Beginning harvest in July and extending harvest through March resulted in reductions in round balers ranging from 50-63%, as compared to the short harvest scenario, on a sub-regional basis. On a regional basis, beginning harvest in July and extending harvest through March resulted in baler reductions up to 58.2%, as compared to the short harvest scenario. For a 9-month harvest, harvesting approximately 50% of total switchgrass harvest in July-September, as compared to harvesting approximately 50% in October-December, resulted in reductions in round balers ranging from 33.3- 43.5%. An extended (9-month) harvest resulted in the lowest annual baler requirements, and on average lower baler utilization rates. The reduced harvest scenarios, when compared to the extended harvest scenarios, resulted in a significant increase in the number of annual balers required for harvest operations. However, among the reduced harvest scenarios (i.e. Scenario 3 and 4), the number of annual balers required for harvest operations showed significantly less variation than between the extended harvest scenarios (i.e. Scenarios 1 and 2). As a result, an increased utilization of the balers in the system, short harvest scenarios resulted in the highest average baler utilization rates. Storage capacity requirements were however found to be greater for short harvest scenarios. For the reduced harvest scenario, employing an October-December harvest window, approximately 50% of harvest was completed by the end of October, and 100% of total harvest was completed by the third month of harvest (i.e. December). / Master of Science