Doctor of Philosophy / Genetics Interdepartmental Program-Agronomy / William T. Schapaugh Jr / The soybean aphid (Aphis glycines Matsumura) has been one of the major pests of soybean [Glycine max (L.) Merr.] in soybean-growing regions of North America since it was first reported in 2000. The objectives of this study were to screen for soybean aphid resistant genotypes, determine the inheritance of resistant genes, and map and validate the resistance gene in the moderate resistant genotype K1621 using simple sequence repeat (SSR) markers. A mapping population of 150 F2:3 families from the cross between K1621 and susceptible genotype KS4202 were evaluated for aphid resistance. Phenotyping was conducted on the basis of total aphid number per plant 7 days following infestation with 4 aphids. Inheritance study indicated that one major dominant gene controls soybean aphid resistance in K1621. After SSR markers for polymorphism were screened between parents, a total of 133 polymorphic markers distributed across the soybean genome were used for genotyping. One quantitative trait loci (QTL) controlling antibiotic resistance was found by using the composite interval mapping method. This QTL localized on chromosome 13 (linkage group F) between markers Sat_234 and S6814 and explained 54% of the phenotypic variation. The putative QTL was further validated by single marker analysis using an independent population derived from the cross of K1621 and Dowling. The locus for soybean aphid resistance in K1621 was named [Rag]_K1621. The markers identified and validated in this study could be useful for marker-assisted selection of [Rag]_K1621.

Doctor of Philosophy / Department of Agronomy / Allan K. Fritz / In this research, a recombinant inbred line (RIL) population derived from cross between a leaf rust- and stripe rust-susceptible spring wheat ‘Avocet S’ and a slow leaf- and stripe-rusting resistant spring wheat ‘Amadina’ was used to postulate and map leaf rust seedling resistance genes, identify quantitative trait loci (QTL) for slow-rusting resistance against leaf and stripe rust, and study slow leaf-rusting components, latent period and infection frequency. Two known Lr genes (Lr23, and Lr26) were identified to be present in ‘Amadina’ through gene postulation, pedigree, cytogenetic, and polymerase chain reaction analyses. One unknown gene associated with seedling resistance was also mapped on chromosome 1BL. In greenhouse experiment, it was estimated that at least five genes conditioning final disease severity (FS) and latent period (LP), and four genes conditioning infection frequency (IF), segregated in the population. Correlations between LP and FS, and LP and IF were moderately negative, and that between IF and FS was moderately positive, indicating inter-dependence of the traits. Two QTL on chromosomes 1BL and 6BL were associated with LP and FS, and three QTL on chromosomes 1BL, 6BL and 2DS were associated with IF. Segregation of the RIL population in field experiment indicated that there were at least four and three adult plant resistance (APR) genes involved in resistance for leaf and stripe rust. Six QTL on chromosomes 3AL, 4AL, 1BL, 5BL, and 7BL were associated with APR for leaf rust, and seven QTL on chromosome 4AL, 5AL, 1BL, 2BL, 4BL, 5BL, 2DL, and 4D were associated with APR for stripe rust. Our results indicated that the major portion of genetic variability for slow-rusting resistance was additive gene action, and, to some extent, epistasis. In this research, we also explored the utility of remote sensing and geographic information systems (GIS) and analytical operations to discriminate leaf rust pustules from other parts of leaf and to accurately determine pustule size in ‘Amadina’ and ‘Avocet S’.

Doctor of Philosophy / Department of Biology / Jyoti Shah / Localized inoculation of a plant with an avirulent pathogen results in the activation of systemic acquired resistance (SAR), a defense mechanism that confers enhanced resistance against a variety of pathogens. The activation of SAR requires the translocation of an unknown signal from the pathogen-inoculated organ to the other organs where defenses are primed to respond faster in response to a future attack by a pathogen. Previous studies with the Arabidopsis thaliana dir1 (defective in induced resistance1) and sfd1 (suppressor of fatty acid desaturase deficiency1) mutants implicated a role for plant lipids in the activation of SAR. DIR1 encodes a putative lipid transfer protein and SFD1 encodes a dihydroxyacetone phosphate (DHAP) reductase involved in plastid glycerolipid metabolism. To further evaluate the role of DHAP reductases and plastid lipids in SAR, the involvement of two additional putative DHAP reductase encoding genes (AtGPDHp and AtGPDHc) and the SFD2 gene, which like SFD1 is involved in plastid glycerolipid metabolism, in SAR was evaluated. Only SFD2 was found to be essential for SAR. Although the lipid profile of the sfd2 mutant was similar to that of the fad5 (fatty acid desaturase 5) mutant, sfd2 is not allelic with fad5 and does not influence FAD5 expression. The SFD2 gene was mapped to an 85 kilo basepairs (kb) region on the third chromosome of Arabidopsis. The lipid composition defect of the sfd2 mutant was partially complemented by two independent recombinant bacterial artificial chromosomes (BACs) that contained genomic DNA spanning the wild type SFD2 locus. The role of plastid synthesized glycerolipids in the activation of SAR was further evaluated by characterizing SAR in additional Arabidopsis mutants that were deficient in plastid lipid metabolism. The requirement of MGD1 (MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE 1), DGD1 (DIGALACTOSYL-DIACYLGLYCEROL SYNTHASE 1) and FAD7 (FATTY ACID DESATURASE 7) genes in SAR, confirmed the essential role of plastid glycerolipids, presumably a galactolipid-dependent factor, in signaling associated with the SAR.

Master of Science / Department of Plant Pathology / Bernd Friebe / Bikram S. Gill / Common, bread, or hexaploid wheat, Triticum aestivum L. (2n=6x=42, AABBDD), has several relatives in the Triticum/Aegilops complex of the Poaceae family in the Triticeae tribe, which are valuable sources for broadening genetic diversity and may provide genes for disease and pest resistance and general wheat improvement. Other wild relatives of wheat also may be exploited for wheat improvement, such as Haynaldia villosa (L.) Schur. (2n=2x=14, VV). It is a diploid species with resistance to powdery mildew, wheat curl mite colonization, cereal eyespot disease, rust diseases, and wheat spindle streak mosaic virus. H. villosa may harbor many other as yet unidentified traits for wheat improvement. The polyploid nature of bread wheat allows tolerance to genomic changes, because homoeologous chromosomes from other genomes compensate for missing wheat chromosomes. In this experiment, we crossed the disomic alien addition line DA4V (2n=6x=44) with a pair of H. villosa chromosomes added to the wheat chromosome complement with wheat monosomic for chromosome 4D (2n=41) to produce 4D/4V double monosomic plants. According to centric breakage-fusion mechanisms, univalents tend to break at their centromeres at meiotic metaphase I producing telocentric chromosomes with unstable or “sticky” ends that can fuse with the sticky ends of other newly formed telocentric chromosomes. This fusion results in Robertsonian whole-arm translocations that may be compensating if a short arm of one chromosome fuses with a long arm of another. Double monosomic plants were screened cytogenetically and further visualized by genomic in situ hybridization (GISH). Five transfers were identified, including T4DS.4VL and T4VS.4DL translocations, and a T4VS-W.W transfer of unknown wheat origin. These results were confirmed by GISH. The T4DS.4VL and T4VS.4DL translocations are genetically compensating and should be exploited in wheat improvement.

Doctor of Philosophy / Department of Entomology / David C. Margolies / James R. Nechols / Foraging traits such as prey consumption rate and the efficiency with which predators convert their prey into offspring are important determinants of local predator-prey dynamics. However, in environments with patchy prey distribution, predator dispersal and aggregation in response to prey-induced volatile cues becomes more critical. My dissertation addressed predator-prey population dynamics in response to variation in four foraging traits in the predatory mite, Phytoseiulus persimilis (Acari: Phytoseiidae): consumption rate, conversion efficiency, dispersal, and olfactory response related to prey. The dispersal response and olfactory sensitivity in predatory mites is modified by prey-related cues. For example, the dispersal response increased with decreasing prey density in a patch and increasing prey-related volatiles from outside the prey patch. The olfactory response of predatory mites also increased with increasing numbers of prey per plant or with the length of time a plant was infested by prey. These results formed the basis for development of bioassays used to examine genetic variation in dispersal and olfactory response of predatory mites. Through artificial selection I documented additive genetic variation in all four traits. After relaxation of selection, high-level phenotypes were stable compared to their low counterparts. There were significant genetic correlations between some of the foraging traits. However, there were no correlations between foraging traits and life-history traits. The existence of genetic variation and covariation among the foraging traits suggests that predatory mites must be able to adopt different foraging strategies in the evolution of prey-finding in a tritrophic system. High consumption, high conversion efficiency and high dispersal response phenotypes interacted differently with prey in a spatially complex landscape. All foraging traits were comparable in terms of predator-prey densities and plant damage; but they were lower than the unselected control. Spatial association and correlation analysis showed that all foraging traits were positively associated with prey; but the strongest association was observed for the high conversion efficiency and dispersal lines. The variability in foraging behaviors of the predatory mite affects its ability to locate patchily distributed prey, thereby influencing foraging efficiency and population dynamics. This research provides new information about the critical link between predator foraging and population dynamics relevant to biological control.

Doctor of Philosophy / Genetics Interdepartmental Program - Biology / Susan J. Brown / Understanding evolutionary processes that structure genetic variation associated with lineage diversification and speciation is a central problem. Shifts in host plant use by insect herbivores and subsequent divergence in mating signals can lead to sexual isolation, especially when selection for specialization on different hosts restricts gene flow among populations. The grasshopper Hesperotettix viridis (Thomas) is an oligophagous grasshopper feeding on plants primarily in the host plant genera Gutierrezia and Solidago in Kansas. I used mitochondrial and microsatellite genetic markers to evaluate the diversification pattern and underlying evolutionary mechanisms of two putative host races of H. viridis. I also quantified host preferences, the degree of sexual isolation among putative host races and divergence in cuticular attributes to identify the nature and origins of initial barriers that isolated populations in the formative stages of divergence. mtDNA data revealed a star-shaped phylogeny, suggesting isolation in a single refugium ~110,000 years ago based on a molecular clock, followed by rapid population expansion. Microsatellite data reveal significant host-based genetic differentiation and structuring in H. viridis populations in Kansas, including a microsatellite locus under strong divergent selection. Neutral microsatellite loci did not reveal a differentiation pattern specific to host plant use. Significant host-based preferences by individuals that fed on two host plant groups were detected in host paired-feeding preference studies. No-choice mate selection experiments revealed preferences for individuals collected from the same host species independent of location with little mating observed between individuals from different host species. Significant differentiation in color and cuticular composition among different host plant races within the study area was also detected. Correlations between host choice, mate choice and phenotypic divergence were observed and this host associated divergence appears to have a genetic basis. Based on the results of this study, I conclude that divergent selection for host plant use underlies observed sexual isolation among populations in this species. Hesperotettix viridis populations in Kansas that fed on Solidago and Gutierrezia species represent two incipient host races, early stages of diversification that could lead to speciation in insect herbivores.

Doctor of Philosophy / Department of Biology / Samantha Wisely / Habitat fragmentation and destruction are the most ubiquitous and serious environmental threats confronting the long-term survival of plant and animal species worldwide. However, some native or exotic species can take advantages of these alterations and expand their range, placing endemic species at risk of extinction by changing the composition of biotic communities and altering ecosystem. Capybara (Hydrochoerus hydrochaeris) are a widely distributed rodent throughout most of South and Central America, but restricted to areas of standing water. As the Gran Chaco ecosystem of Paraguay has been converted from dry tropical forest to pastureland, I hypothesized that this habitat alteration created potential for invasion by capybara into newly fragmented areas. I used ecological niche modeling to generate hypotheses about how the distribution of capybara has been affected by land use change, and tested those hypotheses with phylogeographic analyses. To understand the mechanisms that have allowed the invasion, I investigated home range, habitat use and thermoregulation of capybara via radiotelemetry in a deforested area in which capybara had recently invaded. Genetic analyses confirm a rapid range expansion scenario with evidence of secondary contact between two distinct phylogroups which had previously been disjunct. Modeling results indicated that conversion of forest to pastureland allowed the expansion to occur. Capybara selected water significantly more than it was available to them, and avoided shrub forest. I found a significant positive correlation between body temperature and distance from water, and a significant negative correlation between distance from water and Chaco ambient temperature. Capybara proximity to water appeared to be tightly linked to body thermoregulation. These results suggest that although capybara have expanded into the Chaco forest as it is converted to pastureland, the presence of permanent water sources in those pastures are the mechanism that allow capybara to persist in this habitat. This is the first study to characterize capybara in a xeric habitat without a year round water source, and scarce natural grasslands. My results show how anthropogenic habitat modification has allowed capybara to thrive. Understanding how capybara invade and utilize the deforested Central Dry Chaco will provide valuable information for the future management of the species and the Chaco ecosystem.

Doctor of Philosophy / Genetics Interdepartmental Program-Plant Pathology / James Nelson / The goal of quantitative-trait locus (QTL) mapping is to understand the genetic architecture of an organism by identifying the genes underlying quantitative traits. It targets gene numbers and locations, interaction with other genes and environments, and the sizes of gene effects on the traits. QTL mapping in plants is often done on a population of progeny derived from one or more designed, or controlled, crosses. These crosses are designed to exploit correlation among marker genotypes for the purposes of mapping QTL. Reducing correlations between markers can improve the precision of location and effect estimates by reducing multicollinearity. The purpose of this thesis is to propose an approach for developing experimental populations to reduce correlation by increasing recombination between markers in QTL mapping populations especially in selfing species. QTL mapping resolution of recombinant inbred lines (RILs) is limited by the amount of recombination RILs experience during development. Intercrossing during line development can be used to counter this disadvantage, but requires additional generations and is difficult in self-pollinated species. In this thesis I propose a way of improving mapping resolution through recombination enrichment. This method is based on genotyping at each generation and advancing lines selected for high recombination and/or low heterozygosity. These lines developed are called SA-RILs (selectively advanced recombinant inbred lines). In simulations, the method yields lines that represent up to twice as many recombination events as RILs developed conventionally by selfing without selection, or the same amount but in three generations, without reduction in homozygosity. Compared to methods that require maintaining a large population for several generations and selecting lines only from the finished population, the method proposed here achieves up to 25% more recombination. Although SA-RILs accumulate more recombination than conventional RILs and can be used as fine-mapping populations for selfing species, the effectiveness of the SA-RIL approach decreases with genome size and is most valuable only when applied either to small genomes or to defined regions of large genomes. Here I propose the development of QTL-focused SA-RILs (QSA-RILs), which are SA-RILs enriched for recombination in regions of a large genome selected for evidence for the presence of a QTL. This evidence can be derived from QTL analysis in a subset of the population at the F2 generation and/or from previous studies. In simulations QSA-RILs afford up to threefold increase in recombination and twofold increase in accuracy of QTL position estimate in comparison with RILs. The regional-selection method also shows potential for resolving QTL linked in repulsion. One of the recent Bayesian methods for QTL mapping, the shrinkage Bayesian method (BayesA (Xu)), has been successfully used for estimating marker effects in the QTL mapping populations. Although the implementation of the BayesA (Xu) method for estimating main effects was described by the author, the equations for the posterior mean and variance, used in estimation of the effects, were not elaborated. Here I derive the equations used for the estimation of main effects for doubled-haploid and F2 populations. I then extend these equations to estimate interaction effects in doubled-haploid populations. These derivations are helpful for an understanding of the intermediate steps leading to the equations described in the original paper introducing the shrinkage Bayesian method.

Doctor of Philosophy / Genetics Interdepartmental Program, Plant Pathology / Xiaoyan Tang / Pseudomonas syringae bacteria utilize the type III secretion system (T3SS) to deliver effector proteins into host cells. The T3SS and effector genes (together called the T3 genes hereafter) are repressed in nutrient rich medium but are rapidly induced after the bacteria are transferred into minimal medium (MM) or infiltrated into the plant. The induction of the T3 genes is mediated by HrpL, an alternative sigma factor that recognizes the conserved hrp box motif in the T3 gene promoters. The induction of hrpL is mediated by HrpR and HrpS, two homologous proteins that bind the hrpL promoter. To identify additional genes involved in regulation of the T3 genes, P. s. pv. phaseolicola (Psph) NPS3121 transposon insertion mutants were screened for reduced induction of avrPto-luc and hrpL-luc, reporter genes for promoters of effector gene avrPto and hrpL, respectively. Determination of the transposon-insertion sites led to the identification of genes with putative functions in signal transduction and transcriptional regulation, protein synthesis, and basic metabolism. A transcriptional regulator (AefRNPS3121) identified in the screen is homologous to AefR, a regulator of the quorum sensing signal and epiphytic (plant-associated) traits that was not known previously to regulate the T3 genes in P. s. pv. syringae (Psy) B728a. AefRNPS3121 in Psph NPS3121 and AefR in Psy B728a are similar in regulating the quorum sensing signal in liquid medium but different in regulating epiphytic traits such as swarming motility, entry into leaves, and survival on the leaf surface. The two component system RhpRS was identified in Pseudomonas syringae as a regulator of the T3 genes (Xiao et al. 2007). In the rhpS- mutant, the response regulator RhpR represses the induction of the T3 gene regulatory cascade, but induces its own promoter in a phosphorylation-dependent manner. Deletion and mutagenesis analyses revealed an inverted repeat (IR) element GTATC-N6-GATAC in the rhpR promoter that confers the RhpR-dependent induction. Computational search of the P. syringae genomes for the putative IR elements and Northern blot analysis of the genes with a putative IR element in the promoter region uncovered five genes that were upregulated (PSPTO2036, PSPTO2767, PSPTO3477, PSPTO3574, and PSPTO3660) and two genes that were down-regulated (PSPTO0536 and PSPTO0897) in an RhpR-dependent manner. ChIP assays indicated that RhpR binds the promoters containing a putative IR element but not the hrpR and hrpL promoters that do not have an IR element, suggesting that RhpR indirectly regulates the transcriptional cascade of hrpRS, hrpL, and the T3 genes. To identify additional genes involved in the rhpRS pathway, suppressor mutants were screened that restored the induction of the avrPto-luc reporter gene in the rhpS- mutant. Determination of the transposon-insertion sites led to the identification of rhpR, an ATP-dependent Lon protease, a sigma 70 family protein (PSPPH1909), and other metabolic genes. A lon- rhpS- double mutant exhibited phenotypes typical of a lon- mutant, suggesting that rhpS acts with or through lon. The expression of lon was elevated in rhpS- and other T3-deficient mutants, indicating a negative feedback mechanism. Both the lon- rhpS- and the PSPPH1909- rhpS- double mutant displayed enhanced transcription of hrpL in MM than did the rhpS- mutant.

Master of Science / Department of Agronomy / Jianming Yu / Tannin, a second metabolic product in sorghum, has been directly related to resistance to insects and birds. Tannin also impacts sorghum nutritional value. Previous studies have shown tannin content has a positive correlation with early season cold tolerance, an important agronomic trait. Sorghum contains condensed tannins in testa layer below the pericarp. The testa layer tannin is controlled by two complementary genes B1 and B2: tannins are present when both genes are dominant but absent when only one or none of these two is dominant. The purpose of this research is to identify and map QTLs associated with the presence of condensed tannins, analyze interaction of QTLs, and provide a potential path to dissect the more complex trait of early season cold tolerance in future studies. A population of 109 F6:7 recombinant inbred lines (RILs) developed from the cross of a high tannin sorghum Shan Qui Red (SQR) and non-tannin line Tx430 was used in the mapping study. Two QTLs related to condense tannin presence in testa layer were mapped to chromosome 2 and 4, respectively. Strong epistatic interaction of these two QTLs was detected. The two QTLs together with their interaction explained 74% of the phenotypic variation. Sorghum grain quality traits, including kernel size, kernel hardness, protein and starch content, are complex traits which are directly related to sorghum nutritional value and market value. Association mapping is a promising method for complex quantitative traits analysis and dissection in plant science. Sorghum grain quality trait association analysis research is purposed to analyze large amount of grain quality data based on a diversity panel. A sorghum bicolor panel of 300 lines including germplasm derived from sorghum conversion program and elite commercial lines were established and served as diversity population for the association study. Phenotypic data of grain quality traits were collected by single kernel characterization system (SKCS) and near infrared reflectance spectroscopy (NIRS). Data analysis proved high diversity within the SB panel. A correlation between tannin presence and kernel hardness was also observed. Quality traits showed high consistence across years and environments.