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.

Doctor of Philosophy / Department of Plant Pathology / Justin D. Faris / Bikram S. Gill / The Q gene is largely responsible for the widespread cultivation of durum and common wheat because it confers the square spike phenotype and the free-threshing character. It also pleiotropically influences many other domestication related traits such as glume shape, glume tenacity, rachis fragility, spike length, plant height, and spike emergence time. The objectives of this research were to confirm or reject the hypothesis that a candidate AP2-like gene is Q, confirm the dosage and pleiotropic effects attributed to Q, and begin defining the differences between the Q and q alleles. The identity of the Q gene was verified by analysis of knockout mutants and found to have a high degree of similarity to members of the AP2 family of transcription factors. Southern analysis of multiple Triticum taxa containing either Q or q indicated that the Q locus is not composed of duplicated q alleles. Ectopic expression analysis allowed the observation of both silencing and over-expression effects of Q. Rachis fragility, glume shape, and glume tenacity mimicked the q phenotype in transgenic plants exhibiting transcriptional silencing of the transgene and the endogenous Q gene. Variation in spike compactness and plant height were directly associated with the level of transgene expression due to the dosage effects of Q. Comparisons of Q and q indicated structural differences as well as variation in the level of transcription. One amino acid difference and several base changes within the promoter were identified as possible critical differences between Q and q. Very little genetic variability was found within the sequenced Q alleles suggesting it arose only once and that q is the more primitive allele.

Master of Science / Department of Biology / Jyoti Shah / Plant defense responses are constitutively activated in the Arabidopsis thaliana ssi2 mutant plant. In addition, the ssi2 mutant allele confers a dwarf phenotype. The SSI2 gene encodes a stearoyl-ACP-desaturase, which converts stearic acid (18:0) to oleic acid (18:1), suggesting a role for lipids in plant defense. Microarray analysis identified several genes which encode putative acyl hydrolases/lipases that are expressed at elevated levels in the leaves of ssi2, in comparison to the wild type plant. One gene in particular, At5g14180, was expressed at 60-fold greater level in ssi2 than in the wild type plant. To study the involvement of At5g14180 in plant defense and lipid metabolism, two transgenic lines containing T-DNA insertions within the At5g14180 gene were identified. These two T-DNA insertional alleles of the At5g14180 gene attenuate the ssi2-conferred heightened resistance to a virulent strain of Pseudomonas syringae pv. maculicola in the ssi2 At5g14180 double mutant plant. Furthermore, pathogen growth was enhanced in the At5g14180 single mutant plants, as compared to the wild type plant. Profiling of lipid composition in leaf tissue identified changes in the lipid composition between the At5g14180 mutant and wild type plants, suggesting that the At5g14180 encoded protein may impact lipid metabolism in Arabidopsis leaves.

Master of Science / Department of Biology / Brett K. Sandercock / Samantha Wisely / In birds, the offspring of females in socially monogamous species can be sired not only by their social partner (within-pair mating) but also by other males (extra-pair mating), resulting in broods of mixed paternity. Several hypotheses have been proposed which attempt to explain the adaptive significance of this behavior, including the genetic diversity hypothesis, the good genes hypothesis, the genetic compatibility hypothesis and the fertility insurance hypothesis. I report results of a 5 year population study of the Upland Sandpiper (Bartramia longicauda) at Konza Prairie Biological Station in northeast Kansas. My objective was to determine the genetic mating system of this socially monogamous shorebird, and determine which of the genetic hypotheses best explains the patterns of extra-pair paternity (EPP) in the population. As part of the analysis, I optimized laboratory protocols for genetic sexing of our monomorphic study species. Potential errors in molecular sexing have been previously described but usually result in females being misidentified as males. Here, I report evidence that events in PCR reactions can lead to the opposite error, with males misidentified as females. I recommend the use of multiple primer sets and large samples of known-sex birds for validation when designing protocols for molecular sex analysis. I genotyped birds and tested for the existence of EPP in 58 family groups of Upland Sandpipers. I found 15% of chicks and 30% of broods were the result of extra-pair paternity in this population, which is high in comparison to other socially monogamous shorebirds. Only 2% of chicks and 2% of broods were attended by females unrelated to the young. I tested ecological covariates known to influence EPP in other birds including relatedness of mated pairs, morphology of the within-pair male, and nest initiation date, as well as variables which signify genetic benefits, including morphology of the offspring and offspring heterozygosity, but found no significant relationships. None of the prevailing genetic hypotheses can fully explain the high rates of EPP in this population of Upland Sandpipers. However, the discovery of fine-scale genetic structure in female birds, but not in males, suggests female natal philopatry or male-biased dispersal. This sex-specific genetic structure could be a mechanism of inbreeding avoidance, thereby eliminating the need for females to choose mates based on relatedness. This study provides the first estimates of EPP for the socially monogamous Upland Sandpiper, and provides evidence that the inbreeding avoidance mechanism of engaging in extra-pair copulations does not seem to be as important in Upland Sandpipers as in other socially monogamous shorebirds. Future research should include the identification of extra-pair males and the determination of offspring fitness after departure from the nest.