Doctor of Philosophy / Genetics Interdepartmental Program / Bikram S. Gill / Leaf rust, caused by fungus Puccinia triticina, is an important foliar disease of wheat worldwide. Breeding for race-nonspecific resistant cultivars is the best strategy to combat this disease. Aegilops tauschii, D genome donor of hexaploid wheat, has provided resistance to several pests and pathogens of wheat. To identify potentially new adult plant resistance (APR) genes, 371 geographically diverse Ae. tauschii accessions were evaluated in field with leaf rust (LR) composite culture of predominant races. Accessions from Afghanistan only displayed APR whereas both seedling resistance and APR were common in the Caspian Sea region. Seventeen accessions with high APR were selected for production of synthetic hexaploid wheat (SHW), using ‘TetraPrelude’ and/or ‘TetraThatcher’ as tetraploid parents. Six SHWs were produced and evaluated for APR to LR and resistance to tan spot at seedling stage. Genetic analysis and mapping of APR introgressed from accession TA2474 was investigated in recombinant inbred lines (RIL) population derived from cross between SHW, TA4161-L3 and spring wheat cultivar, ‘WL711’. Genotyping-by-sequencing approach was used to genotype the RILs. Maximum disease severity (MDS) for LR was significantly correlated among all experiments and APR to LR was highly heritable trait in this population. Nine genomic regions significantly associated with APR to LR were QLr.ksu-1AL, QLr.ksu-1BS, QLr.ksu-1BL.1, QLr.ksu-1BL.2, QLr.ksu-2DS, QLr.ksu-2DL, QLr.ksu-5AL, QLr.ksu-5DL and QLr.ksu-6BL. Association of QLr.ksu-1BL.1 with marker Xwmc44 indicated this locus could be slow-rusting APR gene, Lr46/Yr29. QTLs detected on 2DS, 2DL and 5DL were contributed by TA4161-L3 and are novel, along with QLr.ksu-5AL. Tan spot, caused by necrotrophic fungus, Pyrenophora tritici-repentis, has recently emerged as a damaging disease of wheat worldwide. To identify QTLs associated with resistance to Race 1 of P. tritici-repentis, F[subscript]2:3 population derived from cross between SHW, TA4161-L1 and winter wheat cultivar, ‘TAM105’ was used. Two major effect QTLs, QTs.ksu-1AS.1 and QTs.ksu-7AS were significantly associated with tan spot resistance and contributed by TA4161-L1. QTs.ksu-7AS is a novel QTL and explained 17% of the phenotypic variation. Novel QTLs for APR to LR and tan spot identified in SHWs add new variation for broadening the gene pool of wheat and providing resources for breeding of durable resistant cultivars.

Doctor of Philosophy / Department of Plant Pathology / Richard B. Todd / Fungi can be found living in a range of environments, including soil and the ocean, and as pathogens of plants and animals. The ability of fungi to adapt to diverse and changing environments is dependent on their ability to sense and respond to an array of signals, including the presence or absence of nitrogen nutrients. Fungi can utilize a diverse array of nitrogen nutrients and do so in a regulated and preferential manner. When preferred nitrogen nutrients such as ammonium and glutamine are present (nitrogen sufficiency), genes required for the utilization of alternative nitrogen sources are not expressed. In the absence of a preferred nitrogen source (nitrogen limitation) the genes for utilization of alternative nitrogen sources are transcriptionally derepressed and can be induced by the presence of a particular nitrogen nutrient, such as nitrate or proline. In the absence of any nitrogen nutrient (nitrogen starvation) the expression of some genes is further elevated. In filamentous fungi the expression of genes required for the utilization of nitrogen nutrients is coordinated by the orthologs of the conserved Aspergillus nidulans GATA transcription factor AreA, which activates transcription of nitrogen utilization genes. AreA activity is controlled by autogenous transcriptional activation, mRNA transcript stability, regulated nucleo-cytoplasmic distribution, and interactions with NmrA, AreB and TamA. The combined effect of these regulatory mechanisms generally results in AreA being inactive during nitrogen sufficiency and active during nitrogen limitation and nitrogen starvation. However, during nitrogen sufficiency AreA remains active at the promoters of some genes, including gdhA, which encodes the key nitrogen assimilation enzyme NADP-dependent glutamate dehydrogenase. In this work we have used both classical genetics and next generation sequencing approaches to examine regulated gene expression and how AreA activity is modulated, primarily during nitrogen sufficiency. We have studied regulation of gdhA to characterize how AreA evades nitrogen metabolite repression. We identify leucine biosynthesis as being a key regulatory signal involved in gdhA expression and characterize the genes required for leucine biosynthesis. We also show that TamA regulates the gdhA promoter by direct DNA binding, which requires interaction with AreA. We have also characterized repression of AreA to identify a potential mode of NmrA corepressor action. Finally, we have characterized the AreA nuclear export signal and explored mechanisms that control regulated nuclear export of AreA.

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.

Master of Science / Department of Entomology / Yoonseong Park / Neuropeptides are diffusible signal molecules mediating vital physiological processes. We have been interested in a group of neuropeptides and their receptors involved in osmoregulatory neuroendocrine system which has been suggested as a possible target for development of new biopesticides. Since the genome sequence of the T. castaneum has recently been completed, we were able to identify the respective genes encoding three peptide hormones from T. castaneum that were characterized for their diuretic activities in other insects: one calcitonin-like (CT-like DH31) and two corticotropin releasing factor-like (CRF-like DH37 and DH47, the numbers indicates the number of amino acid residues). This peptide is expressed at all developmental stages and in the central nervous system (CNS), Malpighian tubules (MT) and gut. The synthetic peptide TricaDH31 also has been show to be biologically active, inducing significant excretions in adults beetles. When Tcdh31 was silenced using RNAi, adults had deformed wings and abnormal body shape. Mortality in adults was high, the number of eggs laid was reduced as well as the hatchability of the eggs. The two biologically active CRF-like peptides in T. castaneum, are encoded by one gene which undergoes alternative splicing. When Tcdh47 was knocked down, high mortality occurred as well as low oviposition and egg hatchability. Similar effects were observed with silencing of both CRF-like genes. However, RNAi of Tcdh37 transcripts had similar, but less severe effects. Adults also had deformed wings when both CRF-like genes were silenced, but not when just one of them was knocked down. These results indicate that CRF-like genes could have additional biological functions to their roles in dieresis. We tested the in vivo activity of these peptides. TenmoDH47 induced high excretions in adults, whereas TenmoDH37 induces smaller excretions. We identified the respective genes encoding two putative receptors for TricaDH31 as Glean_13321 and Glean_02694 (Trica-ctr1 and Trica-ctr2, respectively) and two receptors for CRF-like peptide as Glean_12799 and Glean_07104 (Trica-crfr1 and Trica-crfr2, respectively). The CT-like receptors are expressed at all developmental stages, in the CNS and MT. RNAi of the receptors revealed that only Trica-ctr2 silencing caused significant mortality and reduction in the number of eggs laid. The CRF-like receptors are expressed at all developmental stages. Adults also had deformed wings and laid fewer eggs after RNAi of Trica-crfr1. RNAi of Trica-crf2 also caused significant mortality. These peptides and receptors seem to fine tune the beetle physiology and may have functions not yet known.

Doctor of Philosophy / Genetics Interdepartmental Program / James C. Nelson / QTL (quantitative-trait locus) analysis aims to locate and estimate the effects of genes that are responsible for quantitative traits, by means of statistical methods that evaluate the association of genetic variation with trait (phenotypic) variation. Quantitative traits are typically controlled by multiple genes with varying degrees of influence on the phenotype. I describe a new QTL analysis method based on shrinkage and a unifying framework based on the generalized linear model for non-normal data. I develop their extensions to multiple-trait QTL analysis. Expression QTL, or eQTL, analysis is QTL analysis applied to gene expression data to reveal the eQTLs controlling transcript-abundance variation, with the goal of elucidating gene regulatory networks. For exploiting eQTL data, I develop a novel extension of the graphical Gaussian model that produces an undirected graph of a gene regulatory network. To reduce the dimensionality, the extension constructs networks one cluster at a time. However, because Fuzzy-K, the clustering method of choice, relies on subjective visual cutoffs for cluster membership, I develop a bootstrap method to overcome this disadvantage. Finally, I describe QGene, an extensible QTL- and eQTL-analysis software platform written in Java and used for implementation of all analyses.

Master of Science / Genetics Interdepartmental Program / Bikram S. Gill / TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput, reverse genetics strategy for scanning mutagenized populations for point mutations in loci of interest. Originally, TILLING was used to investigate gene function in Arabidopsis and has since been similarly applied for gene functional analysis in other organisms. TILLING also allows the generation of novel genetic variation in specific genotypes and, thus, has been implemented as a tool for crop improvement. Ethyl methanesulfonate (EMS) is a widely used mutagen to induce point mutations in most TILLING protocols. M1 plants are then self-pollinated and M2 seed harvested. A single seed is grown from each M2 progeny and tissue taken for DNA isolation. M3 seed is cataloged. DNA is pooled to increase the efficiency and aid in mutation detection. Polymerase chain reaction (PCR) is used to amplify a locus of interest using the M2 DNA pools as a template. The PCR products are digested with an endonuclease that cleaves mismatched, mutant DNA, and the digested products are visualized. The pools for which PCR products are positive for a mutation are deconvoluted to determine which individual plant of the pool was responsible for the mutation. DNA from the positive individual is sequenced to determine the type of mutation (missense, nonsense, synonymous). Individuals with mutations that are more likely to disrupt gene function (nonsense and certain missense) are studied further by growing the corresponding M3 generation. In bread wheat, Triticum aestivum, TILLING is complicated by polyploidy: genes that have homoeologs require that the functionality of each be studied. If functional homoeologs are present for all three genomes, mutants must be identified for each homoeolog, followed by successive intercrossing to produce a triple mutant plant. As a model for wheat genetics, we propose TILLING in diploid wheat. EMS mutant populations were created in diploid wheat (Triticum monococcum ssp. monococcum) and the hexaploid bread wheat cultivar ‘Jagger’. The diploid and hexaploid wheat populations were screened for mutations at the waxy locus, GBSS1, as a validation of our population and for comparative analysis of mutation rates in 2x and 6x wheat. For diploid wheat, GBSSI was screened in 716 M2 plants, and one mutant was found for 1.9 Mb screened. 3 For hexaploid wheat, GBSSI was screened in 518 M2 plants, and 30 mutants were identified within a total of 657 Kb screened, giving a mutation frequency of one mutation per 22 Kb. The reasons for this vast difference in mutation frequency between diploid and hexaploid wheat are discussed. The diploid wheat population was further examined by screening for mutations within four lignin biosynthesis candidate genes, for a total of 2 Mb screened. A single mutant was discovered for both of the lignin genes PAL6 and HCT, giving a mutation frequency of one mutation per 1 Mb screened.

Doctor of Philosophy / Department of Biology / Michael A. Herman / Determining the genetic mechanisms involved in organismal response to environmental change is essential for understanding the effects of anthropogenic disturbance. The composition of the bacterial-feeding nematode community is an excellent biological indicator of disturbance, particularly in grassland ecosystems. We have previously shown that grassland soil nematodes are responsive to perturbations in the field including the addition of nitrogen fertilizer. We are interested in how this perturbation affects the microbial community and downstream effects on the next trophic level, the bacterial-feeding nematodes. To determine the effects of disturbance on soil bacterial communities we used massively parallel sequencing and found that chronic nitrogen addition on tallgrass prairie significantly impacts overall bacterial community diversity and the abundance of specific bacterial taxa. Because native soil nematodes lack well developed genomic tools, we employed Caenorhabditis elegans as a model for native soil nematode taxa and used transcriptional profiling to identify 204 candidate genes regulated in response to altered bacterial diets isolated from grassland soils. To biologically validate our results we used mutations that inactivate 21 of the identified genes and showed that most contribute to fitness or lifespan in a given bacterial environment. Although these bacteria may not be natural C. elegans food sources, this study aimed to show how changes in food source, as can occur in environmental disturbance, has large effects on gene expression and those genes whose expression are affected, contribute to fitness. Furthermore, we identified new functions for genes of unknown function as well as previously well-characterized genes, demonstrating the utility of this approach to further describe C. elegans genome. We also investigated the function of previously well-characterized C. elegans defense pathways in our grassland soil bacterial environments and found that some are environment specific. Additionally, we found that cuticular collagen genes are important for lifespan, and appear to function downstream of known defense pathways. Overall, our results suggest that anthropogenic disturbance in grasslands alters the most basal components of the soil food web, bacteria and bacterial-feeding nematodes through the genes they possess and how they are expressed, and resultant bottom-up effects could have profound consequences on ecosystem health and function.

Master of Science / Department of Plant Pathology / Eduard D. Akhunov / Loretta C. Johnson / Big bluestem (Andropogon gerardii Vitman) is an ecologically dominant grass of the North American grasslands with precipitation-dependent productivity. However, climatic predictions for big bluestem’s dominant range in the Great Plains include increased periods of drought. The main objectives of this research were to determine the extent of neutral and non-neutral genetic differentiation and diversity among putative big bluestem ecotypes using amplified fragment length polymorphism (AFLP) markers. This is the first study of both neutral and non-neutral genetic diversity of big bluestem which also includes source populations of well-described ecotypes studied in reciprocal common gardens. A total of 378 plants were genotyped from 11 source prairies, originating from one of three ecoregions (Central Kansas, Eastern Kansas, and Illinois). Using two AFLP primer sets, 387 polymorphic markers (error rate 9.18%) were found. Un-rooted neighbor joining tree and principle-component analyses showed continuous genetic differentiation between Kansas and Illinois putative ecotypes, with genetic overlap occurring between Kansas ecotypes. Analysis of molecular variance showed high diversity within-prairie sites (80%) relative to across-prairies (11%), and across- ecoregions (9%) (p<0.001). Within-prairie genetic diversity levels were similar among ecoregions (84-92%), with the highest genetic variation maintained in Illinois prairies (92%). Population structure analyses supported K=6 genetic clusters across the environmental gradient, with Kansas prairies belonging to three main genetic groups, and Illinois prairies having largely divergent allele frequencies from Kansas prairies. Interestingly, BAYESCAN analysis of the three putative ecotypes identified eight F[subscript]ST-outlier AFLP loci under potential diversifying selection. Frequency patterns of loci under diversifying selection were further linked to geo-environmental descriptors including precipitation, temperature severity, diurnal temperature variation, prairie location, and elevation. The observed allele frequency divergence between Kansas and Illinois ecotypes suggests tallgrass restorations should consider possible maladaptation of non-local ecotypes and genetic swamping. However, high within-prairie genetic variation may help individual big bluestem populations withstand climatic variability.

Doctor of Philosophy / Department of Biology / Susan J. Brown / RNA interference (RNAi) is a natural gene-silencing phenomenon triggered by dsRNA (dsRNA). While RNAi is an endogenous process that plays essential roles in regulating gene expression it can also be harnessed as a tool for the study of gene function. Introducing dsRNA that is homologous to target mRNA into a cell triggers the RNAi response causing the destruction of the homologous mRNA and a loss of function phenotype. In some organisms, such as the nematode Caenorhabditis elegans, once dsRNA is introduced into the body cavity, the RNAi effect is seen throughout the organism because the dsRNA is taken up by individual cells and is then spread from cell to cell. This process has been termed the systemic RNAi response. For other organisms, such as the fruit fly Drosophila melanogaster, introduction of dsRNA into the body cavity does not result in a systemic RNAi response. This may be due to the cell’s inability to take up dsRNA or spread that dsRNA from cell to cell. For other organisms, including mammals, introduction of dsRNA into the body cavity does not result in a systemic RNAi response because the immune response causes dsRNA destruction before it can be utilized in the RNAi pathway. For organisms that do not exhibit a systemic RNAi response, complex genetic methods are needed to introduce dsRNA into cells to induce the RNAi response. Therefore, one of the challenges in utilizing RNAi as a genetic tool is introducing the dsRNA into individual cells. In recent years, systemic RNAi responses have been documented in both model and non-model organisms, making RNAi an accessible genetic tool. The red flour beetle, Tribolium castaneum is an emerging model organism that has a robust systemic RNAi response. However, the mechanism of systemic RNAi and the specific parameters required to obtain a strong systemic RNAi response in this organism have not been thoroughly investigated. The aim of this work is to provide data that can allow RNAi to be better utilized as a genetic tool in Tribolium and to use this information as a basis for the use of RNAi in other insects in which it can be performed. Specifically we provide data on the essential parameters necessary to achieve an effective systemic response in Tribolium, we describe differences in the systemic RNAi response between Drosophila and Tribolium, we analyze the conservation and function of RNAi machinery genes in Tribolium and we provide information on the genes critical for a systemic RNAi response in Tribolium.

Glycogen synthase kinase-3 (GSK-3) alpha and beta are central regulators of key developmental pathways, including Wnt, Hedgehog and Notch, which control stem cell activities and cellular differentiation. Both forms of GSK-3 are also regulated by receptor tyrosine kinases via the PI3K/Akt growth-promoting pathway and are involved in feedback mechanisms that maintain signaling homeostasis. These signaling systems have critical functions in mammary gland development and aberrations in them have been implicated in breast cancer. However, the role of GSK-3 in breast oncogenesis is unclear. Here, I provide evidence that maintenance of appropriate GSK-3 activity is necessary for normal acinar morphogenesis of mammary cells in the ductal/alveolar epithelium. Inadequate GSK-3 levels result in enlarged structures that often lack hollow lumens and resemble early premalignant breast cancer lesions. A potential contribution for PI3K signaling to this phenotype was identified as a PI3K inhibitor partially reversed the observed morphological defects. Mammary epithelial cell (MEC) identity also requires modulation of signals through the Wnt/beta-catenin pathway. GSK-3-depleted mammary glands not only transdifferentiate into squamous epithelium but also develop highly proliferative adenosquamous carcinomas characterized by activated beta-catenin. Furthermore, beta-catenin appears to be required for both cell fate changes and tumorigenesis in the absence of GSK-3 function. Mammary tissues engineered to enable conditional deletion of beta-catenin in a GSK-3-null background also assumed an epidermoid cell fate with ensuing tumor formation albeit with a significantly longer latency and different histopathology. The metaplastic nature of tumors observed is similar to a rare yet aggressive form of human breast disease, metaplastic breast carcinomas (MBCs). Mammospheres (MS) generated from GSK-3 depleted MECs exhibited a less compact morphology compared to those with activated beta-catenin, which also exhibited an expansion of the CD24:CD49f double positive progenitor population and enhanced self-renewal. No MS were formed by MECs lacking GSK-3 and beta-catenin. ErbB2/Neu-mediated mammary tumor progression has been associated with Wnt/beta-catenin pathway activation. Loss of beta-catenin delayed tumor onset in a constitutively active ErbB2 mouse model but did not alter either the luminal characteristics of the ensuing tumors nor their metastatic potential. Collectively these studies indicate GSK-3 plays important roles in mammary gland function thereby suppressing mammary tumor formation.