Doctor of Philosophy / Department of Biology / Susan J. Brown / How animals polarize and establish the main axis during embryogenesis has been one of the most attractive questions in Biology. Increasing body of work in various model organisms implicates that most metazoans utilize the canonical Wnt signaling pathway to pattern the anterior-posterior (AP) axis, despite the limited evidence from arthropods. In Drosophila, a highly derived insect, canonical Wnt activity is not required for global AP patterning, but in typical insects including Tribolium castaneum, loss of canonical Wnt activity results in posterior truncation. To determine the eff ects of increased canonical Wnt levels, I analyzed the function of axin, encoding a highly conserved negative regulator of the pathway. Tc-axin transcripts are maternally localized to the anterior pole in freshly laid eggs. Parental RNAi for Tc-axin produced progeny phenotypes that ranged from mildly a ffected embryos with cuticles displaying a graded loss of anterior structures, to severely a ffected embryos lacking cuticles and condensing to the posterior pole of the egg without any de finable structures. Altered expression patterns of several blastodermal markers indicated anterior expansion of posterior fates. Epistasis analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the eff ects of Tc-axin depletion are mediated through this pathway and that canonical Wnt activity must be repressed for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt activity must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt signaling in de fining AP polarity and patterning in metazoan development. Additionally, as an anterior structure, the extraembryonic serosa is reduced in Tc-axin RNAi progeny. However, in Tc-pangolin (Tc-pan, a homolog of Wnt downstream component) RNAi progeny, an interesting phenotype was produced that serosa was not only reduced but also separated into distinct anterior and dorsal domains. I carefully recorded this phenomenon with live imaging using a Tribolium transgenic line that expresses GFP in each nucleus. Through careful examination with embryonic fate-map markers, I found that the tissue between separated serosa domains is dorsally extended head lobe. And I also found that in severe phenotype, dorsal serosa was completely gone while anterior serosa not, suggesting independent regulation mechanisms for anterior and dorsal serosa formation. This descriptive data will complement future study in the genetic mechanism underlying serosa formation by providing more details in morphogenesis.

Doctor of Philosophy / Division of Biology / Theodore J. Morgan / Thermal stress impacts animals around the globe and understanding how organisms adapt to changes in temperature is of particular interest under current climate change predictions. My research focuses on the evolutionary genetics involved in cold tolerance and plasticity of cold tolerance using both artificially selected and naturally segregating populations, while tying the genes of interest to their physiological components. First I address cross-tolerance of stress traits following artificial selection to a non-lethal cold tolerance metric, chill-coma recovery. Using these artificial selection populations, we found that stress traits such as desiccation tolerance, starvation tolerance, acclimation, and chronic and acute cold tolerance do not correlate with level of cold tolerance as defined by chill-coma recovery time. We next assessed lifetime fitness of these different cold tolerance lines and found that only at low temperatures did fitness differ among cold tolerance levels. We then analyzed gene expression differences between resistant and susceptible populations at three time points to understand where selection pressures are hypothesized to act on genomic variation. Our gene expression analyses found many differences between resistant and susceptible lines, primarily manifesting themselves in the recovery period following cold exposure. We next utilized a community resource, the Drosophila melanogaster reference panel, to identify naturally segregating variation in genes associated with cold acclimation and fitness. We specifically asked if long- and short-term acclimation ability had overlapping genetic regions and if plasticity values from constant rearing environments were associated with demographic parameters in fluctuating environments. We found that long- and short-term acclimation are under unique genetic control and functionally tested several genes for acclimation ability. We also found that acclimation ability in constant environments and fitness in fluctuating environments do not correlate, but that genotypes are constrained in their fitness abilities between a warm and cool environment. Our analyses describe several novel genes associated with cold tolerance selection and long- and short-term acclimation expanding our knowledge of the complex relationship between demographic components and survivorship as well as a unique investigation of the change in gene expression during cold exposure.

Master of Science / Department of Animal Sciences and Industry / Jennifer M. Bormann / It is important for animals to be feed efficient and reproductively sound to optimize profits for cow- calf producers. The objective of this study was to determine the relationship between feed efficiency and reproductive performance. Feed efficiency measures included residual feed intake (RFI), feed to gain ratio (F:G) and daily dry matter intake (DMI). Reproductive measurements were pregnancy rate, first service conception rate, pregnancy type (AI, natural. open), calving percentage, calving day (CD) and age at first calving. Two data sets which included 136 crossbred Angus females sired by bulls with high or low RFI estimated breeding values (EBV) with multiple parity information and 56 purebred Hereford heifers with their first parity calving information were analyzed. Initially, the crossbred Angus females were analyzed based on their phenotypic RFI values. There was no difference in pregnancy rate between the feed efficiency measures. Second parity pregnant females had lower (F:G) with first service conception (P=0.053), and pregnancy types (P=0.014) than the open (less efficient) females. In parity 5, phenotypically efficient RFI Angus females were pregnant to first service conception versus inefficient RFI Angus females (P=0.052) and those with lower DMI were diagnosed pregnant (P=0.0002). When evaluated as a repeated trait, RFI was not a significant indicator of CD (P=0.514). Crossbred Angus females were analyzed based on their sire’s RFI EBV and grouped accordingly into high (inefficient) or low (efficient) RFI sires. Females sired by high or low RFI (EBV) bulls showed no difference for the reproductive traits, except for a tendency for inefficient sired heifers to have a lower calving percentage in parity 2 (P=0.048). When CD was analyzed as a repeated measure, no difference between sire groups was found (efficient CD of 35.64 d, inefficient CD of 34.23, p = 0.789). In the Hereford heifers, RFI was not an indicator of CD (P = 0.774). There was also no difference in RFI between pregnant and open Hereford females with pregnant females having an LSMean of 0.11 kg/d and the open females having an LSMean of 0.14 kg/d (P = 0.860). Further research needs to be done to confirm any relationships between feed efficiency and reproduction.

Doctor of Philosophy / Genetics Interdepartmental Program - Plant Pathology / Bikram Gill / The diploid D genome species, Aegilops tauschii Coss. (2n=2x=14,DD) has provided numerous genes for resistance to diseases and insect pests that attack bread wheat (Triticum aestivum L. (2n=6x=42, AABBDD). Wheat production is currently threatened by broadly virulent races of the ‘Ug99’ lineage of wheat stem rust caused by the fungus, Puccinia graminis f.sp. tritici Pers. & Eriks. Screening of a large set of Ae. tauschii germplasm for resistance to TTKSK (Ug99) identified potentially novel sources of resistance. To expedite utilization of TTKSK resistance from Ae. tauschii, a direct hybridization approach was established that integrates gene transfer, mapping and introgression into one process. Direct crossing of Ae. tauschii accessions with an elite wheat breeding was used to initiate transfer of resistance. Genetic mapping of resistance was accomplished during gene transfer through development of BC[subscript]2 mapping populations. Bulked segregant analysis of BC[subscript]2F[subscript]1 genotypes at 70 SSR loci across the D genome identified the chromosome locations of stem rust resistance genes and facilitated genetic mapping. Using this approach, TTKSK resistance from CDL4424 and TA1662 was mapped on chromosome arm 1DS flanked distally by Xwmc432 and proximally by Xwmc222 at 4.4 cM, TA10187 on 6DS linked to Xcfd49 at 1.9 cM and TA10171 on 7DS linked Xwmc827 at 0.9 cM. TTKSK resistance from additional Ae. tauschii accessions CDL4366, TA1615, TA1642, TA1693 and TA1718 has been recovered in segregating populations but await mapping. Altogether, TTKSK resistance from eight Ae. tauschii accessions has been introgressed to a hard winter wheat genetic background. Three new stem resistance genes have been tagged with molecular markers for marker assisted breeding and will provide valuable material for stem rust resistance breeding and gene pyramids for effective control of stem rust.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

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 Agronomy / William T. Schapaugh Jr / Crops research moving forward faces many challenges to improve crop performance. In breeding programs, phenotyping has time and economic constraints requiring new phenotyping techniques to be developed to improve selection efficiency and increase germplasm entering the pipeline. The objectives of these studies were to examine the changes in spectral reflectance with soybean breeding from 1923 to 2010, evaluate band regions most significantly contributing to yield estimation, evaluate spectral reflectance data for yield estimation modeling across environments and growth stages and to evaluate the usefulness of spectral data as an optimized phenotyping technique in breeding programs. Twenty maturity group III (MGIII) and twenty maturity group IV (MGIV) soybeans, arranged in a randomized complete block design, were grown in Manhattan, KS in 2011 and 2012. Spectral reflectance data were collected over the growing season in a total of six irrigated and water- stressed environments. Partial least squares and multiple linear regression were used for spectral variable selection and yield estimation model building. Significant differences were found between genotypes for yield and spectral reflectance data, with the visible (VI) having greater differences between genotypes than the near-infrared (NIR). This study found significant correlations with year of release (YOR) in the VI and NIR portions of the spectra, with newer released cultivars tending to have lower reflectance in the VI and high reflectance in the NIR. Spectral reflectance data accounted for a large portion of variability for seed yield between genotypes using the red edge and NIR portions of the spectra. Irrigated environments tended to explain a larger portion of seed yield variability than water-stressed environments. Growth stages most useful for yield estimation was highly dependent upon the environment as well as maturity group. This study found that spectral reflectance data is a good candidate for exploration into optimized phenotyping techniques and with further research and validation datasets, may be a suitable indirect selection technique for breeding programs.

Doctor of Philosophy / Department of Plant Pathology / John F. Leslie / Members of the genus Fusarium are widely distributed in many geographic regions of the world. This genus includes plant pathogens of many important cereal crops, e.g., wheat, maize, rice and sorghum, and of other native and economically important plants. From culture collections at Kansas State University and Universiti Sains Malaysia, strains from Southeast Asia, primarily from Malaysia and Thailand, associated with mango malformation disease, bakanae disease of rice, and stalk rot of sorghum were analyzed in sexual crosses and molecular diagnostics, e.g., Amplified Fragment Length Polymorphisms (AFLPs). Fusarium proliferatum was recovered from all three crops, with each crop also yielding some species unique to the crop, e.g. F. fujikuroi from rice, F. thapsinum from sorghum, and F. mangiferae from mango. These results are consistent with hypotheses that F. proliferatum has a wide host range while other species have much more limited host preferences. The absence from our samples of species associated with these diseases in other parts of the world suggests policies should be developed to reduce the chances of introduction of novel pathogens into Southeast Asia. Fusarium fujikuroi and F. proliferatum are closely related. They usually can be separated by sexual cross-fertility and DNA sequence analysis. However, some strains can cross irregularly and with poor fertility to produce viable interspecific hybrids. From a laboratory cross between F. fujikuroi FGSC8932 and F. proliferatum FGSC7615, 533 progeny were collected. These progeny were characterized for their AFLP genotype, mating type, gibberellic acid production, and pathogenicity on rice, onions, and apples. A recombination-based map from this interspecific cross was constructed. QTLs associated with gibberellic acid production, rice pathogenicity, and onion pathogenicity were identified. Gene segregation amongst the progeny of the F. fujikuroi × F. proliferatum cross was distorted towards F. proliferatum. Both novel and transgressive pathogenicity phenotypes were detected. Overall, this research demonstrates the potential threats that can result from an interspecific cross. These threats include pathogens with novel toxin profiles, new pathogenicity phenotypes, and more virulent strains. The variation observed among the progeny may enable isolation and characterization of genetic factors that have a role in pathogenicity, toxin production, and host specificity.