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Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Jack D. Fry / ‘Meyer’ zoysiagrass (Zoysia japonica Steud.) has been the predominant cultivar in the transition zone of the U.S. since its release in 1952, primarily because of its good freezing tolerance. However, it is slow to establish and recover after sod harvest, and has poor shade tolerance. I evaluated ‘Meyer’, some commonly used cultivars, and 18 progeny from crosses of ‘Emerald’ (Z. japonica × Z. tenuifolia Willd. ex Thiele) × Z. japonica or Z. matrella (L.) Merr. × Z. japonica for stolon growth characteristics; sod tensile strength and recovery after harvest; shade resistance; freezing tolerance and its relationship to autumn color retention; and the potential influence of dehydrin and chitinase gene expression in freezing tolerance. After planting vegetative plugs, rates of stolon initiation (r = 0.66 in 2007, r = 0.94 in 2008) and elongation (r = 0.66 in 2007, r = 0.53 in 2008) were positively correlated (P < 0.05) with zoysiagrass coverage. At 60 days after sod harvest, recovery growth coverage ranged from 17% to 97% and a progeny from Z. matrella × Meyer (97% coverage) demonstrated superior sod recovery growth to Meyer (38% coverage). Under 68% silver maple (Acer saccharinum L.) tree shade, stolon number was reduced 38 to 95% and stolon length 9 to 70% compared to turf in full sun. Several progeny from crosses between Emerald or a Z. matrella x Z. japonica produced more and/or longer stolons than Meyer in the shade, suggesting potential for increased shade tolerance. Autumn color in October and November, 2007 was positively correlated (r = 0.44 and r = 0.58, P < 0.01) with the lethal temperature killing 50% of tillers (LT50) in December, 2007. All grasses except Cavalier and one progeny were equivalent to Meyer in freezing tolerance with LT50s ranging from -0.2 to -12.2 oC. Dehydrin-like (11.9, 23, 44.3, and 66.3 kDa) and chitinase (26.9 kDa) gene expression increased with cold acclimation and was similar among all grasses. In general, some new zoysiagrass progeny exhibited superior growth and/or stress tolerances compared to Meyer, which bodes well for potential release of a new cultivar for use in the transition zone.

Doctor of Philosophy / Department of Biology / Mark Ungerer / Elucidating the molecular basis of adaptive phenotypic variation represents a central aim in evolutionary biology. Using the model plant species Arabidopsis thaliana, I studied the intra-specific variation in freezing tolerance among natural accessions across its native range. Considerable variation in freezing tolerance among 71 selected accessions was observed both with and without a prior cold acclimation treatment, suggesting that both differences in cold-acclimation capacity and in intrinsic physiology contribute to this variation. A highly significant positive relationship was observed between freezing tolerance and latitude of origin of these accessions. This clinal pattern of variation is found to be attributable, at least in part, to relaxed purifying selection on CBF/DREB1 genes in the species’ southern range. These CBF/DREB1 genes encode transcriptional activators that play a critical role in the ability of A. thaliana plants to undergo cold acclimation and thereby achieve maximum freezing tolerance. Relative to accessions from northern regions, accessions of A. thaliana from the southern part of their geographic range exhibit significantly higher levels of nonsynonymous polymorphisms in coding regions of CBF/DREB1 genes. Relaxed selection on the CBF/DREB1s in southern accessions also has resulted in mutations in regulatory regions that lead to abrogated expression. These mutations in coding and regulatory regions compromise the function of CBF/DREB1 transcriptional activators during the cold acclimation process, as determined by reductions in rates of induction and maximum levels of expression in the downstream genes they regulate. These mutations could be selective neutral or beneficial in southern accessions depending on whether there is an allocation cost associated with cold acclimation. The fitness benefit and possible allocation cost of cold acclimation was examined in freezing and freezing-free environments using natural accessions exhibiting contrasting abilities of cold acclimation as well as transgenic CBF gene over-expression or knockdown/knockout lines. The extent to which cold acclimation benefits the plant in presence of freezing temperature is revealed, but a cost of cold acclimation wasn’t detected in the absence of freezing temperature under our experimental design, which suggests that these mutations in CBF genes in southern accessions might be neutral to natural selection.

Tyrimų tikslas. Identifikuoti naujus žieminių kviečių genus, aktyvuojamus žemose temperatūrose bei atlikti šių genų sekų ir genetinės raiškos analizę. Tyrimų uždaviniai: 1. Ištirti žieminių kviečių genotipų tolerantiškumą žemoms neigiamoms temperatūroms natūraliomis ir dirbtinėmis sąlygomis. 2. Nustatyti skirtingose grūdinimosi stadijose diferenciškai ekspresuojamus genus (kDNR-AFLP analizė) ir atlikti DNR sekų analizę. 3. Optimizuoti mutagenezės metodą ir sukurti dviejų perspektyvių žieminių kviečių linijų mutagenines populiacijas. 4. Nustatyti skirtingu intensyvumu ekspresuojamų genų mutacijas HRM metodu, įvertinti nustatytų mutacijų įtaką genų ekspresijai grūdinimosi metu ir žemų neigiamų temperatūrų tolerancijai. / Research objective: To identify new genes of winter wheat induced at low temperatures, to perform sequence analysis of these genes and to quantify their expression. Experimental tasks: 1. To evaluate freezing tolerance of various winter wheat genotypes under field and controlled conditions. 2. To identify differentially expressed genes at subsequent time points of cold acclimation and to perform analysis of their DNA sequences. 3. To optimize mutagenesis method and to create Targeting Induced Local Lesions in Genomes (TILLING) population of two winter wheat lines. 4. To identify mutations in differentially expressed genes by High Resolution melting (HRM) method, to estimate the impact of mutations on the gene expression during cold acclimation and tolerance to freezing.

Research objective: To identify new genes of winter wheat induced at low temperatures, to perform sequence analysis of these genes and to quantify their expression. Experimental tasks: 1. To evaluate freezing tolerance of various winter wheat genotypes under field and controlled conditions. 2. To identify differentially expressed genes at subsequent time points of cold acclimation and to perform analysis of their DNA sequences. 3. To optimize mutagenesis method and to create Targeting Induced Local Lesions in Genomes (TILLING) population of two winter wheat lines. 4. To identify mutations in differentially expressed genes by High Resolution melting (HRM) method, to estimate the impact of mutations on the gene expression during cold acclimation and tolerance to freezing. / Tyrimų tikslas. Identifikuoti naujus žieminių kviečių genus, aktyvuojamus žemose temperatūrose bei atlikti šių genų sekų ir genetinės raiškos analizę. Tyrimų uždaviniai: 1. Ištirti žieminių kviečių genotipų tolerantiškumą žemoms neigiamoms temperatūroms natūraliomis ir dirbtinėmis sąlygomis. 2. Nustatyti skirtingose grūdinimosi stadijose diferenciškai ekspresuojamus genus (kDNR-AFLP analizė) ir atlikti DNR sekų analizę. 3. Optimizuoti mutagenezės metodą ir sukurti dviejų perspektyvių žieminių kviečių linijų mutagenines populiacijas. 4. Nustatyti skirtingu intensyvumu ekspresuojamų genų mutacijas HRM metodu, įvertinti nustatytų mutacijų įtaką genų ekspresijai grūdinimosi metu ir žemų neigiamų temperatūrų tolerancijai.

This thesis evaluated the role of Serine/Arginine-rich proteins, also known as SR proteins, in addition to LAMMER kinases in the cold acclimation response using Brassica napus and Arabidopsis thaliana. Transcription profile analyses of SR and LAMMER kinase genes in Brassica napus and BnCBF overexpressor lines showed that exposure to low temperatures led to increased transcript levels for nine SR genes and two kinases. BnCBF overexpression was found to exacerbate this response. This was associated with increases in SR protein abundance and phosphorylation status, suggesting that SR proteins play an essential role in cold acclimation. These findings prompted further studies to assess the role of SR proteins and kinases in the cold acclimation induced adjustment of photosynthesis, the acquisition of freezing tolerance and the transcriptional profile of CBF, SPS and COR genes, which play an important role in the transcriptional cascade allowing plants to undergo cold acclimation. Using Arabidopsis loss-of-function mutants of SR proteins and AME3 LAMMER kinase, it was shown that At-RSZ22 and At-SR45 are indispensable in the regulation of photosynthesis under non-acclimated and cold acclimation conditions. At-RSZ22a, At-SCL30 and At-RS41 were then proposed to play a crucial role in the cold acclimation induced adjustment of photosynthetic performance. Moreover, the deletion of At-AME3 kinase not only jeopardized the cold acclimation induced adjustment of photosynthetic performance, but also the acquisition of freezing tolerance. This was associated with attenuation of the transcription profile of key cold responsive genes and protein abundance of COR15 A/B and dehydrins. These findings prompted further physiological characterization of ame3 mutants, and the elucidation of Serine/Arginine-rich proteins capable of interacting with this LAMMER kinase of interest. Under cold stress and acclimation conditions, the deletion of At-AME3 LAMMER kinase impeded Photosystem I physiology and state-1 state-2 transitions. These findings were associated with decreases in Photosystem II and Photosystem I protein abundance. Yeast 2-hybrid assays showed that six SR proteins are capable of physically interacting with AME3. Taken together, the results of this study demonstrate that At-RSZ22 and At-SR45 are essential in the photosynthetic performance of Arabidopsis, that At-RSZ22a, At-SCL30 and At-RS41 play an essential role in the cold acclimation induced recovery of photosynthetic performance, and that At-AME3 plays an essential role in the cold acclimation response.

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Little is known about the tick populations found within the central Appalachian/southwestern region of Virginia. The main focus of this research was to better assess local tick communities in the central Appalachian-Southwestern region of Virginia, which was addressed by determining species diversity, habitat associations, seasonal phenology, pathogen prevalence, and ecological factors that influences tick presence and abundance. A field study was conducted from June 2019 - November 2020 across 8 counties and 3 habitat types. Forested habitats exhibited greater tick species diversity than pasture and urban habitats. Each tick species was observed to be associated with particular habitats. The presence of B. burgdorferi sensu stricto (causative agent of Lyme disease), the human variant of A. phagocytophilum (causative agent of human granulocytic anaplasmosis), and Powassan virus (the causative agent of Powassan encephalitis) were detected in collected field specimens, suggesting a significant threat to public health. The detection of Powassan virus RNA in local Ixodes scapularis ticks is the first evidence of this viral pathogen within the region. The overwintering abilities of ticks, whose populations are expanding or becoming more invasive, including Haemaphysalis longicornis, Amblyomma americanum, and Amblyomma maculatum, were examined through a combination of laboratory and field experiments. Amblyomma americanum and H. longicornis nymphal ticks had a lower supercooling temperature than adult ticks, suggesting their potential to overwinter better; A. maculatum nymphs had similar average supercooling temperatures as the other two species at nymphal stage. Via a field experiment, A. americanum, H. longicornis, and A. maculatum were subjected to natural elements of a Virginian winter in a two-factor design investigating elevation and potential insulation coverage. Elevation and insulation coverage were found to have no significant impact on the overwintering survival of H. longicornis and A. americanum. However, the life-stage of the tick was determined to be a significant factor that dictated the survival of ticks of these species. Overwintering survival of Amblyomma maculatum nymphs was influenced by insulation (proxied by leaf litter); which may be attributed to this tick's preference of drier climate. Low overwintering survival suggests that a tick may not be able to establish a permanent population within the area. / Master of Science in Life Sciences / Ticks can transmit many pathogens, bacteria or viruses that cause disease, to both humans and animals. Diseases associated with ticks can include Lyme disease, Human Anaplasmosis, Ehrlichiosis, Rocky Mountain spotted fever, as well as viral illnesses. The pathogens that cause these diseases are vectored by specific tick species. Currently, there are no published reports of tick species present within the central Appalachian/southwestern region of Virginia. A study to determine local tick populations presence and the factors influencing their maintenance in the environment was conducted. Additionally, field collected specimens were screened to assess pathogen prevalence within the region. I confirm the presence of Borrelia burgdorferi sensu stricto (causative agent for Lyme disease), Anaplasma phagocytophilum (causative agent for Human Granulocytic Anaplasmosis), and Powassan virus within Ixodes scapularis, commonly referred to as black-legged ticks. Habitat (forest, urban, pasture) was a significant influencing factor on the presence of I. scapularis, Amblyomma americanum, and Haemaphysalis longicornis (e.g., the latter species was predominantly found in pasture habitats). Secondly, I investigated how well three sympatric tick species would overwinter within the region. Survival of a tick is heavily dependent upon their ability to withstand desiccation, obtain a blood meal from a host, mate, and reproduce. Certain ticks may be limited to certain regions based on elevation, temperature, and relative humidity. I investigated the influence of elevation and insulation coverage (presence of leaves vs. no leaves) on A. americanum (Lone star tick), H. longicornis (Asian longhorned tick), and A. maculatum (Gulf Coast tick). Elevation and insulation coverage had no significant impact on the overwintering survival of the Lone star and Asian longhorned ticks. Tick survival was mainly dictated by what life-stage they were in; Lone star ticks survived better as adult than at other life stages. Asian longhorned ticks survived better as nymphs than larva and adults. Insulation coverage influenced the overwintering survival of the Gulf Coast tick. These ticks survived better with no leaves, which may be associated with their preference of drier climates.