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Meeting the food, feed, and fiber needs of a growing world population represents one of the signature challenges of this century. The UN FAO estimates that food production alone must increase by 70 percent by 2050 to meet the needs of a projected nine billion people. Demand will also increase for livestock feed, biofuel feedstocks, fiber for paper products, and construction materials. Meeting these demands will require implementation of advanced technologies, sustainable management of natural resources, and coordination of political forces. We must think beyond the boundaries of traditional agriculture, to integrate breakthroughs in plant science, engineering, environmental sciences, and other disciplines...

Michaux's sumac (Rhus michauxii) is a federally endangered rhizomatous shrub endemic to the southeastern United States, with two of the largest populations located at Maneuver Training Center--Fort Pickett, VA (Fort Pickett), and a nearby property, Deepwater. Michaux's sumac requires soil disturbance and fire to maintain healthy populations. Before being added to the endangered species list, 47% of populations were extirpated due to habitat loss, fire suppression, and hybridization with smooth sumac (Rhus glabra). Concerns with hybridization include hybrid swamping if hybrids are fertile, or outbreeding depression if hybrids display reduced fitness. I used genotyping-by-sequencing to estimate the extent of hybridization at Fort Pickett and Deepwater, and to assess how such hybridization may impact survival of Michaux's sumac as a distinct species at each of these locations. Additionally, population structure was examined using DAPC (discriminant analysis of principal components) and Admixture analyses to determine whether the colonies at Fort Pickett and Deepwater make up separate populations, meta-populations, or one large population. Analysis of 107,344 SNPs (single nucleotide polymorphisms) using Introgress and Admixture software suggested widespread hybridization at both Fort Pickett and Deepwater, with hybrids present in most of the sampled colonies. Population structure analyses revealed differentiation between the Fort Pickett and Deepwater populations, but little evidence of separate populations among the colonies sampled at Fort Pickett. These results are important for conservation planning to ensure the long-term survival of Michaux's sumac at Fort Pickett and Deepwater and can be used to help inform future management decisions. Master of Science Michaux's sumac (Rhus michauxii) is a small, federally endangered shrub endemic to the southeastern United States, with populations remaining in North Carolina, Virginia, and Georgia. To date, the largest known colonies of Michaux's sumac are in Virginia at Maneuver Training Center--Fort Pickett, VA (Fort Pickett), and at a nearby privately owned property called Deepwater. Michaux's sumac requires soil disturbance and fire to reduce competition and maintain healthy populations. It currently faces threats from habitat loss due to agricultural land use and fire suppression, and hybridization with a closely related species, smooth sumac (Rhus glabra). Hybridization is a threat to Michaux's sumac at Fort Pickett and Deepwater because it co-occurs with smooth sumac throughout the area. This study determined how much smooth sumac and Michaux's sumac are hybridizing in these locations and assessed whether hybridization is a threat to the long-term survival of the populations at each site. A secondary goal of the study was to gain a better understanding of how genetically similar the colonies within and between locations are to one another. Understanding the level of hybridization and the population structure of Michaux's sumac is important for making management decisions to protect the species. I found widespread hybridization between Michaux's and smooth sumac, with hybrid individuals at nearly all the colonies sampled. Additionally, there is evidence that Fort Pickett and Deepwater comprise two distinct populations, but the colonies inside each area are likely not separate populations. These results will inform future conservation management decisions for the species.

Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Thus, methods to accelerate genetic improvement to improve woody biomass yield, quality and optimal growth on marginal lands are of considerable interest. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, co-orthologs of Arabidopsis SECONDARY CELL WALL NAC DOMAIN2 (SND2), which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, members of the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN) gene family that act as flowering repressors in Arabidopsis and many plants. I studied INRA 717-1B (P. tremula x P. alba) transgenics with an artificial microRNA (AmiRNA) downregulating PopNAC154 and PopNAC156 (AmiSND2 trees). In a field trial, AmiSND2 trees showed higher mean height and diameter than wild-type (WT). We also observed that AmiSND2 transgenics showed delayed leaf senescence and leaf drop. After conducting controlled environment studies with AmiSND2 trees, I was able to confirm that downregulation of PopNAC154 and PopNAC156 genes does not alter the short daylength-induced bud set and growth cessation but it delays the low temperature induced leaf senescence and leaf drop. Further I was able to show that down regulation of the PopNAC154 and PopNAC156 genes resulted in significantly higher mean plant heights and delayed bud set compared to the WT plants under low soil nutrient conditions. Wood chemistry data analysis of field grown AmiSND2 trees showed that they have a significantly higher cellulose content a lower lignin content compared to that of the WT. Thus, these results show that downregulating the PopNAC154 and PopNAC156 genes has the potential to increase biomass yield and quality. In a previous study, simultaneous downregulation of PopCEN1 and PopCEN2 genes using RNA interference (RNAi) method caused poplar trees to flower only after two years of growth in the field. I used CRISPR/CAS9 method to knock-out each paralog individually as well as the related gene, PopBFT. The popcen1 mutant trees developed flowers even under in vitro conditions, but popcen2 mutants did not show an obvious phenotype. popbft mutant trees also did not show an obvious phenotype under standard growing conditions. However, when soil nutrient availability was allowed to deplete, the popbft mutants showed lower mean plant height compared to the WT and also showed lower root length and root volume under low Nitrogen conditions in an in vitro assay compared to the WT. These results prove that PopCEN1 gene is directly involved in repressing flowering in poplar and allele-specific mutation should be tested as an approach to accelerate breeding. PopCEN2 and PopBFT might not have a role in regulation of flowering time, and though additional studies are needed, PopBFT appears to have a role in regulating growth in response to nutrient availability. Master of Science Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Genes control the woody biomass yield, and quality among all the other characteristics, thus, methods to accelerate genetic improvement to improve these characteristics is paramount. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, genes which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, genes that act as flowering repressors in Arabidopsis and many plants. I studied a genetically modified poplar, where PopNAC154 and PopNAC156 genes’ function were reduced. After subjecting these plants to shorter daylength periods and cold temperatures in controlled conditions I was able to show that decreased activity of the PopNAC154 and PopNAC156 genes causes the trees to slowdown the leaf senescence and retain their leaves under low temperature conditions compared to the genetically unaltered wildtype (WT) poplar. I was also able to show that decreased activity of the PopNAC154 and PopNAC156 genes allow the poplar plants to grow better than the WT plants under low soil nutrient conditions. I used gene editing to switch off PopCEN1, PopCEN2, and PopBFT genes. When PopCEN1 gene was switched off individually the young mutant trees flowered whereas normally trees take six to eight years to flower. However, when the PopCEN2, and PopBFT genes were switched off individually the resulting mutant plants did not show any signs of flowering. Hence, I was able to show that PopCEN1 gene is directly involved in repressing the flowering in poplar plants. In conclusion, my work identified PopNAC154 and PopNAC156 as candidate genes for manipulation by breeding or biotechnology to increase wood yield, and suggested ways to induce flowering to accelerate breeding through manipulation of PopCEN1.

Development of winter wheat (Triticum aestivum L.) cultivars capable of more efficient uptake and utilization of applied nitrogen (N) has the potential to increase grower profitability and reduce negative environmental consequences associated with N lost from the plant-soil system. The first study sought to evaluate genotypic variation for N use efficiency (NUE) and identify lines consistently expressing high or low NUE under two or more N rates in a total of 51 N-environments. The results indicated that significant genotype by N rate interactions were frequently observed when trials utilized at least three N rates and identified wheat lines with high and stable yield potential that varied in performance under low N conditions. In addition, NUE was associated with above-ground biomass at physiological maturity were found to be both highly heritable across multiple N supplies. In the second study, two bi-parental mapping populations having a common low ('Yorktown') and two high (VA05W-151 and VA09W-52) NUE parents were characterized to dissect the genetics underlying N response. The populations were evaluated in eight N-environments and genotyped using single-nucleotide polymorphism data derived from a genotyping-by-sequencing protocol to identify quantitative trait loci (QTL) associated with high NUE. Six QTL for NUE were identified on chromosomes 1D, 2D, 4A, 6A, 7A, and 7D that were associated with N use efficiency. The QTL on 2D and 4A co-localized with known loci governing photoperiod sensitivity and resistance to Fusarium head blight (caused by the fungal pathogen Fusarium graminearum Schwabe), respectively. Three of the identified QTL (6A, 7A, and 7D) were associated with NUE in previous investigations, while the QTL on 1D was novel. The final experiment employed a small panel of soft red winter wheat lines to study the effects of photoperiod alleles on chromosome 1D (Ppd-D1) on yield-related traits under three or five N rates that were variably split over two growth stages in eight environments. The results validated the effect of a photoperiod sensitive allele (Ppd-D1b) that was associated with increased grain yield across N rates in half of the Virginia testing environments and under low N rates in all Ohio testing sites at the expense of grain N content. Yield advantages conferred by the Ppd-D1b allele were attributable to increased floret fertility and kernel number per spike. The findings from these studies have direct application for winter wheat breeding programs targeting NUE improvements. Doctor of Philosophy Wheat (Triticum aestivum L.) products account for a significant percentage of the total dietary calories and protein consumed globally. To meet production demands, wheat requires efficient nitrogen (N) management to ensure continued grower profitability and to reduce negative environmental impacts of N lost from agricultural systems. This dissertation sought to evaluate variation among wheat lines for N use efficiency (NUE), assess the performance of wheat lines under multiple N supplies, validate traits that are associated with NUE, investigate the role of photoperiod sensitivity genes on N response, and identify regions of the wheat genome associated with high N use efficiency. These studies were conducted using panels of winter wheat lines grown under two or more N conditions over a combined 32 location-years. Results of Chapter I identified variation in cultivar response to N rates was more frequently observed when a greater number of N rates were used in trials of wheat N response. The first chapter also identified variation among wheat lines for NUE and identified lines that consistently produce high grain yields over N-location-years. In addition, above-ground biomass at physiological maturity was found to be strongly associated with grain yield under all N rates and was highly heritable in both studies. Chapter II utilized a combination of genetic and observable trait data to perform genetic analysis in two bi-parental populations grown in eight Nlocation-years. The study identified reproducible and significant genetic markers associated with NUE for application in wheat breeding programs. Upon analysis of photoperiod sensitive versus insensitive wheat lines in Chapter III, photoperiod sensitive wheat lines had a significant yield advantage under N-limited conditions in Ohio and across N treatments in half of the Virginia testing location-years. This resulted from an increased number of kernels per spike and fertile florets in photoperiod sensitive wheat lines. Results from this dissertation suggest that active breeding and selection for N response may be achieved through the employment of high NUE genes and the continued identification of adapted high NUE wheat parental lines.

The adaptation of trees to temperate and boreal climates depends on their ability to respond to environmental signals that are markers of seasonal changes in order to survive winter and maximize growth. The genus Populus (poplars) is a model system for identifying the genes and molecular mechanisms that regulate growth and dormancy transitions. Photoperiod and temperature regulate both vegetative and floral phenology. FLOWERING LOCUS T (FT) and FLOWERING LOCUS D (FD) are key regulators of flowering time in Arabidopsis and other plants. The distinct functions of three poplar FD-LIKE (FDL) genes and two FTs were explored through gain-of-function, dominant repression, and CRISPR/CAS9-mediated gene editing. We studied trees in controlled environments, including manipulation of daylength and temperature to mimic an annual seasonal growth and dormancy cycle. Our studies showed that the FDL proteins share less than complete functional equivalency. Among the three paralogs, only FDL2.2 promoted precocious flowering, whereas FDL1 and FDL3 appear to have distinct roles in vegetative growth and phenology. Whereas overexpression of any FDL gene delays short day-induced growth cessation and bud set, only FDL3 coordinately altered leaf development and the transition to secondary growth in a photoperiod-dependent manner. For the first time, we demonstrate distinct functions of the two FT paralogs in vegetative phenology. Study of ft1ft2 double mutants and ft1-specific mutants showed that FT1 promotes dormancy release, whereas FT2 is necessary to sustain growth. Collectively, our results reveal that poplar FTs and FDLs have distinct roles in controlling different aspects of vegetative phenology and woody shoot development. PHD The adaptation of trees to temperate and boreal climates depends on their ability to respond to environmental signals that are markers of seasonal changes in order to survive winter and maximize growth. The genus Populus (poplars) is a model system for identifying the genes and molecular mechanisms that regulate growth and dormancy transitions. Photoperiod and temperature regulate both vegetative and floral phenology. FLOWERING LOCUS T (FT) and FLOWERING LOCUS D (FD) are key regulators of flowering time in Arabidopsis and other plants. The distinct functions of three poplar FD-LIKE (FDL) genes and two FTs were explored through gain-of-function, dominant repression, and CRISPR/CAS9-mediated gene editing. We studied trees in controlled environments, including manipulation of daylength and temperature to mimic an annual seasonal growth and dormancy cycle. Our studies showed that the FDL proteins share less than complete functional equivalency. Among the three paralogs, only FDL2.2 promoted precocious flowering, whereas FDL1 and FDL3 appear to have distinct roles in vegetative growth and phenology. Whereas overexpression of any FDL gene delays short day-induced growth cessation and bud set, only FDL3 coordinately altered leaf development and the transition to secondary growth in a photoperiod-dependent manner. For the first time, we demonstrate distinct functions of the two FT paralogs in vegetative phenology. Study of ft1ft2 double mutants and ft1-specific mutants showed that FT1 promotes dormancy release, whereas FT2 is necessary to sustain growth. Collectively, our results reveal that poplar FTs and FDLs have distinct roles in controlling different aspects of vegetative phenology and woody shoot development.

Disease resistance is critical in soft red winter wheat (Triticum aestivum L.) cultivars. Leaf rust caused by Puccinia triticina Eriks and stripe rust caused by Puccinia striiformis Westend. f.sp. tritici Eriks. are destructive pathogens of wheat. From 2014 to 2015 phenotypic data was collected at diverse locations for resistance to leaf rust (North Carolina, Texas, and Virginia) and stripe rust (Arkansas, North Carolina, Georgia, Texas, and Virginia) in a Pioneer ‘25R47’ /‘Jamestown’ (P47/JT) population composed of 186 F5:9 recombinant inbred lines (RILs). Analysis of the P47/JT population identified two quantitative trait loci (QTL) for leaf rust resistance on chromosome 5B and two QTL for stripe rust resistance on chromosomes 3B and 6A. Phenotypic variation (%) explained by the putative leaf rust resistance QTL of Jamestown on 5B was as high as 22.1%. Variation explained by the putative stripe rust resistance QTL of Jamestown on 3B and 6A was as high as 11.1 and 14.3%, respectively. Jamestown is postulated to contain gene Lr18. Seedlings of 186 F5:9 recombinant inbred lines from the P47/JT population and 200 F2 seedlings from eight other crosses including Jamestown and/or the Lr18 host differential line RL6009 (Thatcher*6/Africa 43) were screened with P. triticina race TNRJJ. Genetic analysis of the populations was conducted to validate the presence of Lr18 in Jamestown. Results of linkage analysis identified SNP maker IWB41960 linked within 5 cM of gene Lr18 in all three populations. From 2016 to 2017 phenotypic data was collected at diverse locations for resistance to leaf rust (Illinois, North Carolina, and Virginia) in a ‘2013412’ (PI 667644) / VA10W-21 (PI 676295) population (412/21) composed of 157 doubled haploid (DH) lines. The 412/21 DH lines were genotyped via genotyping by sequence (GBS). Analysis of the 412/21 population identified one quantitative trait loci (QTL) region associated with adult plant resistance to leaf rust on chromosome 1B. Phenotypic variation (%) explained by the putative leaf rust resistance QTL of 2013412 on 1B was as high as 40.1%. Kompetitive allele-specific (KASP) markers KASP_S1B_8414614 and KASP_S1B_8566239 were developed as markers for use in marker assisted selection. Ph. D.

The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield. Ph. D.

Forest trees are an important pool of biodiversity at the gene, individual and an ecosystem level. This variation is a result of complex environmental interactions, as well as neutral and selective forces acting on populations. Patterns of standing genetic variation are the result of adaption to past and contemporary climate change, but also historical demographic events, and disentangling the role of these forces is a central problem in population genomics. The overall goal of this study is to characterize the relative effects of demography and selection in the genome of Populus trichocarpa, a riparian deciduous tree species of North America. Specifically, I used a variety of methods to summarize patterns of genetic diversity and population structure in P. trichocarpa, and to reconstruct its demographic history. I subsequently incorporated these demographic insights to guide the application of several methods to identify genome-wide targets of natural selection within and among rangewide populations adapted to heterogeneous selection regimes. Results of this study provide insights into the history of divergence and differentiation in P. trichocarpa populations and help us identify the functional genetic variants contributing to phenotypic divergence and fitness of the individuals in it. Ph. D.

Forest trees represent a unique group of organisms combined with ecological and economic importance. Owing to their random mating system and widespread geographical distribution, they harbor abundance genetic variation both within and among populations. Despite their importance, research in forest trees has been underrepresented majorly due to their large and complex genome and scarce funding. However, recent climate change and other associated problems such as insect outbreaks, diseases and stress related damages have urged scientists to focus more on trees. Furthermore, the advent in high-throughput sequencing technologies have allowed trees to be sequenced and used as reference genome, which provided deeper understanding between genotype and environment. Whole genome sequencing is still not possible for organisms having large genomes including most tree species, and it is still not feasible economically for population genomic studies which require sequencing hundreds of samples. To get around this problem, genomic reduction is required. Sequence capture has been one of the genomic reduction techniques enabled studying the subset of the DNA of interest. In this paper, our primary goal is to outline challenges, provide guidance about the utility of sequence capture in trees, and to leverage such data in genome-wide association analyses to find the genetic variants that underlie complex, adaptive traits in spruce and pine, as well as poplar. Results of this research will facilitate bridging the genomic information gap between trees and other organisms. Moreover, it will provide better understanding how genetic variation governs phenotype in trees, which will facilitate both marker assisted selection for improved traits as well as provide guidance to determine forest management strategies for reforestation to mitigate the effects of climate change. Ph. D.

Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe, is a devastating fungal disease that affects barley production worldwide through damaged kernels, low yields, and production of mycotoxins. The most effective means of control is through the use of FHB resistant cultivars that are developed through gene pyramiding and incorporation of various sources of resistance. Resistance identified in winter barley cultivar Eve has been identified in Virginia Tech's Small Grains Breeding Program. The objectives of this study are to validate resistance and identify additional resistance QTL, and identify tightly linked and diagnostic markers for use in MAS programs. A population of F5:7 RILs derived from the cross Eve/Doyce were evaluated for FHB resistance. FHB incidence (Inc), FHB severity (Sev), Fusarium damaged kernels (FDK), and deoxynivalenol (DON) were assessed over 2014-15 and 2015-16 at Blacksburg, VA, Mount Holly, VA, Kinston, NC, and Lexington, KY. A QTL on chromosome 6H associated with FHB Sev, FDK, and DON was identified. The QTL accounted for as high as 14.5% of the phenotypic variation for DON accumulation, 8.3% for FHB Sev, and 11.87% to 17.63% for FDK. The QTL marker region spans 56.5 to 66.6 cM with flanking markers SCRI_RS_147342 and Bmag0613. QTL for morphological traits, heading date and height, were found in the same region as the FHB resistance traits. Diagnostic SNP makers associated with the FHB resistance QTL identified can be used for FHB resistance identification in MAS breeding programs to incorporate and pyramid resistance QTL and genes into other plant material. Master of Science

In multiple species, genome-wide association (GWA) studies have become an increasingly prevalent method of identifying the quantitative trait loci (QTLs) that underlie complex traits. Despite this, relatively few GWA analyses using high-density genomic markers have been carried out on highly quantitative traits in wheat. We utilized single-nucleotide polymorphism (SNP) data generated via a genotyping-by-sequencing (GBS) protocol to perform GWA on multiple yield-related traits using a panel of 329 soft red winter wheat genotypes grown in four environments. In addition, the SNP data was used to examine linkage disequilibrium and population structure within the testing panel. The results indicated that an alien translocation from the species Triticum timopheevii was responsible for the majority of observed population structure. In addition, a total of 50 significant marker-trait associations were identified. However, a subsequent study cast some doubt upon the reproducibility and reliability of plant QTLs identified via GWA analyses. We used two highly-related panels of different genotypes grown in different sets of environments to attempt to identify highly stable QTLs. No QTLs were shared across panels for any trait, suggesting that QTL-by-environment and QTL-by-genetic background interaction effects are significant, even when testing across many environments. In light of the challenges involved in QTL mapping, prediction of phenotypes using whole-genome marker data is an attractive alternative. However, many evaluations of genomic prediction in crop species have utilized univariate models adapted from animal breeding. These models cannot directly account for genotype-by-environment interaction, and hence are often not suitable for use with lower-heritability traits assessed in multiple environments. We sought to test genomic prediction models capable of more ad-hoc analyses, utilizing highly unbalanced experimental designs consisting of individuals with varying degrees of relatedness. The results suggest that these designs can successfully be used to generate reasonably accurate phenotypic predictions. In addition, multivariate models can dramatically increase predictive accuracy for some traits, though this depends upon the quantity and characteristics of genotype-by-environment interaction. Ph. D.

Forest tree breeding methods rank among the most efficient ways to increase productivity and quality of forests. With the advent of high-throughput genotyping technology, genome-enabled breeding has started to gain importance and may overcome some weaknesses of traditional tree breeding. Genomic Selection (GS), which involves using genome-wide markers to predict breeding values of individuals in a population, has been proposed for animal and plant breeding programs. GS enables very accurate selection decisions through estimation of genomic estimated breeding values (GEBVs). While the goal of GS is to predict phenotype from genotype, it does not identify the underlying genes that have important roles in a trait. Genome-Wide Association Studies (GWAS) approaches are therefore complementary to GS, enabling identification of these genes, which may be useful for marker-assisted selection in some traits. In this study, we first estimated heritability for several adaptive traits (cold hardiness, dbh, bud flush, height, and bud set) in a population of Populus trichocarpa and for height, diameter, and stem straightness in Pinus taeda. GEBVs accuracies were estimated using a ridge regression–best linear unbiased prediction (rrBLUP) model, and these accuracies were compared with estimated heritabilities. GWAS was also performed for the both imputed and non–imputed data of P. taeda population using TASSEL (Trait Analysis by aSSociation Evolution and Linkage) software, as well as rrBLUP and FFBSKAT (Fast Family-Based Sequence Kernel Association Test) packages in R. Heritabilities ranged from 0.34 to 0.56 for P. trichocarpa and 0.14 to 0.37 for P.taeda. GWAS identified 3244 associations for dbh, 4077 associations for stem straightness, and 5280 SNPs for height (p≤0.05) in TASSEL using the reduced model (marker data only), whereas 2729, 3272 and 3531 associations were found with the full model where we also included population structure as a covariate. FFBSKAT showed a similar number of SNP associations (2989, 3046 and 3058). There was an inflation of SNP associations (~20k) found in rrBLUP, which suggests population structure was not effectively controlled. The GEBVs accuracies ranged from 0.09 and 0.22 for P.trichocarpa and 0.09 to 0.23 for P.taeda using rrBLUP method. Testing the effect of repetation on the accuracy of GEBV for poplar showed that there was no significant difference between the number of cycles. Also, there was no significant difference the accuracy of GEBVs in pine between two different imputation methods, the marker mean value and Beagle software. Master of Science

Temperate tree species exhibit seasonal growth cycling, and the timing of such transition varies with local climate. Under anthropogenic climate change, the local pattern of growth and dormancy in tree populations is expected to become uncoupled with shifting seasonal environmental signals, particularly temperature. Thus, an understanding of the genetic underpinnings of local adaptation is key to predicting the fate of tree populations in the future. In this thesis, we coupled sampling of range-wide natural accessions of P. trichocarpa with adaptive trait phenotyping and genome-wide genotyping to uncover relationships between genotype, phenotype, and environment. We detected strong correlations between adaptive phenotypes, climate, and geography, which suggested climatic selection driving adaptation of these populations to local environments. We subsequently combined genotype-phenotype association tests with sliding window analysis and identified regions strongly associated with these adaptive traits. We also compared adaptive markers identified in two independent GWAS on samples across latitude and altitude transects and found a set of associated variants shared across both transects. We further scanned the genome with three selection tests to identify regions showing evidence of recent positive and divergent selection. By comparing candidate selection regions across altitude and latitude, we detected a set of overlapping regions showing differentiation across gradients of the same climate variables. We validated the functional imortance of these selection regions by combining GWAS and showed that selection regions contain a strong signature of phenotypic associations. We also studied the distribution of deleterious allels across genome and natural populations, and found that deleterious alleles preferentially accumulate in regions of low recombination and hithihking regions. Finally, marginal populations contained more deleterious alleles compared with central populations, which is likely due to ineffective selection in small populations and recent bottlenecks associated with postglacial recolonization. These findings provide new insights into the genomic architecture underlying climatic adaptation and how selection drives adaptive evolution of tree species. Ph. D.

Almost every precursor mRNA (pre-mRNA) in a eukaryotic organism undergoes splicing, in some cases resulting in the formation of more than one splice variant, a process called alternative splicing. RNA-Seq provides a major opportunity to capture the state of the transcriptome, which includes the detection of alternative spicing events. Alternative splicing is a highly regulated process occurring in a complex machinery called the spliceosome. In this dissertation, I focus on identification of different splice variants and splicing factors that are produced during Arabidopsis and soybean embryo development. I developed several data analysis pipelines for the detection and the functional characterization of active splice variants and splicing factors that arise during embryo development. The main goal of this dissertation was to identify transcriptional changes associated with specific stages of embryo development and infer possible associations between known regulatory genes and their targets. We identified several instances of exon skipping and intron retention as products of alternative splicing. The coding potential of the splice variants were evaluated using CodeWise. I developed CodeWise, a weighted support vector machine classifier to assess the coding potential of novel transcripts with respect to RNA secondary structure free energy, conserved domains, and sequence properties. We also examined the effect of alternative splicing on the domain composition of resulting protein isoforms. The majority of splice variants pairs encode proteins with identical domains or similar domains with truncation and in less than 10% of the cases alternative splicing results in gain or loss of a conserved domain. I constructed several possible regulatory networks that occur at specific stages of embryo development. In addition, in order to gain a better understanding of splicing regulation, we developed the concept of co-splicing networks, as a group of transcripts containing common RNA-binding motifs, which are co-expressed with a specific splicing factor. For this purpose, I developed a multi-stage analysis pipeline to integrate the co-expression networks with de novo RNA binding motif discovery at inferred splice sites, resulting in the identification of specific splicing factors and the corresponding cis-regulatory sequences that cause the production of splice variants. This approach resulted in the development of several novel hypotheses about the regulation of minor and major splicing in developing Arabidopsis embryos. In summary, this dissertation provides a comprehensive view of splicing regulation in Arabidopsis and soybean embryo development using computational analysis. Ph. D.

Microsatellites (MSTs) are short tandem repeats of motifs, 1 — 6 nucleotide in length, and are considered mutational 'hot-spots' and show a greater degree of somatic variability and population polymorphisms than surrounding DNA sequences. MSTs provide for a unique computational alignment problem for many commonly used algorithms, and therefore required software tools to be developed to specifically address these issues. For this work we developed a novel approach to extract MSTs from next-gen sequencing data that can robustly detect signatures of MST mutation bias and somatic variation occurring in next-gen data including a high frequency of in-phase indels. Somatic variability, novel genomic polymorphisms that arise within a cell population not found in the progenitors, plays a critical role in cellular reprogramming leading to the development and progression of cancer. MST mutation rates are between 10 and 1000 time higher than that of surrounding DNA. MSTs are found ubiquitously throughout the genome including in nearly all transcribed regions and 10-20% of coding genomic regions. Currently the only established DNA repair defect that that has been directly linked to MST instability is replication coupled mismatch repair (MMR). An initial analysis of the utility of the software was conducted with DNA repair impaired cell lines. The results demonstrated the utility in identifying the consequences of DNA repair impairments on genomic stability. There were major objectives of the finding including 1) complimenting genomics of matched DNA samples with in-sample quantification of variation and 2) demonstrating that DNA repair proficient cells and those with different defects in DNA repair can have different somatic MST variability (SMV) profiles that may be potential markers for these defects. DNA repair disorders are debilitating conditions that result in physical and neurological abnormalities robbing the individual of a normal quality of life and life span. The various conditions that fall into this class are known as progeroid disorders and they provide a very important glimpse into the aging process on a genomic level. The conditions for four cohorts analyzed here were; Cockayne's syndrome, caused by the loss of the ERCC8 gene, also known as CSA; xeroderma pigmentosum, caused by the loss of the XPA or XPB genes; Werner syndrome, caused by the loss of the RecQL2 gene; and Rothmond-Thomson syndrome, caused by the loss of the RecQL4 gene. The goal of this project was to determine if impaired excision repair genes CSA or global XPA and B or excision repair supporting helicases BLM or RecQL4 leads to MST destabilization. Comparing cohorts from excision repair disorders with a co-sequenced normal cohort we found that CSA both RecQ helicases had an effect on the exome somatic variability of MSTs. On the other hand the effects of XPA/B were inconclusive. MST instability (MSI), defined as acquired/lost primary alleles in tumors for a small set of microsatellite loci, has been implicated and is a clinically relevant marker for colorectal cancer. Conversely, no clinically actionable genetic markers have been found for liver cancer, a cancer with a very high mortality rate. Here we explore the use SMV defined as the presence of minor alleles at MST loci, as a complementary measure of MSI as a genetic marker for colorectal and liver cancer by analyzing Illumina sequenced genomes from The Cancer Genome Atlas. Our data shows that SMV may distinguish a subpopulation of African American patients with colorectal cancer, ~33% of the population in this study. Further, for liver cancer, a higher rate of SMV may be indicative of earlier age of onset. In conclusion, the work presented here suggests that MSI should be expanded to include SMV, not only instability. Ph. D.

Local adaptation to climate in temperate forest trees involves the integration of multiple physiological, morphological, and phenological traits. Latitudinal clines for the relevant component traits are frequently observed for species that have a north-south distribution, but these relationships do not account for climatic variation within a given latitudinal band, which may be reflected in adaptive traits. We used black cottonwood (Populus trichocarpa) as a model to characterize the interplay between geography, climate, and adaptation to abiotic factors. Twelve traits (height, diameter, volume index, crown diameter, number of branches, number of sylleptic branches, relative number of branches, Relative canopy depth, Bud set, Bud flush, cold index of injury, carbon isotope ratio) were measured in a range-wide sample of 124 P. trichocarpa genotypes grown in a common garden. Heritability's were moderate to high (0.24 to 0.55) and significant population differentiation (QST > 0.3) suggested adaptive divergence. When climate variables were taken as predictors and the 12 traits as response variables in a multivariate regression tree analysis, aridity (Eref) explained the most variation, with subsequent splits grouping individuals according to mean temperature of the warmest month, frost-free period (FFP), and mean annual precipitation (MAP). This grouping matches relatively well the splits using geographic variables as predictors: the northernmost groups (short FFP and low Eref) had the lowest growth performance, and the highest cold hardiness. The groups spanning the south of British Columbia (low Eref and intermediate temperatures) displayed an average growth and cold hardiness. The group from the coast of California and Oregon (high Eref and FFP) had the best growth performance and the lowest cold hardiness. The southernmost and high-elevated group (with High Eref and low FFP) performed poorly, had a low cold hardiness and a significantly lower WUE. Master of Science

Gametophytic self-incompatibility limits the ability to derive inbred lines of potato through self-pollination and is prevalent in diploid potato. Within a population of F1 hybrids between two genotypes used in potato genome sequencing, we observed fruit set on many greenhouse-grown plants. Subsequently, after controlled self-pollinations, we confirmed fruit set in 32 of 103 F1 plants. Our goal was to identify genes responsible for self-compatibility in this population and to advance selfed progeny to develop highly homozygous inbred lines. The F1 population was genotyped using a single nucleotide polymorphism (SNP) array. Polymorphic and robust SNPs were analyzed by Fisher\'s Exact Test to identify allelic states segregating with the self-compatible phenotype. Filtering 1966 SNPs to retain only those with p-values less than 0.0001 yielded 95 highly significant SNPs, with all SNPs on anchored scaffolds located on chromosome 12. Candidate genes encoding for multiple notable proteins including an S-protein homologue were identified near highly significant SNPs on the Potato Genome Browser. Seeds obtained after self-pollination of self-compatible individuals were used to advance the population for three generations. SNP chip genotyping of the S3 generation revealed entirely different SNPs segregating for self-compatibility on nine different chromosomes. Comparison of the allelic state of SNPs in the F1 and S3 generations revealed a heterozygosity reduction by 80%, with fixation of many SNPs including those surrounding the S-protein homologue. We conclude that the genes responsible for segregation of self-compatibility in the S3 generation are different from those in the F1 generation. Master of Science

We are using the yeast two-hybrid (Y2H) system to identify novel protein-protein interactions (PPI) relevant to wood formation. Bait proteins for Y2H binary assays and screening against a xylem cDNA prey library were selected from approximately 400 Populus trichocarpa genes that are at least 8-fold more highly expressed in differentiating secondary xylem versus phloem-cambium, and designated here as poplar biomass (PB) genes. Here we report some of the interactions involving selected PB proteins and efforts to characterize their functions in Populus and Arabidopsis. Members of the ROP GTPase family, PB15 in poplar and ROP11 in Arabidopsis, interact with the domain of unknown function (DUF) 620 (DUF620) proteins (e.g., PB129 in poplar). Ectopic co-expression of PB15 and PB129 in Arabidopsis caused outgrowths at the base of flower pedicels and altered leaf morphology. Interestingly, the co-expression phenotype could not be observed in transgenic plants that are only expressing either one of the interacting partners separately. Transgenics altered in expression of PB15 and/or PB129 were prepared in Populus and characterization of transgenic trees will be performed in greenhouse and field. In addition to DUF620 family proteins, ROP11 also interacts with the COP9 subunit CSN5A in Arabidopsis. We confirmed the interaction of ROP11 and CSN5A in Y2H and employed available mutants for ROP11 and CSN5A in Arabidopsis to genetically characterize this interaction. Surprisingly, loss of ROP11 was found to rescue the csn5a-2 pleiotropic phenotype. Ectopic expression of a ROP11 dominant negative mutant in the csn5a-2 background also complemented the stunted growth phenotype. Transcript analysis and gel blot assays showed that CSN5A transcript levels remained unchanged in all rescue lines, whereas CSN5A protein levels increased relative to WT. Taken together, we concluded that ROP11 negatively regulate CSN5A protein level in plant by some as yet unknown mechanism. Ph. D.

With the advent of sequencing technologies, a variety of methods have been available day by day. Each of these methods have helped scientists to for a deeper understanding of the biological function and evolutionary constraints on the relevant genes, which can be gained through the use of modern computational approaches. Numerous approaches have being developed to advance these goals, and interaction network mapping is one of them. This method has been employed to study a variety of organisms to illustrate shared (conserved) or individual (unique) properties, and is mainly based on identifying and visualizing modules of co-expressed genes. As being a very strong candidate for such tools, co-expression gene network was used in this study to indentify the genes in wood formation of Populus trichocarpa with the help of the other novel bioinformatics tools such as Gene Ontology and Cytoscape. In order to booster the accuracy of the findings, we have combined it with an evolutionary approach, synonymous and non-synonymous ratio (dN/dS) of the proteins to show the selective patterns of the genes in a comparative fashion between woody and non-woody plants. This thesis is proposed to help plant scientists to gain insights into the genes that are involved in wood formation. By taking advantage of the computational studies have been done on this paper, one can validate the experiments along with reducing the cumbersomeness of the lab trials on the topic of wood formation in plants Master of Science