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Item The Genomic Alteration Landscape of Pancreatic Duct Adenocarcinoma(North Dakota State University, 2022) Adeleke, DavidBy 2030, PDAC is projected to be the second leading cause of cancer-related death in the US. PDAC is a multifactorial disease driven by genomic alterations. Understanding this alteration landscape will both refine the knowledge of disease etiology and enhance disease stratifications, drug design, and targeted treatment. This study aimed to identify novel genetic alterations that are associated with pancreatic cancer biology and prognosis to further refine the genetic focus for therapy development, disease subtyping, and risk assessment in PDAC. To this end, SNV, CNV, and clinical data for PDAC patients were downloaded from the ICGC data portal and analyzed for somatic mutations and recurrent copy number variations. This study showed that KRAS, TP53, and TTN are not only highly mutated but also associated with poor survival in PDAC. Also, this study showed that CN-LOH TP53, KRAS, SMAD4, and RYR3 were associated with reduced risk of death from PDAC.Item Comparative Analysis of Zinc Oxide Nanoparticles Induced Transcriptomic Responses in Arabidopsis(North Dakota State University, 2019) Alasvand Zarasvand, AzitaThe impact that zinc oxide nanoparticles (ZnONP) have on plant physiological responses was evaluated by comparing gene expression changes after Arabidopsis thaliana plants were exposed to ZnONP, in comparison with ionic Zn2+ (ZnSO4) and non-treated controls. After treatment with ZnONP (concentration10 μg L−1), ionic Zn2+ (applied as ZnSO4 at a concentration of 19.7 μg/ L −1), expression analyses via RNA sequencing revealed that 369 genes were down regulated and 249 were upregulated (p < [FDR] 0.05, expression difference < 3). The downregulated genes in ZnONP treated seedlings compared to the Zn +2 ion and untreated controls were mainly abiotic stress (oxidative stress, low temperature) and biotic stress such as defense responsive genes based on the gene ontology analysis. The upregulated genes in response to ZnONP treated plants compared to the Zn +2 ion control plants were mainly photosynthesis, light harvesting complex, and hormone responsive genes such as abscisic acid.Item Variation in Core and Accessory Parts of Genome of Escherichia Coli Isolated from Soil from Riparian Areas in New York State(North Dakota State University, 2016) Maistrenko, OleksandrEscherichia coli is commensal bacteria and is a symbiont of the digestive system of vertebrates. Due to frequent deposition of E. coli into extrahost habitats (soil, water), approximately half of its population exists as free living organisms. It is unclear what genome-wide variation stands behind adaptation for extrahost habitat. This thesis applies a genome-wide association study approach to find genetic variation in core and accessory parts of genome of E. coli that is associated with 1) forest or agricultural field soil habitats and 2) with survival phenotype in soil microcosm. Gene composition analysis suggests that pan-genome of environmental E. coli is unlimited. Core and accessory genome contained variation associated with survival phenotype and with forest or field habitat.Item Genomic Analysis of Septoria nodorum Blotch Susceptibility Genes Snn1 and Snn2 in Wheat(North Dakota State University, 2019) Seneviratne, WSJM Sudeshi LakmaliSeptoria nodorum blotch is a disease of wheat caused by the necrotrophic fungus Parastagonospora nodorum. In the wheat-P. nodorum pathosystem, recognition of pathogen-produced necrotrophic effectors (NEs) by dominant host genes leads to host cell death, which allows the pathogen to gain nutrients and proliferate. To date, nine host gene-NE interactions have been reported in this pathosystem. Among them, the Snn2-SnTox2 interaction has shown to be important in both seedling and adult plant susceptibility. A saturated genetic linkage map was developed using a segregating population of recombinant inbred lines and a high-resolution map was then developed using F2 plants derived from a cross between the SnTox2-insensitive wheat line BR34 and the SnTox2-sensitive line BG301. Over 10,000 gametes were screened for high-resolution mapping and the Snn2 gene was delineated to a genetic interval of 0.10 cM that corresponds to a physical segment of approximately 0.53 Mb on the short arm of wheat chromosome 2D. A total of 27 predicted genes present in this region and thirteen of them were identified as strong candidates. Seven EMS-induced Snn2-insensitive mutants were generated for gene validation. Results of this study provide the foundation for cloning of Snn2. The host sensitivity gene Snn1, which confers sensitivity to SnTox1, was previously cloned. Here, allelic diversity of Snn1 was studied to identify causal polymorphisms, and to develop markers useful for marker assisted selection (MAS). Twenty-seven coding sequence haplotypes that correspond to 21 amino acid haplotypes were identified. Three SNPs were identified as the possible mutations that caused the insensitive allele in wild emmer to become the sensitive allele in domesticated wheat. In addition, four SNPs that changed the sensitive allele into insensitive alleles were identified. SNP-based markers that could detect three of those SNPs were developed. Results of this study help to increase our knowledge in wheat-NE interactions and host sensitivity gene evolution.Item Genomic Characterization of Necrotrophic Effector Sensitivity Genes in Wheat(North Dakota State University, 2022) Running, KatherineThe necrotrophic fungal pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis cause the diseases septoria nodorum blotch (SNB) and tan spot, respectively, reducing yield by decreasing the photosynthetic area of the plant. The pathogens produce necrotrophic effectors (NEs) that target host genes to induce cell death. The Tsc1-Ptr ToxC and Tsn1-Ptr ToxA interactions contribute to plant susceptibility to tan spot, while the Tsn1-SnToxA and Snn5-B1-SnTox5 interactions contribute to SNB susceptibility. The three main goals of this dissertation were to clone susceptibility genes Tsc1 and Snn5 and develop robust genetic markers for use in marker-assisted elimination of Tsn1. A genetic linkage map was developed delineating the Tsc1 region to 184 kb. Structural and gene content comparisons of the identified Tsc1 and Tsn1 regions in using the sequenced wheat genomes revealed gene content variation correlating with host phenotypes, reducing the Tsc1 candidate gene list to just two genes. Comparative sequence analysis in two generated mutant populations revealed the identity of Tsc1, which has protein kinase and leucine-rich repeat domains. The structural and gene content comparison of the sequenced genomes in the Tsn1 region identified two conserved haplotypes in accessions with presence/absence variation corresponding with ToxA sensitivity. Genetic markers flanking Tsn1 were designed in segments syntenic between Tsn1+ and Tsn1- accessions, allowing the codominant detection of Tsn1. The Tsn1 markers were validated on over 1,500 wheat accessions, demonstrating a near perfect ability to determine if an accession would be insensitive to ToxA. The application of these markers in wheat breeding programs can effectively reduce susceptibility to ToxA-producing pathogens. Snn5-B1 candidates were identified in the Chinese Spring genome and validated using random mutagenesis, targeted mutagenesis, and the Cadenza TILLING mutants. Snn5-B1 contains protein kinase and major sperm protein domains. Furthermore, a second SnTox5 sensitivity locus, designated Snn5-B2, was mapped to the short arm of chromosome 2B in durum wheat. The cloning of susceptibility genes Tsc1 and Snn5-B1 allows for the development of molecular markers based on causal polymorphisms and for gene-disruption though gene-editing methods for the selection or creation of nonfunctional alleles that cannot be targeted by NE to induce cell death and disease.Item Validation of the Pyrenophora teres f. teres PttBee1 Region and Mapping of Resistance to P. teres f. maculata in Durum(North Dakota State University, 2021) Skiba, Ryan MichaelPyrenophora teres is a fungal pathogen of barley and other closely related grass species. Two forms of the pathogen, P. teres f. teres and P. teres f. maculata, are the causative agents of net form net blotch and spot form net blotch of barley, respectively. Genetic and bioinformatic approaches were used to identify eight candidate effectors in the P. teres f. teres PttBee1 region. Genes were validated using CRISPR-Cas9 mediated gene disruptions. As no transformants displayed alterations in virulence, additional markers were implemented into a P. teres f. teres genetic map to refine the locus. As P. teres f. maculata has recently emerged as a pathogen of wheat, a quantitative trait loci analysis and genome wide association study were performed with this pathogen using a durum wheat biparental mapping population and a subset of the Global Durum Wheat Panel, respectively, both showing an association with resistance/susceptibility on chromosome 2A.Item Leveraging Genomics and Transcriptomics for Gene Discovery in Dry Pea(North Dakota State University, 2024) Johnson, JosephineOver the past two decades, there has been a significant increase in the utilization of DNA marker-based mapping studies to genetically map and further improve complex quantitative traits. A major caveat of this approach is that genetic mapping of the underlying genes conferring target phenotypes is challenging often due to the extent of long-range linkage disequilibrium (LD) in the genome, particularly in self-pollinated crops. Recent technologies allow us to examine expression-phenotype associations using transcriptome-wide association studies (TWAS) which is independently affected by LD, unlike in the case of genetic markers. This is of greatest utility in species where linkage disequilibrium is extensive such as dry pea, where genes can be prioritized for association with a trait because their expression patterns are independent. The goal of this study is to use gene expression collected from the developing pods of pea and the TWAS approach for mapping and prioritizing likely causal genes underlying seed protein content and yield. As the effective population size (Ne) of the USDA (United States Department of Agriculture) diversity panel provided substantial genetic variation, we utilized 300 USDA pea lines from within the collection and performed a comprehensive single-tissue, multi-environment TWAS across six diverse environments (2 years * 2 locations) in the major pea growing regions in the USA. As we compared the results of TWAS with genome-wide association studies (GWAS), we detected more common and unique set of strongly associated genes. In all TWAS models, the significant genes exhibited clear differentiation, unlike in the case of GWAS. A joint analysis of GWAS and TWAS results using the fisher’s combined test (FCT) increased the power of detecting more trait-associated genes including RGB. Using GWAS, TWAS and FCT models, we detected 45 genes for protein, 60 genes for yield, and 20 genes that were common to both traits. These results highlight the complex interaction between genetic factors and environmental influences in shaping the genetic architecture of seed yield and protein. Our study proved that multi-omics strategy increases the gene mapping resolution by surpassing the GWAS and/or TWAS approach, and highlights the potential phenotypic consequences of regulatory variation in dry pea.Item Genetic Characterization of Dormancy in Durum Wheat(North Dakota State University, 2012) Dilawari, MridullTwo populations derived by crossing LDN x LDN Dic-3A (Population I) and LDN x LDN Dic-3B (Population II) were genetically characterized for the seed dormancy present on chromosome 3A and 3B of durum wheat. The genes for seed dormancy in these two populations were contributed by the wild parent T. dicoccoides. Although the populations showed transgressive segregants for both dormant as well as nondormant parent, the populations were similar to the dormant parent at Langdon and Prosper 2006 field locations for Population I and at Langdon 2007 and Autumn greenhouse season for Population II. Genotypic and phenotypic analysis over the combined populations showed an environmental effect on expression of the trait. Different QTL were identified for both field and greenhouse season for the population derived from the cross between LDN x LDN Dic-3A. Five QTL for seed dormancy were identified on chromosome 3A for the QTL analysis performed over combined field locations. One QTL ranging between marker interval Xcfa2193 and Xcfd2a was consistently present for the 30 day period of seed germination and was also found to be linked to red grain color trait. The QTL analysis performed on the population derived from the cross between LDN x LDN Dic-3B identified only one major QTL on the long arm of chromosome 3B between the marker interval Xbarc84 and Xwmc291. This QTL was consistently present for all the field and spring greenhouse season for the seed germination period of 30 days. The QTL x E effect was also observed for this QTL, however it was very small.Item Identification of Stem Rust Resistance Quantitative Trait Loci in Durum Wheat Populations(North Dakota State University, 2022) Lund, KennedyStem rust (Puccinia graminis f. sp. tritici) negatively impacts durum wheat production worldwide. Resistance loci from four resistant landrace durum wheat lines were identified in biparental F¬5¬ recombinant inbred line (RIL) populations after crossing with the susceptible line ‘Rusty’. The populations were tested with foreign race of stem rust from Eastern Africa and Europe (JRCQC, TRTTF, TTRTF, and TTKSK) and local races from United States Upper Midwest (MCCF and RKQQ), followed by genotyping and linkage map construction to identify stem rust resistant quantitative trait loci (QTLs). At least one stem rust resistance QTL was identified in each population with a total of twelve QTLs identified overall. Seven of the identified QTL regions validated previously published stem rust resistance genes and the other five identified potentially novel stem rust resistance genes. Various resistance mechanisms were determined from QTL regions that provide stem rust resistance to the four durum wheat RIL populations.Item Unraveling the Genetic Architecture of Agronomic Traits and Developing a Genome Wide InDel Panel in Common Bean (Phaseolus vulgaris)(North Dakota State University, 2015) Mafimoghaddam, SamiraCommon bean (Phaseolus vulgaris) is an economically important legume. The agronomic characteristics of this crop such as days to flower, growth habit and seed yield affect breeding strategies. However, little is known about the genomic regions controlling these traits. Therefore, discovering the genetic architecture underlying important agronomic traits can accelerate breeding via marker assisted selection (MAS) in addition to providing genomic and biological information. Genome wide association studies (GWAS) are currently the method of choice to find the genomic regions associated with traits of interest using a population of unrelated individuals. It takes advantage of the historic recombinations that exist in the population to map the traits at a higher resolution. The availability of a reference genome in common bean has paved the way for higher throughput and more accurate genomic research including the discovery of new knowledge and development of new tools. In the first experiment we conducted GWAS using a panel of 280 diverse genotypes from the Middle American gene pool and about 15,000 SNPS with minor allele frequency of 5% and greater to map seven important agronomic traits in common bean. We were able to detect known and new genomic regions with strong candidate genes associated with these traits. In the second experiment we used sequence data from 19 genotypes from different bean market classes to develop a panel of insertion-deletion (InDel) markers that can be used for MAS as well as other genetic and genomic studies. These user-friendly, cost-effective, and co-dominant markers were tested for their efficiency and application. They demonstrated utility in a medium throughput genetic map construction and diversity analysis.