Genomics & Bioinformatics

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Bioinformatic Analysis to Identify and Understand Aberrant DNA Methylation Pattern Associated with Pancreatic Cancer
(North Dakota State University, 2021) Zamani, Mariam
In this study, we searched for significant hypo and hyper methylation CpG (5'-C-phosphate-G-3') probes from The Cancer Genome Atlas (TCGA) datasets. First, the relationship between hypo and hypermethylation pattern in significantly expressed genes associated in pancreatic ductal adenocarcinoma (PDAC) was analyzed using computational methodologies in R package. This was done by combining DNA methylation (DM) and gene expression (GE) information, and their corresponding metadata (i.e., clinical data and molecular subtypes) and saved as R files. Next, examination of differentially methylated CpG sites (DMCs) between two groups (normal vs tumor) was identified gene sets. From this analysis, we found nine (09) overexpressed hypomethylated and six (06) under expressed hypermethylated genes near significant CpG probes. Results from this work will shed light on the relationship between CpG methylation and gene expression associated with PDAC.
Comparative Analysis of Zinc Oxide Nanoparticles Induced Transcriptomic Responses in Arabidopsis
(North Dakota State University, 2019) Alasvand Zarasvand, Azita
The 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.
Genetic Characterization of Dormancy in Durum Wheat
(North Dakota State University, 2012) Dilawari, Mridull
Two 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.
Genome-Wide Association Mapping of Host Resistance to Stem Rust, Leaf Rust, Tan Spot, and Septoria Nodorum Blotch in Cultivated Emmer Wheat
(North Dakota State University, 2015) Sun, Qun
Cultivated emmer wheat (Triticum turgidum ssp. dicoccum) is a good source of genes for resistance to several major diseases of wheat. The objectives of this study were to use genome-wide association analysis to detect genomic regions in cultivated emmer germplasm harboring novel resistance genes to four wheat diseases: stem rust, leaf rust, tan spot, and Septoria nodorum blotch (SNB). A natural population including 180 cultivated emmer accessions with a high level of geographic diversity was assembled as the association-mapping panel. This cultivated emmer panel was evaluated phenotypically by scoring reactions to stem rust, leaf rust, tan spot, and SNB and was genotyped using a 9K SNP Infinium array. After filtering for missing data points and minor allele frequency (MAF), 4,134 SNPs were used for association analysis using 178 emmer accessions. Based on principle component (PC) analysis, five subpopulations strongly associated with geographic origins were suggested by the first three PCs. Genome-wide association analysis revealed that 222, 42, 146, and 42 SNPs were significantly associated with resistance to stem rust, leaf rust, tan spot, and SNB, respectively, at the significant level of 1 percentile. Among the significant SNPs at the significant level of 0.1 percentile, ten, one, nine, and one co-located with known genes or QTL associated with resistance to the four diseases, respectively. The remaining significant SNPs were located in the genomic regions where no known resistance genes have been identified for the four diseases. This evidence suggests that some of the emmer wheat accessions carry novel genes conferring resistance to the four diseases. Additionally, 14, three, eight, and five LD blocks harboring at least one significant SNP were identified and might harbor putative QTL related to resistance to the four diseases, respectively. These studies provide information about the genomic regions in cultivated emmer that are associated with resistance to stem rust, leaf rust, tan spot, and SNB. Results from these studies provide guidance for selecting emmer accessions when decisions are being made about the parents that will be used for the development of new resistant germplasm and mapping populations for identifying novel genes conferring resistance to major wheat diseases.
Genome-wide Scan for Loci Affecting Iron Deficieny Chlorosis in Soybean.
(North Dakota State University, 2010) Chikara, Shireen
Iron deficiency results in iron deficiency chlorosis (IDC) in soybean grown in the north central regions of the United States. Soybean plants display a variety of symptoms, ranging from slight yellowing of the leaves to interveinal chlorosis, and sometimes IDC is followed by stunted growth. In severe cases IDC may even lead to cell death. The objective of this project was to employ a whole genome association mapping approach to uncover the genomic regions associated with the iron deficiency trait in soybean. Golden gate assay technology was applied to expedite the screening of 1,536 single nucleotide polymorphisms in two different sets of soybean populations belonging to the year 2005 and 2006. The two soybean populations were screened for IDC at multiple locations in replicated field trials. The experiment only considered marker loci with a minor allele frequency greater than 0.1. Probability-probability plot helped in selecting the appropriate general linear models, which controlled for only population structure, and mixed linear models, which controlled for both the population structure and the ancestry. For the 2005 population, three statistical approaches (PCA, PCA+K and PCA+K*) identified twelve marker/trait associations, and for the 2006 population, five statistical models (Q, PCA, Q+K, Q+K * and PCA+K*) resulted in the discovery of twenty-two such associations. Although none of the markers significantly associated with JDC was common to both the populations under study, similar regions of significance were observed between the two years. When the phenotypic and the genotypic data of the two populations were combined, 10 markers were significantly (pFDR < 0.01) associated with the IDC trait using the PCA and PCA+K* statistical models. Out of the 10 markers, six selected markers showed a significant phenotypic mean difference for the tolerant and susceptible alleles. A detailed analysis revealed that using a smaller set of combinations from these six markers can effectively identify IDC tolerant genotypes. The next step would be to verify the reproducibility of the selected set of marker combinations in another set of populations.
The Genomic Alteration Landscape of Pancreatic Duct Adenocarcinoma
(North Dakota State University, 2022) Adeleke, David
By 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.
Genomic Analysis of Domestication-Related Traits and Stem Rust Resistance in Tetraploid Wheat
(North Dakota State University, 2017) Saini, Jyoti
Modern durum and common wheat cultivars were developed from ancient wheat ancestors by natural and artificial selection of agronomic and domestication traits, which ultimatey decreased their genetic diversity and made them more susceptible to various biotic and abiotic stresses. At present, new sources of resistance need to be introgressed into future wheat cultivars to combat the effect of the disease stem rust caused by the biotrophic fungal pathogen Puccinia graminis f.sp. tritici (Pgt). In this dissertation, I first analyzed the domestication-related traits in a tetraploid recombinant inbred line (RIL) population developed from a cross between the durum wheat line Rusty and the cultivated emmer accession PI 193883 (referred to as the RP883 population). Second, the RP883 population and a double haploid (DH) population (referred to as the LP749 population) derived from a cross between the durum cultivar Lebsock and the Triticum. turgidium ssp. carthlicum accession PI 94749, and nine durum wheat cultivars were screened with Pgt races TMLKC, TTKSK, TRTTF, and TTTTF. Domestication-related trait analysis in the RP883 population showed vernalization (Vrn-A1) and domestication (Q) genes had a pleiotrophic effect on spike length, spikelet per spike, spike compactness, and threshability. Additionally, an interaction and dosage effect of three free-threshing trait governing loci, teneacious glume Tg2A and Tg2B, and q, revealed that mutation in all three loci are required to attain complete free threshability. The stem rust analysis done in the RP883 population showed two Sr gene regions conferring resistance against TMLKC, TTKSK, and TRTTF: one novel gene region on chromosome 2BL (Sr883) and likely a new allele or gene residing in close proximity to the Sr13 gene on 6AL. The second stem rust study using the LP749 population and nine durum wheat cultivars showed that most likely the U.S. durum germplasm carries the four major Sr genes, Sr7a (4AL), Sr8155B1 (6AS), Sr13 (6AL), and likely Sr9e (2BL) against TTKSK, TRTTF, and TTTTF. In conclusion, results obtained from this domestication study provide knowledge about different stages in wheat evolution. Both stem rust studies revealed genetic diversity in the tetraploid wheat gene pool and indicate their utility in future breeding programs.
Genomic Analysis of Septoria nodorum Blotch Susceptibility Genes Snn1 and Snn2 in Wheat
(North Dakota State University, 2019) Seneviratne, WSJM Sudeshi Lakmali
Septoria 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.
Genomic Analysis of Tan Spot and Stagonospora Nodorum Blotch Resistance in Wheat
(North Dakota State University, 2011) Abeysekara, Nilwala Shyamen
Host-selective toxins, or necrotrophic effectors, are important determinants of disease in both wheat-Stagonospora nodorum and wheat- Pyrenophora tritici-repentis pathosystems. This study describes the identification, validation, and genomic analysis of compatible host gene-effector interactions in these systems. In the wheat-S. nodorum system, the Snn4-SnTox4 interaction was identified and validated using the hexaploid wheat ( Tri ti cum aestivum L.) recombinant inbred populations 'Arina' x 'Forno' ( AF) and Salamouni x 'Katepwa' (SK), respectively. The single dominant gene, Snn4, which mapped to the short arm of chromosome 1 A in both populations, governs sensitivity to the proteinaceous effector, SnTox4, which is estimated to be 10-30 kDa in size. The compatible Snn4-SnTox4 interaction played a significant role in disease development in both the AF and SK populations accounting for 41 % and 23.5% of the phenotypic variation, respectively. Effects of the additional minor QTL were largely additive in both genetic backgrounds. Molecular mapping in the SK population using microsatellites and markers developed using bin-mapped expressed sequence tags (ESTs), and from ESTs identified based on colinear studies with rice (Oryza sativa L.) and Brachypodium, delineated Tsc2 to a 3.3 cM interval and confirmed its location on 2BS of hexaploid wheat. The compatible T.~c2-Ptr ToxB interaction accounted for 54% of the disease variation in the SK population. The marker XBE44454 l, which co-segregated with Tsc2 is diagnostic of the gene and will be useful in marker-assisted selection (MAS).
Genomic Characterization of Necrotrophic Effector Sensitivity Genes in Wheat
(North Dakota State University, 2022) Running, Katherine
The 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.
Identification of Stem Rust Resistance Quantitative Trait Loci in Durum Wheat Populations
(North Dakota State University, 2022) Lund, Kennedy
Stem 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.
Mapping and Characterization of Yield Component Traits and Septoria Nodorum Blotch Susceptibility in Wheat
(North Dakota State University, 2021) Peters Haugrud, Amanda Rose
Wheat, a major global economic crop and food source, is currently threatened by climate change and the cascading effects, including increased disease pressure. Additionally, wheat yields have not increased significantly for decades, which may impact future food supply. Compared to other crop species, relatively few genes related to wheat yield have been mapped and cloned, with the vast majority in bread, or hexaploid, wheat. In this dissertation, I used three tetraploid wheat populations, Ben × PI 41025 (BP025), Divide × PI 272527 (DP527), and Rusty × PI 193883 (RP883) which were derived from crossing durum cultivars with cultivated emmer accessions. These three populations were evaluated under field conditions in three seasons for 11 traits related to yield. Additionally, the DP527 population was evaluated under greenhouse conditions for these same 11 traits. The known genes ELF3, Ppd-B1, Vrn-A1, Q, Vrn-B1, WAPO-A1, FT-1, GNI-A1, GRF4 and Vrn2 were associated with numerous yield traits. For multiple QTL, the cultivated emmer parent contributed the increased effects. Findings from this study and the identified markers may be useful for breeders who are interested in introgressing the beneficial genes I identified into their germplasm. Here, I also report on the progress and markers developed for fine mapping of a kernels per spike gene that was first mapped in the BP025 population. The work I have done provides a foundation for the cloning of this kernels per spike gene. Lastly, in this dissertation, I screened a global winter wheat panel for genetic regions associated with susceptibility to the necrotrophic pathogen Parastagonospora nodorum, the causal agent of septoria nodorum blotch. I identified the previously cloned genes Tsn1 and Snn3-B1 to be associated with disease caused by the isolates Sn2000 and Sn4, respectively. I also report the first time a panel has been screened for sensitivity to the necrotrophic effectors SnTox267 and SnTox5, along with the prevalence of SnToxA, SnTox1, and SnTox3 sensitivity in this panel. In conclusion, results obtained from these studies provides knowledge of genes/markers which are available to breeders that may provide useful in breeding programs and the overall goal of increasing wheat yield.
Mapping Ur-6, a Bean Rust Resistance Gene in Common Bean
(North Dakota State University, 2020) Beerbower, Peter Edward
Bean rust, caused by the fungus Uromyces appendiculatus (Pers.:Pers) Unger, is a disease of common bean (Phaseolus vulgaris) prevalent in the Americas and Africa. The most cost-effective countermeasure to bean rust is genetic resistance. While 17 dominant rust resistance genes (named with Ur- symbol) have been identified in common bean, not all of these genes have been genetically fine-mapped. To expand our knowledge of rust resistance genes in common bean, Ur-6 was mapped in the common bean genome. A GWAS analyses suggested that Ur-6 is present on chromosome Pv07 of P. vulgaris. Two InDel markers tightly linked to Ur-6 were developed by F2 bi-parental mapping and may prove effective for marker-assisted selection in bean breeding programs in the future. Further, 25 candidate genes were identified and are the potential focus of future gene validation research.
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, Samira
Common 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.
Using Machine Learning and Text Mining Algorithms to Facilitate Research Discovery of Plant Food Metabolomics and Its Application for Human Health Benefit Targets
(North Dakota State University, 2020) Mathew, Jithin Jose
With the increase in scholarly articles published every day, the need for an automated systematic exploratory literature review tool is rising. With the advance in Text Mining and Machine Learning methods, such data exploratory tools are researched and developed in every scientific domain. This research aims at finding the best keyphrase extraction algorithm and topic modeling algorithm that is going to be the foundation and main component of a tool that will aid in Systematic Literature Review. Based on experimentation on a set of highly relevant scholarly articles published in the domain of food science, two graph-based keyphrase extraction algorithms, TopicalPageRank and PositionRank were picked as the best two algorithms among 9 keyphrase extraction algorithms for picking domain-specific keywords. Among the two topic modeling algorithms, Latent Dirichlet Assignment (LDA) and Non-zero Matrix Factorization (NMF), documents chosen in this research were best classified into suitable topics by the NMF method validated by a domain expert. This research lays the framework for a faster tool development for Systematic Literature Review.
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 Michael
Pyrenophora 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.
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, Oleksandr
Escherichia 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.

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