Whole genome duplication, or polyploidy, is a common process in plants by which failures in meiosis or fertilization result in offspring with twice the number of chromosomes. This doubles the number of copies of every gene, an effect thought to generate new ‘raw material' upon which natural selection can act. Few studies exist examining the consequences of polyploidy for plant physiological traits. Doubling the number of gene copies may have unknown effects on leaf structure and function. In this study, I compare diploid, tetraploid, and hexaploid species within the genus Helianthus (wild sunflowers). Forty different accessions of wild sunflowers were grown under standardized greenhouse conditions and phenotyped for both leaf functional traits and leaf hyperspectral reflectance. Interestingly, I find that whole genome duplication can have effects on leaf functional traits relevant to both size and ecophysiology, and thus that polyploidy may lead to functional trait differentiation between polyploids and their diploid progenitors.

Rice is one of the most important cereal crops for the world population. Flowering time and seed development of rice are directly related to plant regional and ecological adaptions, and productive yield. In this dissertation, to gain knowledge of seed development in rice, the status of post-translational modifications (PTMs) in developing rice seeds was investigated. Numerous modified lysine sites in developing rice seeds were identified utilizing antibody-based affinity enrichment approaches and nano-HPLC/MS/MS analyses of acetylated, succinylated, crotonylated and 2-hydroxyisobutyrylated peptides. Functional annotation analyses indicated that a wide variety of vital biological processes were targeted by lysine PTMs. A number of modified histone and non-histone proteins were found to harbor multiple PTMs, and our findings showed that many modified histone sites were conserved across plant, human, and animal systems. Comprehensive analyses of lysine modification sites illustrated that the sites were highly sequence-specific for distinct motifs. Overall, this study provides a systematic analysis of lysine PTM proteome in plants, which will serve as the basis for future investigations of the regulatory mechanisms and functions of lysine PTMs. The mechanisms of flowering time variances in response to different photoperiods were further studied in the rice mutant, HSS. QTL-seq analysis identified a major effect on chromosome 6 responsible for the phenotypic divergence between Nipponbare (wild-type) and HSS rice. Sequence and mRNA expression analyses confirmed that allelic variants of Hd1 make HSS plants less sensitive to photoperiod by altering expression level of Hd3a. Diurnal expression pattern analyses revealed that DTH8 transcripts were largely affected by Hd1 expression level in both LD and SD. This result suggested that Hd1 may able to regulate DTH8 and DTH8-Hd1 complex abundance in response to day length in rice flowering time regulation. In addition, we discussed the functions of PTMs in flowering time regulation in rice.

Avian pathogenic Escherichia. coli (APEC) and Campylobacter are pathogenic threats to poultry and human health, respectively. In this study, the prevalence of these pathogens in Mississippi broilers and their antimicrobial resistance (AMR) properties were investigated, and a multidrug-resistant APEC strain (APEC-O2-MS1170) was further explored by whole-genome sequencing (WGS). The efficacy of in ovo injection of Lactobacillus in reducing the APEC in broilers was evaluated. Results revealed a high prevalence of APEC and Campylobacter in broilers and broiler products. A lot of isolates were resistant to antibiotics of different sorts. Moreover, the in ovo administration of Lactobacillus did not reduce the incidence of APEC. The WGS of APEC-O2-MS1170 revealed its detailed AMR and virulence properties and alerted a potential zoonotic risk. In conclusion, the Lactobacillus did not reduce the incidence of APEC in broilers, and the prevalence and AMR of APEC and Campylobacter are still challenges faced by the poultry industry.

This research investigates the application of machine learning techniques in computational genomics across two distinct domains: (1) the predicting the source of bacterial pathogen using whole genome sequencing data, and (2) the functional annotation of genes using single- cell RNA sequencing data. This work proposes the development of a bioinformatics pipeline tailored for identifying genomic variants, including gene presence/absence and single nu- cleotide polymorphism. This methodology is applied to specific strains such as Salmonella enterica serovar Typhimurium and the Ralstonia solanacearum species complex. Phylo- genetic analyses along with pan-genome and positive selection studiesshow that genomic variants and evolutionary patterns of S. Typhimurium vary across sources, which suggests that sources can be accurately attributed based on genomic variants empowered by machine learning. We benchmarked seven traditional machine learning algorithms, achieving a no- table accuracy of 94.6% in host prediction for S. Typhimurium using the Random Forest model, underscored by SHAP value analyses which elucidated key predictive features. Next, the focus is shifted to the prediction of Gene Ontology terms for Arabidopsis genes using single-cell RNA-seq data. This analysis offers a detailed comparison of gene expression in root versus shoot tissues, juxtaposed with insights from bulk RNA-seq data. The integration of regulatory network data from DAP-seq significantly enhances the prediction accuracy of gene functions. / Master of Science / This work applies machine learning techniques to two areas in computational biology: pre- dicting the hosts of bacterial pathogens based on their genome data, and predicting the func- tions of plant genes using single-cell gene expression data. The first part develops a method to analyze genome sequences from bacterial pathogens like Salmonella enterica serovar Ty- phimurium and the Ralstonia solanacearum species complex, identifying genomic variants, including gene presence/absence and single nucleotide polymorphism, which are variations in genetic code. By studying the evolutionary relationships and genetic diversity among dif- ferent strains, the motivation for using machine learning models to predict the sources (e.g., poultry, swine) of the pathogen genomes is established. Several machine learning models are then trained on these datasets, and the most important factors contributing to the predic- tions are identified. The second part focuses on predicting the functions of genes in the model plant species Arabidopsis thaliana using the gene expression data measured at the single-cell level to train machine learning models for identifying standardized gene function descrip- tions called Gene Ontology (GO) terms. By comparing results from single-cell and bulk tissue data, the study evaluates whether the higher resolution of single-cell data improves gene function prediction accuracy. Additionally, by incorporating information about gene regulation from a specialized experiment, the role of gene expression control in determining gene functions is explored.

Soybeans are an economically important plant, with an annual crop value that consistently exceeds 20 billion dollars in the United States alone. A recent increase in demand for soybeans, stemming from its diverse applications in products such as animal feed, oil, and biofuel, has created an emphasis for soybean breeders in value added cultivars. These cultivars, have improved, or altered, agronomic or seed composition traits, allowing them to be efficiently utilized in a specific niche of the processing industry. Facilitating the development of such cultivars requires a thorough understanding of the genetic factors that affect the manifestation of value added traits. Value added traits investigated in this study include seed sucrose, raffinose, stachyose, and phytate content, seed weight, and maturity. The objective of the first part of this project was to characterize the source of low seed stachyose in soybean line PI200508. Two F2 populations, developed from PI200508 and soybean introductions which exhibited higher seed stachyose content were utilized in a QTL analysis approach that incorporated the use of the Williams82 whole genome shotgun (WGS) sequence (http://www.phytozome.org) in a candidate gene mapping approach. A predicted soybean galactosyltransferase gene was established as a candidate gene due to its observed segregation with the single low stachyose QTL observed on molecular linkage group (MLG) C2 in both populations. Sequencing of this putative gene revealed a unique 3 bp deletion in PI200508. A marker developed to exploit this deletion accounted for 88% and 94% of the phenotypic variance for seed stachyose content in the two experimental populations, highlighting its potential for use in marker assisted selection of the PI200508 source of low raffinose and stachyose. The second part of this project involved QTL analysis of seed sucrose, raffinose, stachyose, and phytate content, as well as seed weight in a linkage map for a F8 RIL population developed from the Glycine max line V71-370 and the Glycine soja introduction PI40712. Analysis across all 20 soybean MLG identified 25 QTL for these traits on MLG A1, A2, C2, D1b, D2, F, G, H, I, L, M, O. Nine of these QTL were supported across multiple environments, indicating that they, and their associated markers, could be useful to breeders working with these traits. The third part of this project used the same F8 RIL linkage map to investigate time to maturity (Reproductive stage R8). V71-370 and PI407162 differ in time to maturity when grown in Virginia, and the RILs developed from this cross displayed a wide range in maturity. Two major QTL were identified on MLG H and L. Examination of the Williams82 WGS sequence in these QTL regions revealed two predicted genes with homology to Arabidopsis thaliana light response and photoperiodism genes which were investigated as candidate soybean maturity genes. Markers developed from these predicted genes showed close association with the observed QTL, and could facilitate the further investigation of this complex trait. / Ph. D.

Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit. / Ph. D.

The first case of field evolved acetolactate synthase (ALS) inhibiting herbicide resistance in the model plant, mouse-ear cress, was reported in winter wheat fields in Westmoreland County, Virginia. A putative resistant (R) mouse-ear population was assessed for ALS resistance relative to a putative susceptible (S) and a susceptible lab population Columbia (C). Results indicated that the R population needed 23 to >2400 fold rate of thifensulfuron relative to S or C population, and it has evolved cross-resistance to sulfonylureas (SU), triazolopyrimidine sulfonanilides (TP), and sulfonylaminocarbonyltriazolinones (SCT). Further studies sequenced the whole genome for four field populations, representing two locations and two resistance levels (high and low) per location, to characterize the genetic mechanism of ALS resistance. The results revealed that all populations contained mutations in the ALS gene at the Pro197 site, although the Pro was substituted by Phe in one location and Thr in the other. Also, both high- and low-level resistant plants at one location had additional mutations (Trp574Leu or Asp376Glu) known to confer resistance to ALS inhibiting herbicides. Patterns of herbicide cross-resistance also varied among the populations. Additionally, research was conducted to assess preemergent (PRE) and postemergent (POST) alternative herbicide options for control of ALS resistant mouse-ear cress and its interference with winter wheat. Results indicate flumioxazin, pyroxasulfone, and metribuzin can be used for effective PRE control whereas 2,4-D, dicamba, and metribuzin can be effective post control options. No mouse-ear cress interference with winter wheat was observed at density of more than 300 plants m-2. / Master of Science in Life Sciences

Epigenetics are stable heritable traits that are not a result of the DNA sequence. Epigenetic modification of DNA cytosine plays a role in development and disease. The covalent bonding of a methyl group or a hydroxymethyl group to the 5-carbon of cytosine epigenetically modifies cytosine to 5-methylcytosine or 5-hydroxymethylcytosine. Upon PCR amplification, the bisulfite treatment of DNA converts unmethylated cytosine to thymine, while 5-methylcytosine, 5-hydroxymethylcytosine, and other bases remain unchanged. The resulting sequences can be mapped to a reference genome; however, this can be challenging due to sequencing technology complexity, low sequence complexity, and biases and errors introduced with bisulfite treatment. Once the short read is mapped, the identity of 5-methylcytosine or 5-hydroxymethylcytosine can be determined by comparing the mapped read to the aligned reference genome. Bisulfite DNA read mapping is characterized by mapping performance as low as 40%. This research improves bisulfite short read mapping quality. First, reads generated from the bisulfite hairpin PCR protocol are used to study mapping failure and solutions. A read may not map to the genome; it may map uniquely, or it may map to multiple locations. Sequence complexity correlates with these mapping categories. The hairpin protocol allows for a recovery, in some cases, of the original untreated read, and mapping this read with the regular read mapper Bowtie2 improved mapper performance by 10%. New bisulfite read mapping software called BisPin was created that calls BFAST (BLAT-like Fast Accurate Search Tool) for mapping. BisPin resolves ambiguously mapped reads with a rescoring strategy, which yields a statistically significant improvement. BFAST-Gap for Ion Torrent reads was developed, since Ion Torrent machines are less expensive than Illumina machines and since Ion Torrent reads are longer. There are few mappers for Ion Torrent data. BFAST-Gap uses homopolymer run length for contextual gap penalty functions, since homopolymer runs cause errors in Ion Torrent reads. In conjunction with BisPin, this software performed well on real and simulated bisulfite Ion Torrent data and Illumina data. InfoTrim, a read trimmer with an entropy term, was developed with competitive results. / Ph. D.

No / Gram-positive bacterial lipoproteins are a functionally diverse and important class of peripheral membrane proteins. Recent advances in molecular biology and the availability of whole genome sequence data have overturned many long-held assumptions about the export and processing of these proteins, most notably the recent discovery that not all lipoproteins are exported as unfolded substrates through the general secretion pathway. Here, we review recent discoveries concerning the export and processing of these proteins, their role in virulence in Gram-positive bacteria and their potential as vaccine candidates or targets for new antimicrobials. / Biotechnology and Biological Sciences Research Council (grant numbers F009224/1, F009429/1, EGH16082), the Medical Research Council (MRC), the Commission of the European Community (grant LSHG-CT-2004–005257) and The Royal Society.

Yes / Burkholderia spp. rely on N-acyl homoserine lactone as quorum-sensing signal molecules which coordinate their phenotype at the population level. In this work, we present the whole genome of Burkholderia sp. strain A9, which enables the discovery of its N-acyl homoserine lactone synthase gene. / UM High Impact Research Grants (UM-MOHE HIR grant UM C/625/1/HIR/MOHE/CHAN/01, H-50001-A000001 and UMMOHE HIR Grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001- A000027)