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.

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.

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 / The first case of field evolved acetolactate synthase (ALS) inhibiting herbicide resistance in 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). The ALS resistance was confirmed in greenhouse and the R population exhibited cross-resistance to three ALS herbicide chemical families. Further studies sequenced the whole genome for four field populations collected from Essex and Westmoreland Counties, Virginia to characterize the genetic mechanism of ALS resistance. The results revealed that all populations contained target site mutations. All populations had a mutation at a commonly implicated point within ALS gene; however, substitutions varied by location. Populations from one location had multiple target site mutation contrary to populations from second location which had only one mutation. Patterns of ALS 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 wheat yield loss was observed from mouse-ear cress interference at a density of more than 300 plants m⁻².

Pseudomonas aeruginosa (PA) is a gram-negative bacillus well known for colonizing human respiratory airways and causing opportunistic infections. Children with neuromuscular disease (NMD) including cerebral palsy (CP) and severe upper airway obstruction who get infected with PA, their chances of experiencing a severe illness, being admitted to a pediatric intensive care unit, and extended or repeated hospital stays increase dramatically. These patients often need a surgical procedure called tracheostomy which act as a channel for microbes to enter lower respiratory tract and increase infections, despite its well documented impact as an opportunistic pathogen comprehensive investigation into the diversity of PA in such vulnerable populations is limited. To fill this gap in knowledge we perform whole genome sequencing (WGS) and phenotypic analysis of 40 PA isolates from the respiratory tract of this susceptible population with and without tracheotomies. Pangenome analysis showed highly variable genome content with 16,212 total genes of which 2326 are core genes. MLST revealed diverse sequence types (STs) among the studied population with 21 known and 10 new STs. Genotypic analysis revealed moderate variations in the antimicrobial resistance determinants and virulence factors among all isolates. In total 8 serogroups were identified, with serogroups O6 and O11 accounting for 70% of all the isolates. Genotypic diversity was observed in overall population however comparative analysis among tracheostomized and non-tracheostomized patient groups showed significant similarity which aligns with the phenotypic analysis revealing significant similarity with minor differences in biofilm formation, motility, hemolysis production, and pigment production. Last, we explored putative healthcare transmission and identified three potential transmission events. These findings provide insight into how WGS along with phenotypic analysis can help us better understand population dynamics, epidemiology, virulence profile and antibiotic resistance profile of PA contributing to respiratory infections which has valuable therapeutic implications for epidemiology and disease management.

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.

Yes / Trypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission. / Funding was received from the Research Foundation Flanders (FWO, grants 1501413N and 1101614N) and the European DG Health and Food Safety (SANTE). We thank the Center of Medical Genetics at the University of Antwerp for hosting the NGS facility.

Yes / Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting. / ERC grant [INTERRUPTB; no. 311725] to BdJ, FG and CJM; an ERC grant to TS [PhyPD; no. 335529]; an FWO PhD fellowship to PM [grant number 1141217N]; the Leibniz Science Campus EvolLUNG for MM and SN; the German Centre for Infection Research (DZIF) for TAK, MM, CU, PB and SN; a SNF SystemsX grant (TBX) to JP and TS and a Marie Heim-Vögtlin fellowship granted to DK by the Swiss National Science Foundation. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government – department EWI.

Yes / BACKGROUND: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis allows rapid, accurate inferences about the sources, location and timing of transmission. However, in an era of heightened concern for personal privacy and science distrust, such inferences could result in unintended harm and undermine the public´s trust. METHODS: We held interdisciplinary stakeholder discussions and performed ethical analyses of real-world illustrative cases to identify principles that optimise benefit and mitigate harm of M. tuberculosis WGS-driven TB source investigations.RESULTS: The speed and precision with which real-time WGS can be used to associate M. tuberculosis strains with sensitive information has raised important concerns. While detailed understanding of transmission events could mitigate harm to vulnerable patients and communities when otherwise unfairly blamed for TB outbreaks, the precision of WGS can also identify transmission events resulting in social blame, fear, discrimination, individual or location stigma, and the use of defaming language by the public, politicians and scientists. Public health programmes should balance the need to safeguard privacy with public health goals, transparency and individual rights, including the right to know who infects whom or where.CONCLUSIONS: Ethical challenges raised by real-time WGS-driven TB source investigation requires public health authorities to move beyond their current legal mandate and embrace transparency, privacy and community engagement.

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. / DNA, deoxyribonucleic acid, is a large molecule comprised of four molecular bases: adenine, cytosine, thymine, and guanine, and it determines heritable traits in living organisms. Sequencing DNA determines the sequential arrangement of bases. A read is a small sequence of DNA bases. Epigenetics are stable heritable traits that are not a result of the DNA sequence. Chemical groups called methyl and hydroxymethyl can be attached to cytosine. These groups are an epigenetic modification of cytosine, and they play a role in disease and development. The chemical bisulfite is used to discover these chemical groups. The bisulfite sequencing of DNA is a process where bisulfite is introduced to DNA, and then the DNA is sequenced. Bisulfite treatment converts cytosines without the methyl and hydroxymethyl chemical groups into thymine. Software is then used to align and match the resulting DNA strands to a large reference DNA strand called a reference genome to distinguish between cytosines that have these chemical groups. This process is called mapping or alignment, and its performance can be as low as 40% for bisulfite data. This research improves this performance. The hairpin protocol is a known bisulfite sequencing method that sequences two opposing DNA strands, where the original untreated strand can sometimes be recovered. Mapping the recovered strands improved performance by 10%. Using hairpin data, sequence complexity, a measure of DNA sequence randomness, correlated with mapping performance. BisPin mapping software was created that implements the hairpin recovery approach. BisPin rescores DNA strands that map to multiple locations on the reference genome, and it supports multiple sequencing technologies. BFAST-Gap, a modified mapping program callable by BisPin, uses a context sensitive function to better align Ion Torrent reads, which tend to have errors in regions of repeated bases. BFAST-Gap was developed, since Ion Torrent sequencing machines are less expensive than Illumina machines and since Ion Torrent reads tend to be longer and have more information. The read trimmer InfoTrim was developed to trim the lengths of short DNA sequences to improve the quality of alignments. These programs were validated on real and simulated DNA data and performed well.