The genetic interactions among 9 soybean [<i>Glycine max</i> (L.) Merr.] cultivars and 6 strains of soybean mosaic virus (SMV) were investigated. The objectives were to identify genes and/or alleles conditioning resistant and necrotic reactions to SMV and to determine the genetic relationships among resistance genes from cultivars exhibiting differential responses to the SMV strains. Seven SMV-resistant (R) cultivars (‘PI 486355’, ‘Suweon 97’, ‘PI 96983’, ‘Ogden’, ‘York’, ‘Marshall’, and ‘Kwanggyo’) were crossed in all combinations among each other and with susceptible (S) cultivars ‘Essex’ and ‘Lee 68’. F₂ populations and F₂-derived F₃ lines were inoculated in field with the SMV type strain Gl and in the greenhouse with the virulent strains G4, G5, G6, G7, and G7A. All F₂ populations from R x S and necrotic (N) x S crosses having PI 96983, Ogden, York, Marshall, and Kwanggyo as either resistant or necrotic parents segregated 3R:1S and 3N:1S, respectively. F₂-derived F₃ progenies from R x S crosses exhibited an F₂ genotypic ratio of 1 homogeneous R : 2 segregating (3R:1S) : l homogeneous S. The results indicate that each of these five resistant parents has a single, dominant or partially dominant gene conditioning the resistant and necrotic reactions to SMV. No segregation for SMV reaction was evident in F₂ and F₃ generations from R x R, N x N, and S x S crosses among the five differential cultivars, indicating that the resistance genes in the five cultivars are alleles at a common locus. The alleles in PI 96983 and Ogden were previously labeled <i>Rsy</i> and <i>rsy^t</i>, respectively. Gene symbols, <i>Rsy^y</i>, <i>Rsy^m</i>, and <i>Rsy^k</i> are proposed for the resistance genes in York, Marshall, and Kwanggyo, respectively. It is also proposed that the gene symbol <i>rsy^t</i> be changed to <i>Rsy^t</i> to more accurately reflect its genetic relationship to the susceptible allele. The R x S crosses with PI 486355 and Suweon 97 as resistant parents segregated 15R:1S in the F₂ and 7 (all R) : 4 (3R:1S) : 4 (15R:1S) : 1 (all S) in the F₃, indicating that each has two independent genes for resistance to SMV. The F₂ plants of PI 486355 x Suweon 97 showed no segregation for SMV reaction, suggesting that they have at least one gene in common. The crosses among all 7 resistant parents produced no susceptible segregates when inoculated with strain G1. It is concluded that the 7 resistant cultivars each have a gene or allele at the <i>Rsy</i> locus. Data from the experiments furnished conclusive evidence that the necrotic reaction in segregating populations is highly associated with plants that are heterozygous for the resistance gene. / Ph. D.

The field of functional genomics aims to understand the complex relationship between genotype and phenotype by integrating genome-wide approaches, such as transcriptomics, proteomics, and metabolomics. Large-scale "-omics" research has been made widely possible by the advent of high-throughput techniques, such as next-generation sequencing and mass-spectrometry. The vast data generated from such studies provide a wealth of information on the biological dynamics underlying phenotypes. Though functional genomics approaches are used extensively in human disease research, their use also spans organisms as miniscule as mycoplasmas to as great as sperm whales. In particular, functional genomics is instrumental in agricultural advancements for the improvement of productivity and sustainability in crop and livestock production. Improvement in soybean production is especially imperative, as soybeans are a primary source of oil and protein for human and livestock consumption, respectively. The research presented here employs functional genomics approaches – transcriptomics and metabolomics – to discern the transcriptional regulation and metabolic events underlying two economically important agronomic traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. At normal levels, seed phytic acid content inhibits mineral absorption in humans and livestock, acting as an antinutrient and contributing to phosphorus pollution; however, the development of low phytic acid soybeans has helped mitigate these issues, as their seeds increase nutrient bioavailability and reduce environmental impact. Despite these desirable qualities, low phytic acid soybeans exhibit poor seed performance, which negatively affects germination rates and yield and has prevented their large-scale commercial production. Thus, part of the focus of this research was investigating the effects of mutations conferring the low phytic acid phenotype on seed germination. Comparative studies between low and normal phytic acid soybean seeds were carried out and revealed distinct differences in metabolite profiles and in the transcriptional regulation of biological pathways that may be vital for successful seed germination. The final part of this research concerns Rsv3-mediated extreme resistance, a unique mode of resistance that is effective against the most virulent strains of Soybean mosaic virus. The molecular mechanisms governing this type of resistance are poorly characterized. Therefore, the research presented here attempts to elucidate the regulatory elements responsible for the induction of the Rsv3-mediated extreme resistance response. Utilizing a comparative transcriptomic time series approach on Soybean mosaic virus-inoculated Rsv3 (resistant) and rsv3 (susceptible) soybean lines, this final study provides gene candidates putatively functioning in the regulation of biological pathways demonstrated to be crucial for Rsv3-mediated resistance. / Doctor of Philosophy / Soybeans are a crop of great economic importance, being a primary source of oil and protein for human and livestock consumption, respectively. Increasing demand for soybean calls for improvement in its production. An emerging field that has had tremendous impact on this endeavor is the field of functional genomics. Functional genomics approaches generate large-scale biological data that can aid in discerning how specific processes are regulated and controlled in an organism. The research presented in this work utilizes functional genomics approaches to elucidate the biological mechanisms underlying two economically important traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. Phytic acid is a compound found in soybean seeds that causes nutrient deficiencies and phosphorus pollution. Soybeans with reduced to phytic acid content have been developed to mitigate these problems; they have poor seed germination and emergence. The studies in this work employ functional genomics approaches to compare unique sets of low and normal phytic acid soybeans to help establish the relationship between seed phytic acid content and seed performance. These studies resulted in new and promising hypotheses for future studies on investigating the low phytic acid trait. The final focus of this work used a functional genomics approach to discern the molecular mechanisms underlying a unique mode of resistance to Soybean mosaic virus. The study identified genes in soybean that are potentially critical to resistance against Soybean mosaic virus.

Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. The complete nucleotide sequences of the genomic RNAs of the naturally occurring partial diploid strain IL-Cb1 were determined. Intermolecular RNA1 recombinants were isolated from strain IL-Cb1 and characterized at the molecular level. Structurally similar recombinant RNA1 was also generated after four passages in soybean derived from plants previously inoculated with a mixture of infectious RNA1 transcripts from two distinct strains. BPMV was developed as a plant viral vector that is appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The foreign gene was inserted between the movement protein (MP) and the large coat protein (L-CP) coding regions. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. Double infection of soybean by BPMV and SMV triggers a synergistic interaction leading to a serious disease. To investigate the underlying mechanism, helper componentprotease (HC-Pro) genes from several SMV strains and TEV were expressed from BPMV vectors. The recombinant BPMV vectors carrying the HC-Pro genes from SMV strain G7 or TEV induced very severe symptoms on soybean whereas constructs containing the HC-Pro gene from SMV isolate P10, a mild strain with an apparent defect in synergism, induced only very mild symptoms. Transient agroinfiltration assays using GFP-transgenic Nicotiana benthamiana showed that HC-Pro from SMV isolate P10 was not a RNA silencing suppressor, whereas those of SMV strain G7 and TEV exhibited strong suppressor activities. Analysis of chimeric HC-Pro genes and point mutations indicated that a positively charged amino acid at position 144 is critical for the suppressor function of not only SMV HC-Pro but also other potyvirus HC-Pro proteins. Although amino acid substitution at position 144 resulted in changes in small RNA profile, it did not affect HC-Pro stability.

Soybean is a dominant oilseed cultivated worldwide for its use in multiple sectors such as food and feed industries, animal husbandry, cosmetics and pharmaceutical sectors, and more recently, in production of biodiesel. Increasing demand of soybean, changing environmental conditions, and evolution of pathogens pose challenges to soybean production in limited acreage. Genetic research is the key to ensure the continued growth in soybean production, with enhanced yield and quality, while reducing the losses due to diseases and pests. This research is focused on the understanding of transcriptional regulation of two economically important agronomic traits of soybean: low seed phytic acid and resistance to Soybean mosaic virus (SMV), using the 'transcriptomics' and 'genomics' approaches. The low phytic acid (lpa) soybean is more desirable than conventional soybean, as phytic acid is an anti-nutritional component of seed and is associated with phosphorus pollution. Despite the eco-friendly nature of the lpa soybean, it shows poor emergence, which reduces soybean yield. This research is mainly focused on addressing the impact of lpa-causing mutations on seed development, which is suspected to cause low emergence in lpa soybeans. The differences in transcriptome profiles of developing seeds in lpa and normal phytic acid soybean are revealed and the biological pathways that may potentially be involved in regulation of seed development are suggested. The second research project is focused on Rsv3-type resistance, which is effective against most virulent strains of Soybean mosaic virus. The Rsv3 locus, which maps on to soybean chromosome 14, contains 10 genes including a cluster of coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) protein-encoding genes. This dissertation employed a comparative sequencing approach to narrow down the list of Rsv3 gene candidates to the most promising CC-NB-LRR gene. The evidence provided in this study clearly indicates a single CC-NB-LRR gene as the most promising candidate to deliver Rsv3-type resistance. / Ph. D.

Soybean mosaic virus (SMV) is the most common member of the viral genus Potyvirus to infect soybeans (Glycine max [L.] Merr.) worldwide. SMV has been traditionally controlled by the deployment of single dominant, strain specific resistance genes, referred to as Rsv genes. Rsv1 is the most widely used form of SMV resistance with nine different alleles conferring resistance only to the lower numbered less virulent strains, G1 to G3. Rsv3 gives resistance to higher numbered more virulent strains G5 to G7. Soybean lines containing Rsv4, are resistant to all seven currently recognized North American SMV strains. In this study, the recently released soybean whole genome sequence was used to design molecular markers for fine mapping Rsv3 to a ~150 kb genomic region containing four coiled-coil nucleotide-binding leucine-rich repeat proteins. In a related study a large population segregating at the Rsv3 locus was screened for resistance to facilitate future characterization of this region. The markers identified in this study will allow for more accurate marker-assisted selection of Rsv3. / Master of Science

Soybean mosaic virus (SMV), a potyvirus detected worldwide, can cause serious diseases in soybean (Glycine max L. Merr.). Host resistance to SMV conferred by a single dominant gene, Rsvl, was studied as a model to gain insights of plant virus resistance genes, and to facilitate the breeding of resistant cultivars. DNA restriction fragment length polymorphisms (RFLPs) and microsatellites (or simple sequence repeats, SSRs) were used as genetic markers to identify the chromosomal location of Rsvl1 in a cross between PI 96983 (resistant) and a susceptible cultivar. Twenty five RFLP and three SSR loci polymorphic between the parental lines were analyzed in 107 F, individuals. Genotypes of Rsv1 were determined by inoculating F2.3 progeny with SMV-G1. Genetic analysis revealed that one SSR (HSP176L) and two RFLP (pA186 and pK644a) markers are closely linked to Rsv1, with a distance of 0.5, 1.5, and 2.1 cM, respectively. The tight linkages of the three markers to Rsv1 were confirmed by SSR and RFLP analysis of three near isogenic lines (NILs) of Rsv1 derived from PI 96983 or Marshall. The three Rsv1-linked markers were then used to screen 67 diverse soybean types. These marker loci showed a remarkably high level of polymorphism, indicating a possible association between disease resistance and rapid sequence divergence. At each Rsv1-linked marker locus, one SSR allele or RFLP haplotype is highly correlated with SMV resistance. These resistance markers, especially the SSR allele at HSP176L which can be detected by the polymerase chain reaction (PCR), may be useful for germplasm screening. The grouping of the 67 accessions according to their Rsv1-linked multilocus marker haplotypes agrees with available pedigree information. A set of differential cultivars known to contain Rsv1 clustered into putative Rsv1- carrying groups. Based on molecular marker analysis and previous inheritance studies, 37 of the 45 resistance accessions probably derive their resistance from Rsv1. The remaining eight accessions include Columbia (Rsv3), and the other potentially diverse resistance sources. A heat shock protein (HSP) multigene family, HSP176L included, was analyzed for its positional proximity to the Rsv1 gene cluster. A technique termed amplified sequence length polymorphism (ASLP) was developed to convert known DNA sequences to PCR-based genetic markers. Among six pairs of HSP primers used, two (HSP175E and 185C) detected ASLPs between the parents, and segregated in the F₂ population with a size of 174. HSP175E was found to be closely-linked (0.7 cM) to HSP176L, both of which are Class I small HSP genes. HSP185C, however, was mapped to a different linkage group, suggesting that it may belong to another family. ADR11, a member of auxin down-regulated (ADR) multigene family, is known to be linked to HSP173B, also a Class I gene but not mappable in this population. ASLP analysis of ADR11 in a set of Rsv1 NILs indicates that it is linked to Rsv1, and ADR11 co-segregates with HSP175E in the F, population. Thus, the Class I small HSP multigene family including HSP176L, 175E, and 173B, and possibly a family of ADR genes, is located near the Rsvi resistance gene cluster. / Ph. D.