Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the most devastating pathogen of soybeans, Glycine max (L.) Merr., causing over $1 billion in yield losses annually in the United States alone. Currently, planting of genetically resistant cultivars is the most commonly employed management strategy. Due to an overuse of genetic resistance derived from the soybean variety ‘PI 88788’, many populations of soybean cyst nematodes are becoming virulent on previously resistant cultivars, urging the understanding and discovery of alternative mechanisms of SCN resistance. In this study, we will delve into the history and epidemiology of Heterodera glycines, learn about the molecular etiology underlying SCN pathogenesis, begin to understand the mechanism of resistance by Peking-type soybeans, and look to discover a novel mechanism of resistance by establishment of a mutagenized population of the soybean variety ‘PI 567516C’.

Soybean [Glycine max (L.) Merr.] is the world’s most widely grown protein/oilseed crop and provides about 70% of global protein meal and 53% of vegetable oil in the United States. Soybean seed oil contains five major fatty acids, from which palmitic acid and stearic acid are two saturated fatty acids, oleic acid improves oxidative stability and linolenic acid is an essential fatty acid for human health. Soybean seed protein and oil are two important quality indices for soybean germplasm breeding. Soluble carbohydrates present in soybean meal provide metabolizable energy in livestock feed. To develop soybean germplasm with improved seed composition traits, it is important to discover novel source of seed fatty acid, protein, and carbohydrates traits. This dissertation aims to develop novel functional genomic technology coupled with an integrated approach for facilitating molecular soybean breeding. In this study, the first objective is to develop a high-throughput TILLING (Targeting Induced Local Lesions IN Genomes) by Target Capture Sequencing (TbyTCS) technology to improve the efficiency of discovering mutations in soybean. The robustness of this technology underlies the high yield of true mutations in genes controlling complex traits in soybean. Soybean mutagenized lines with modified fatty acids composition have been successfully developed to meet the different needs of end users. Altered fatty acids phenotypes have been associated with induced mutations in 3-ketoacyl-acyl carrier protein (ACP) synthase II (GmKASII), Delta-9-stearoyl-acyl carrier protein desaturase (GmSACPD), omega-6 fatty acid desaturase 2 (GmFAD2), and omega-3 fatty acid desaturase (GmFAD3) genes identified through TbyTCS. The second objective is to characterize the soybean acyl-ACP thioesterase gene family through a comprehensive analysis. The additional members have been discovered belonging to 16:0-ACP fatty acid thioesterase (GmFATB) gene family. The mutations at oleoyl-ACP fatty acid thioesterase (GmFATA1A) have been revealed to result in the high seed oleic acid content. The novel alleles of GmFATB genes have also been identified to confer low palmitic acid and high oleic acid phenotypes in soybean seeds. The third objective is to assess the phenotypic variations and correlation among seed composition traits in mutagenized soybean populations. Correlation analyses have been conducted among soybean carbohydrates, protein, and oil content of soybean mutagenized populations and germplasm lines. Chemical mutagenesis played an essential role in soybean breeding to generate novel and desired seed composition traits.

Master of Science / Genetics Interdepartmental Program / Bikram S. Gill / TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput, reverse genetics strategy for scanning mutagenized populations for point mutations in loci of interest. Originally, TILLING was used to investigate gene function in Arabidopsis and has since been similarly applied for gene functional analysis in other organisms. TILLING also allows the generation of novel genetic variation in specific genotypes and, thus, has been implemented as a tool for crop improvement. Ethyl methanesulfonate (EMS) is a widely used mutagen to induce point mutations in most TILLING protocols. M1 plants are then self-pollinated and M2 seed harvested. A single seed is grown from each M2 progeny and tissue taken for DNA isolation. M3 seed is cataloged. DNA is pooled to increase the efficiency and aid in mutation detection. Polymerase chain reaction (PCR) is used to amplify a locus of interest using the M2 DNA pools as a template. The PCR products are digested with an endonuclease that cleaves mismatched, mutant DNA, and the digested products are visualized. The pools for which PCR products are positive for a mutation are deconvoluted to determine which individual plant of the pool was responsible for the mutation. DNA from the positive individual is sequenced to determine the type of mutation (missense, nonsense, synonymous). Individuals with mutations that are more likely to disrupt gene function (nonsense and certain missense) are studied further by growing the corresponding M3 generation. In bread wheat, Triticum aestivum, TILLING is complicated by polyploidy: genes that have homoeologs require that the functionality of each be studied. If functional homoeologs are present for all three genomes, mutants must be identified for each homoeolog, followed by successive intercrossing to produce a triple mutant plant. As a model for wheat genetics, we propose TILLING in diploid wheat. EMS mutant populations were created in diploid wheat (Triticum monococcum ssp. monococcum) and the hexaploid bread wheat cultivar ‘Jagger’. The diploid and hexaploid wheat populations were screened for mutations at the waxy locus, GBSS1, as a validation of our population and for comparative analysis of mutation rates in 2x and 6x wheat. For diploid wheat, GBSSI was screened in 716 M2 plants, and one mutant was found for 1.9 Mb screened. 3 For hexaploid wheat, GBSSI was screened in 518 M2 plants, and 30 mutants were identified within a total of 657 Kb screened, giving a mutation frequency of one mutation per 22 Kb. The reasons for this vast difference in mutation frequency between diploid and hexaploid wheat are discussed. The diploid wheat population was further examined by screening for mutations within four lignin biosynthesis candidate genes, for a total of 2 Mb screened. A single mutant was discovered for both of the lignin genes PAL6 and HCT, giving a mutation frequency of one mutation per 1 Mb screened.