Soybean (Glycine max (L.) Merr.) cyst nematode (SCN) (Heterodera glycines Ichinohe), an obligate sedentary endoparasite, is the most economically destructive pathogen in soybean production and causes over $1 billion in annual losses in the United States. Planting resistant cultivars is the primary management method to control SCN for the long-term purpose, but the nature of genetic resistance is little known. The Rhg1 (Resistance to H. glycines) locus on chromosome 18 is found as a major quantitative trait locus (QTL) that contributes resistance to SCN. The chemical mutagen ethylmethane sulfonate (EMS) can be utilized to induce genetic mutations in soybean populations, which screened by an efficient reverse genetic strategy known as Targeting Induced Local Lesions IN Genomes (TILLING) for functional gene analyses. The objective of this study was to analyze the function of SNAP gene (Glyma18g02590) at rhg1 allele from `Forrest' (`Peking'-derived SCN resistant cultivar) using TILLING. Soybean cultivar `Forrest' seeds were mutagenized with EMS and grown to generate M1 plants. M1 plants were self-pollinated to produce approximately 3000 M2 plants. Genomic DNAs were extracted from young leaves of individual M2 plants and quantified to normalize concentration of DNAs. The DNA samples were then pooled eight-fold in 96-well plates for mutations screening by TILLING. Moreover, 12 phenotypic traits including chlorophyll deficiency, leaf shape, branch architecture, seed color, seed weight, fatty acid phenotype were identified in the mutagenized population, analyzed and archived in this study.

Soybean is one of the most important grain legumes grown in US and worldwide, and is a major component of human and animal protein diets. Despite improvements in management practices, and the introduction of improved soybean cultivars, soil borne pathogens continue to cause tremendous yield loss in soybean production each year. Among soil borne pathogens; Soybean Cyst Nematode (SCN) or Heterodera glycines together with Sudden Death Syndrome (SDS) induced by Fusarium virguliforme are responsible for the most damages in soybean fields. The most effective way to control these two pathogens is to develop resistant cultivars. Resistance to any population (HgType) of H. glycines, requires a functional allele at rhg1/Rfs2 locus. The rhg1/Rfs2 gene encodes a receptor-like kinase (RLK) protein. By analysing near isogenic lines (NIL) segregating for rhg1/Rfs2, rhg1-like loci were found at other locations most conservedly on LG B1. While the nature of rhg1 allele was thought to be recessive, heterozygous NIL segregating at the rhg1 locus showed that the resistant allele was dominant. Rhg1 was also inferred to be multigeneic due to absence of recombination between the RLK and other 2 genes. Functional and structural analyses were conducted on the leucine rich repeat (LRR) from RLK protein encoded by GmRLK18-1 within the Rhg1/Rfs2 locus. The LRR of GmRLK18-1 showed a high binding affinity to CLE-like peptides found in both nematode secretions and plant developmental control. Crosslinking assays and native gel analysis of GmRLK18-1-LRR validated its model as a crystal homo-dimer. Larger proteins were also shown to bind the LRR domain, in far-Western analyses both methionine synthase and cyclophilin bound strongly to the LRR domain. Homology and ab-initio modeling of the LRR domain of the GmRLK18-1 was predicted as both a monomer and a homodimer containing intrinsically unstructured regions. Amino acid substitutions found among GmRLK18-1 allotypes A87V, Q115K and H274N were predicted to play crucial roles in protein function and stability. The receptor like kinase (RLK) GmRLK18-1 within the Rhg1/Rfs2 locus underlies a pleiotropic resistance to both SCN and SDS. The resistance allele was shown to be dominant in both heterozygous NILs at Rhg1/Rfs2 and transgenics (hetero- or hemi-zygous). The RLK was found to provide a partial resistance to SCN and importantly a nearly complete resistance to both root and leaf symptoms of SDS. In the presence of Rhg4, the RLK-transgenic plants developed nearly full resistance to SCN. Therefore the RLK was proven to underlie a major portion of the Rhg1/Rfs2 locus.