Task 1: By the end of the project year we will determine whether the large-scale use of copy number assays can be used to identify plants with increases in copy number and will have initiated tests to determine if this results in increased SCN resistance.

Task 2: By the end of the project year we will provide information regarding variable DNA methylation at the Rhg1 locus and its correlation with observed differences in the SCN resistance of different soybean lines that carry similar Rhg1 haplotypes. We will develop and begin testing of assays for Rhg1 methylation and RNA expression that can be used in breeding.

Task 3: Although it may take two years to complete the task, we hope by the end of the proposed project year to have at least preliminarily identified the relevant SCN resistance-conferring gene at one of the PI 468916-derived loci cqSCN006 or cqSCN007.

Task 4: Findings presented at scientific meetings in spring and summer 2017.

Updated November 8, 2017:
• Research has been focused on identifying plants with crossover events in the regions where the SCN resistance QTL cqSCN-006 and cqSCN-007 are located. Overall 16 plants were identified, 5 with crossovers in cqSCN-006 and 11 close to cqSCN-007. The positions of the crossover events in the selected plants were mapped with markers and the selected plants are now being grown to maturity. Seed harvested from some of these plants will be grown in SCN tests to determine the position of the QTL relative to the crossover events.
• The chromosome walking for the SCN-cq006 and 007 loci is in final phases with small gaps to be closed. In addition to the large deletion located in the SCN-cq007 interval, a new candidate polymorphism has been identified in a gamma-SNAP protein, a key candidate gene in the SCN-cq006 interval. Although the polymorphism lies outside the coding region of the gene as annotated in the reference genome, the polymorphism may affect the coding sequence of an alternatively spliced mRNA expressed in the resistant genotype.
• Continued work with WCIC (former Monsanto soybean transformation facility, now UW-Madison facility), to generate transgenic CRISPR soybean lines for each candidate gene in the cqSCN-006 and cqSCN-007 intervals. For each of 17 candidate genes we have constructed and are now modifying plasmids for CRISPR mutagenesis.
• Ten plants that show a higher copy number than Fayette were selected along with five plants that show a lower copy number. Once the plants are mature, progeny from the plants will be tested for SCN resistance and any lines that show changes in resistance will be evaluated with other genetic methods to confirm their copy number.
• Regarding epigenetic status of Rhg1 locus: Several different experiments have been initiated: these include high resolution DNA methylation data for Rhg1 locus in 5 soybean varieties, studies of the Rhg1 chromatin status, and studies of the effect of hypo methylation on Rhg1 expression.

The knowledge that the number of copies of the gene Rhg1 may have bearing on the magnitude of SCN resistance within a variety will make a difference in the future of how varieties are bred for improved SCN resistance.