Results from North Dakota State University:
From the results of a large scale screening and SNP analysis of data generated in 2005-2006, we discovered nine SNP loci that were associated with IDC in soybean. Our goal this year was to determine the extent to which these were useful in additional years. The material we tested was from trials grown in 2008, 2009, and 2010. Collectively, these trials consisted of ~1000 independent lines from mostly private (some public) breeding programs. Their IDC scores were generated from a large screening trial funded by another soybean funding source. We isolated DNA from those 1000 lines, and each of the lines was scored with the nine different SNPs. We used the commercial KSPaR assay for scoring. That data was combined with the 2005 and 2006 to provide a broad perspective regarding the markers under evaluation.

Among the nine SNP loci, the phenotype of the two alleles differed by varying degrees. Of particular relevance was one SNP, BARC-039383-07310, that showed a significant difference between the two alleles in each of the five years. Individual genotypes with IDC tolerance allele (C nucleotide) at this locus also had the lowest IDC score averaged over all of the years. The SNP locus with the next best score was BARC-05973-16418. Here the A allele gave the lowest IDC scores. IDC tolerance is a very complex trait that involves genes that control soil conditions, and uptake and transport of iron. Therefore, it is reasonable to consider pairs of loci. Therefore we looked at various combinations of SNP marker genotypes. We discovered that those individuals with the tolerance form of the two SNPs mentioned above had the lowest IDC over all five years. In addition, when we looked at the data on a location-by-location basis, we discovered that this bi-locus SNP genotype was the lowest in 20 of the 22 locations from which the data was collected. Finally, we discovered that genes known to be involved in iron metabolism are located near these two SNPs. We will continue to evaluate these SNPs by analyzing additional materials from a broader geographical locale. We also plan to map these regions in much greater detail to see if we can discover the actual gene associated with these IDC responses.

Results from USDA/Ames, Iowa:
This location has focused on a pair of isogenic lines that differ for IDC tolerance. These have been extensively evaluated using a hydroponic based assay that contains reduced levels of iron. The assay this year followed up on the discovery of transcription factors associated with a major IDC locus found on chromosome Gm3. (This same region was found to be significantly associated with IDC tolerance using the association mapping techniques used at North Dakota State University.) Results showed that the expression of several transcription factors located in this region was increased under iron stress. Two of these genes are similar to a gene in the model plant species that controls the expression of an iron stress responsive gene AtFIT. From a different perspective, a gene involved with DNA replication, repair, and recombination (Replication protein A = RPA), was discovered to be down regulated under iron stress in the iron efficient genotype Clark, while the expression pattern was opposite for the iron inefficient genotype IsoClark. We hypothesized that low expression levels of RPA genes were beneficial during iron stress. To test this hypothesis, two copies of RPA3 were silenced simultaneously in IsoClark during iron stress. Upon RPA3 silencing, IDC visual score and chlorophyll content improved. The differential expression pattern between the two NILs well as improved IDC symptoms upon RPA3 silencing in IsoClark suggest a link between DNA replication and the iron stress response.

Results from USDA/St. Paul, Minnesota:
Using the hydroponic system described for USDA/Ames, IA, 14 RNA samples were collected from Clark and IsoClark, and high throughput sequence data were collected from the samples. Bioinformatic analysis revealed genes associated with several pathways were induced or repressed. Of particular interest are genes involved in metal transport, carbohydrate and cell wall metabolism, secondary metabolism, and gene regulation. The regulators included receptor kinases and transcription factors representing members of the WRKY, AP2, and GRAS families. These results supported the development of transgenics to assess the effect of these genes on improving IDC tolerance. To date, transgenic lines containing Vit, Nas, and ABC transporter gene constructs were developed. We have been unable to obtain transgenic lines containing FRO and ODD. The T1 lines for Vit and Nas have been planted but germination is poor. However, we have enough plants at this time that we should be able to obtain the T2 generation for theses lines. We are in the process of genotyping the T1 Vit, Nas, and ABC transgenic lines. Interestingly the T1 homozygous ABC transporter line designed for reduced expression shows reduced growth and chlorosis. The T2 lines will be genotyped shortly, and plans are in place to thoroughly characterize these T2 plants.

Major technical accomplishments include:
• Discovery that multiple genes are involved in the response to iron stress and several of these may be targets for gene manipulation;
• Discovery of two loci closely associated with IDC tolerance that have the potential to be used as markers for breeding purposes
• Discovery that transcription factors and replication associated proteins are closely involved in the IDC response, genes that may prove useful for assessing the potential of a line to exhibit IDC tolerance