1a.Objectives (from AD-416):
1. Develop molecular markers (SNPs) that distinguish iron efficient and iron inefficient soybean. 2. Screen - 350 genotypes representing public and private IDC breeding lines and cultivars. 3. Score - 30 previously untested Accessions and Plant Introductions for iron efficiency and screen them against the markers developed in #1 above. 4. Correlate molecular marker scores with iron efficiency scores. 5. Identify the markers optimal for selecting iron efficient germplasm and make the markers publicly available.
1b.Approach (from AD-416):
Use the soybean whole genome sequence to search two major iron QTL regions to identify all recognizable regulatory genes and all 'candidate' genes residing within the QTL region. Regulatory genes and selected candidate genes will be re-sequenced in eight genotypes representing iron efficient and iron inefficient soybean types (Efficient: Clark, A15, Hawkeye, and PI 437654; Inefficient: Anoka, BSR 101, Pride B216, T203). From this resequencing single nucleotide polymorphisms will be identified and SNP markers will be designed. The SNPs will be screened against approximately 350 genotypes representing midwest breeding lines. Screening will be done using the 'Sequenome' technology at Iowa State University. Statistical methods will be employed to identify those markers with prediction capabilities in IDC breeding programs. Markers selected will be made available to the public.
A Single Nucleotide Polymorphism (SNP) analysis identified mutations within Glyma03g28610 that were highly correlated with iron efficiency in test populations. Further analysis showed that this gene is a transcription regulator, homologous to genes in Arabidopsis thaliana known to function upstream of genes involved in iron reduction and transport in the roots. We carried out gene expression analysis that showed this gene is up-regulated during iron stress, further suggesting its relevance in the iron acquisition pathway. Introgression mapping was performed using two related near-isogenic lines in which a region located on soybean chromosome three, required for iron efficiency was identified. The region corresponds to the previously reported iron efficiency QTL. The location was further confirmed through QTL mapping conducted in this study. Transcriptome sequencing and qRT-PCR identified Glyma03g28610 and a homolog of itself, encoding transcription factors within the region that were significantly induced in soybean roots under iron stress. The two induced transcription factors were identified as homologs of the subgroup lb bHLH genes in that are known to regulate the strategy I response in A. thaliana. Re-sequencing of these differentially expressed genes unveiled a significant deletion within a predicted dimerization domain. We hypothesize this deletion disrupts the FIT / bHLH heterodimer that has been shown to induce known iron acquisition genes. This work reveals one significant mechanism by which soybean responds to iron deficiency, a nutritional disease causing million of dollars in lost production each year.