Confirmation and Functional Characterization of Candidate Genes for Iron Toxicity Tolerance in Rice (Oryza sativa L.)

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Iron toxicity is a major nutrient imbalance affecting rice production in paddy fields with low soil redox potential. Uptake of excess ferrous iron causes oxidative stress in rice leaves due to hydroxyl radical production via the Fenton reaction. The aim of this project is to contribute to the development of rice varieties adapted to iron toxic conditions by (i) characterizing candidate genes underlying tolerance quantitative trait loci (QTL) determined previously in hydroponic screening experiments, and (ii) testing selected donors of tolerant alleles in field and pot experiments in Madagascar, a potential target country for the proposed research. Bi-parental QTL mapping and a genome wide association study (GWAS) conducted previously in the applicants work group identified several loci, in which five candidate genes were nominated based on their chromosomal positions, functional annotations, and preliminary physiological experiments. Potential knock-out and activation-tagged mutants for these genes were compiled from public gene banks and will be genotyped and tested in hydroponic iron stress experiments. Tolerance or sensitivity will be routinely evaluated by measurements of visible symptom and biomass formation, lipid peroxidation, and iron concentrations. In addition, specific physiological hypotheses underlying each of the proposed candidate genes will be tested, and naturally occurring sequence polymorphisms will be determined. To allow for further testing of the most promising candidate genes in variable genetic background, transformation vectors will be constructed for both over-expression using a constitutive promoter, and gene knock-out using the CRISPR/cas9 genome editing technology. These molecular investigations will be accompanied by testing of selected genotypes from the originally used mapping populations in natural and semi-natural environments in Madagascar. Field and greenhouse-based pot experiments using soil from an iron toxic field site will be conducted using 25 selected genotypes. These will be grown under iron toxic conditions using either no fertilizer, or fertilizer according to the recommendations of local agricultural research institutions. Stress response will be evaluated by determination of symptom formation, biomass and yield components, iron concentrations, and physiological factors associated with shoot redox balance. Together these investigations are expected to comprehensively characterize allelic variants of genes that show consistent effects in different iron toxic environments, and can therefore be used for marker-assisted breeding of rice varieties adapted to iron toxicity