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

铁中毒是低土壤氧化还原电位稻田影响水稻生产的主要营养失衡。过量亚铁的吸收导致水稻叶片氧化应激，这是由于芬顿反应产生羟基自由基。该项目的目的是通过(i)鉴定先前在水培筛选实验中确定的耐受性数量性状位点（QTL）的候选基因，以及（ii）在马达加斯加（拟议研究的潜在目标国家）的田间和盆栽试验中测试耐受性等位基因的选定供体，从而促进开发适应铁毒性条件的水稻品种。双亲本QTL定位和基因组全关联研究（GWAS）先前在申请人工作组中进行过，确定了几个位点，其中五个候选基因根据其染色体位置，功能注释和初步生理实验被提名。从公共基因库中编译这些基因的潜在敲除和激活标记突变体，并将在水培铁胁迫实验中进行基因分型和测试。耐受性或敏感性将通过测量可见症状和生物量形成、脂质过氧化和铁浓度进行常规评估。此外，将测试每个候选基因的特定生理假设，并确定自然发生的序列多态性。为了在可变遗传背景下进一步测试最有希望的候选基因，将构建转化载体，使用组成型启动子进行过表达，并使用CRISPR/cas9基因组编辑技术进行基因敲除。在进行这些分子调查的同时，将对马达加斯加自然和半自然环境中最初使用的作图人群中选定的基因型进行测试。田间和温室盆栽试验将利用铁中毒场地的土壤进行25个选定的基因型。这些作物将在铁中毒的条件下种植，要么不施肥，要么根据当地农业研究机构的建议施肥。胁迫反应将通过测定症状形成、生物量和产量成分、铁浓度和与茎部氧化还原平衡相关的生理因素来评估。总之，这些研究有望全面表征在不同铁毒性环境中表现出一致效应的基因的等位基因变异，并因此可用于适应铁毒性的水稻品种的标记辅助育种

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