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

在土壤氧化还原潜力较低的稻田，铁毒是影响水稻生产的主要营养失衡问题。过量的亚铁吸收会通过Fenton反应产生羟基自由基，从而导致水稻叶片的氧化胁迫。该项目的目的是通过(I)确定先前在水培筛选试验中确定的耐性数量性状座位(QTL)的候选基因，以及(Ii)在马达加斯加的田间和盆栽试验中测试选定的耐性等位基因供体，从而促进适应铁毒条件的水稻品种的发展。马达加斯加是拟议研究的潜在目标国家。之前在申请者工作组进行的双亲QTL定位和基因组广泛关联研究(Gwas)确定了几个基因座，其中五个候选基因是根据它们的染色体位置、功能注释和初步的生理实验提名的。这些基因的潜在敲除和激活标记突变体是从公共基因库汇编的，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可见症状和生物量形成、脂质过氧化和铁浓度的测量来常规评估。此外，还将测试每个候选基因背后的特定生理假设，并确定自然发生的序列多态。为了在可变的遗传背景下进一步测试最有希望的候选基因，将使用构成启动子构建过度表达的转化载体，并使用CRISPR/Cas9基因组编辑技术构建基因敲除转化载体。在进行分子调查的同时，还将在马达加斯加的自然和半自然环境中对最初使用的绘制人口图的人群进行选定的基因类型测试。大田和温室盆栽实验将使用25个选定的基因类型，使用来自铁中毒田间地点的土壤。根据当地农业研究机构的建议，这些植物将在铁中毒的条件下种植，要么不施肥，要么施肥。胁迫反应将通过确定症状形成、生物量和产量构成因素、铁浓度以及与地上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环境中表现出一致效应的基因的等位变异，从而可用于适应铁毒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