小麦广谱抗性基因lm的精细定位、候选基因筛选以及育种利用策略

Development of crops carrying the genes controlling broad-spectrum and durable resistance is economically important for improving eco-adpatability and yield stability of crops in diverse ecoregions, and also for benefiting surrounding environment by reducing fungicides application. Our previous investigations demonstrated that the resistance gene, designated lm, from Chinese wheat cv. Ning7840 confers high level of resistances, probably by mediating spontaneously occurring hypersensitive reaction-like lesion mimic (LM) phenotype at the adult plant stage, to both rust and powdery mildew diseases (caused by biotrophic pathogens),and to type II resistance to fusarium head blight(caused by hemi-biotrophic pathogen). We have mapped the gene lm to the long arm of 1B chromosome within the deletion bin of C1BL6-0.32 and have identified the associated molecular markers such as SNPs and SSRs etc. In the current study, we are going to further map the gene lm to a chromosomal region at a fine scale and to screen its candiate genes by association of genotypes with phenotypes in different genetic stocks such as recombinant inbred lines (RIL), near-isogenic lines (NIL) and the specially-designed segregating populations. More markers used for fine mapping of the gene of interest will be developed based on published 1BL-specific physical and genetic maps as well as survey sequences, and particularly on the differentially expressed genes screened both at RNA (by transcripts de novo sequencing approach) and proteomic levels (by 2-D gel electrophoresis and MALDI-TOF/TOF approaches) in the NILs and pools contrasting in LM phenotypes. The mechanisms underlying the occurrence of LM will be deciphered at physiological, biochemical, cellular and molecular levels, respectively.The genetic effects of the gene alone in the background of cv. Yangmai15 (susceptible to both powdery mildew and rust diseases) and the additive and epistatic effects in the genetic background of cv.Amadina (carrying resistance gene Lr46) will also evaluated under field conditions.The results of both our previous work on LM and the current investigations are promising to facilitate isolation of lm, to help understand the mechanisms underlying broad-spectrum and durable resistance, and will also be essentially useful in developing, by marker assisted breeding approach, wheat cultivars with a high level of resistances to a wide array of isolates of a pathogen or/and multiple pathogens.

广谱持久抗性基因的挖掘和育种利用对提高作物的适应性、稳产性和减少农药对环境的污染至关重要。我们的前期研究表明：小麦品种宁7840携带的隐性单基因lm很可能通过控制抽穗前后叶片上自发出现的过敏性类似反应介导广谱成株期抗病性，能够极显著降低锈病和白粉病危害，也能显著增强赤霉病扩展抗性。已将该基因定位于小麦1BL染色体的C1BL6-0.32区域，并找到了关联的SNP等标记。本项目基于RIL、NIL和次级分离大群体等材料，借助1BL的物理-遗传图谱等信息和通过对NIL等的转录本de novo测序及差异蛋白质谱分析，开发新的标记，结合表型鉴定和连锁作图，精细定位lm并筛选候选基因；从理化和分子水平剖析其可能的作用机理；分析目标基因在不同遗传背景中和大田条件下的遗传效应，并结合前期研究结果，提出目标基因的育种利用策略。该项研究对克隆目标基因、解析广谱抗病机制和培育广谱持久抗性小麦品种都具有重要意义。

广谱持久抗性基因的挖掘和育种利用对提高作物的适应性、稳产性和减少农药对环境的污染至关重要。小麦叶片上自发出现的过敏性类似反应介导广谱成株期抗病性。我们研究发现，在低氮条件下，小麦品系P7001的叶片表现类过敏反应性状（HRL，也称之为lesion mimic），并高抗白粉病，该性状在充足氮供应条件下消失，对白粉病的抗性也显著降低。表明P7001携带的类过敏反应性状受N素调节，因此利用P7001与另一在低氮和高氮条件下均不表现HRL性状的小麦品系P216进行杂交构建了F5作图群体，结合可靠的表型鉴定、混池RNA-seq的SNP分析以及基于新开发的SSR标记分析，我们将这一氮依赖的类过敏性反应基因（命名为Ndhr11）定位于小麦2B染色体短臂，该基因与dCAPs标记7hrdc2共分离，该基因可能是一个新的lesion mimic基因。Pathway分析表明，植物-病原菌互作、N代谢、玉米素合成以及植物激素信号途径基因在目标性状的相对池间差异极其显著。该基因的发现有助于解析HRL-施氮量-抗病性三者的关系、探索HRL性状对氮素的响应机理及其调节抗病性的关键信号路径和基因。目前正在扩大分离群体和开发更多标记，图位克隆P7001中携带的Ndhr11基因。此外，小麦品种宁7840也携带lesion mimic性状，我们对宁7840进行EMS诱变，创建了lesion mimic缺失的突变体dlm1和dlm2，并对突变体和野生型进行了转录组de novo测序，分析了差异表达基因、突变SNP位点和信号路径，验证了突变位点（SNP）与表型性状的共分离，在dlm2突变体中筛选到潜在的候选基因，对该基因的后续深入研究有助于解析细胞死亡、类过敏反应和广谱抗性机制。因此我们按计划完成了项目中的主要研究内容并取得比期望更好的进展。