Rapid identification disease resistance genes from plant genomes by resistance gene enrichment sequencing (RenSeq) of EMS-derived susceptible mutants

植物病害在农业中造成显著的产量损失。小麦和马铃薯是世界上最重要的两种作物，包括印度和英国。小麦最具破坏性的病害是锈病。条锈病发生在作物生长的任何地方，在某些地区造成平均每年高达10%的产量损失。在绿色革命之前，茎锈病与经常性的作物歉收和饥荒有关。当时引入的耐药性现在已经被14年前开始出现在非洲的新菌株所打破。马铃薯晚疫病是19世纪40年代爱尔兰马铃薯大饥荒的原因，至今仍是马铃薯种植的严重障碍。农药可以控制这些疾病，但价格昂贵，与农业的可持续集约化不一致，在发展中国家和自给农民中，农药根本负担不起。引入这种天然的抵抗力是一种优雅的疾病管理方式。然而，用于引入R基因的传统方法通常涉及长育种轨迹以避免连锁拖累，即同时引入有害性状。此外，当一次使用一个R基因时，R基因往往在几个季节内被病原体克服。我们的长期策略是通过分子克隆分离尽可能多的新R基因，并使用转基因方法将它们组合引入。分子克隆技术使得将几个新基因放在基因组的同一位置上成为可能，而且确实很简单，使育种者能够将它们作为“单一”基因进行研究，避免连锁反应。此外，从第一原则来看，具有不同特异性的R基因金字塔应该更持久，然而，传统的基于图位的R基因克隆仍然具有挑战性。首先，由于缺乏重组，大部分植物基因组无法进行基于图位的遗传学。第二，大多数R基因属于称为NB-LRR的基因结构类别，其倾向于驻留在复杂的簇中，并且数百个NB-LRR填充典型的植物基因组。因此，科学家经常划定一个地图区间包含多个NB-LRR，必须找出哪些赋予感兴趣的阻力。一种已成功用于将候选列表缩小到单个NB-LRR的方法是诱变和筛选易感突变体。这创造了离散的变化，从而一个简单的比较突变体和野生型可以确定R基因。我们提出了一种策略，将显着提高R基因的识别率。在我们工作流程的第一步，我们将筛选大量诱变植物的敏感突变体。在第二步中，我们将使用我们实验室最近实施的最先进的测序技术来选择性地捕获和测序植物基因组中的所有NB-LRR。这将使我们能够快速和廉价地比较野生型和突变体，以识别和克隆抗性基因。这项研究的成果将是三倍：（i）使用已知的对照，我们将实施我们的通用策略，从复杂的基因组中分离R基因，（ii）我们将应用该策略从马铃薯和小麦中鉴定新的R基因（分别针对晚疫病和小麦锈病），以及（iii）我们将在印度和英国环境中测试我们的关键小麦锈病R基因。我们设想我们的策略不仅将显著加速R基因克隆，它也可用于追踪不符合标准遗传学的R基因，例如在基因组的低或非重组发生区域，包括着丝粒、外来渗入片段和易位。在小麦中，这将允许访问过多的有用的R基因，这些基因目前由于与有害的产量抑制等位基因的连锁而无法使用。

Plant disease causes significant yield losses in agriculture. Wheat and potato are two of the most important crops worldwide, including India and the UK. Among the most damaging diseases of wheat are the rusts. Stripe rust occurs wherever the crop is grown causing average yearly yield losses of up to 10% in some regions. Stem rust was until the green revolution associated with regular crop failures and famine. The resistance introduced then has now been broken by new strains of the fungus, which started appearing in Africa 14 years ago. The potato late blight disease, the cause of the Great Irish Potato famine in the 1840s, is still a serious impediment to potato cultivation today. Pesticides can control these diseases but they are expensive, at odds with sustainable intensification of agriculture, and in developing countries and for subsistence farmers, they are simply unaffordable.Wild relatives of domesticated crops contain many useful disease resistance (R) genes. Introducing this natural resistance is an elegant way of managing disease. However, traditional methods for introducing R genes typically involve long breeding trajectories to avoid linkage drag, i.e. the simultaneous introduction of deleterious traits. Furthermore, R genes tend to be overcome by the pathogen within a few seasons when deployed one at a time.Our long-term strategy is to isolate, by molecular cloning, as many new R genes as possible, and introduce them in combinations using GM methods. Molecular cloning makes it possible, indeed straightforward, to put several new genes together in the same location in the genome, allowing breeders to work with them as a "single" gene and avoiding linkage drag. Moreover, from first principles, a pyramid of R genes with distinct specificities should be more durable.Traditional map-based cloning of R genes, however, is still challenging. First, large tracts of plant genomes are inaccessible to map-based genetics due to lack of recombination. Second, most R genes belong to a structural class of genes called NB-LRRs, which tend to reside in complex clusters, and many hundreds of NB-LRRs populate a typical plant genome. The scientist therefore frequently delimits a map interval containing multiple NB-LRRs and must find out which confers the resistance of interest. An approach, which has been successfully used to narrow down the candidate list to a single NB-LRR, is mutagenesis and screening for susceptible mutants. This creates discrete variations whereby a simple comparison of mutant and wildtype can identify the R gene.We propose a strategy that will significantly increase the rate of R gene identification. In a first step of our workflow, we will screen large numbers of mutagenized plants for susceptible mutants. In a second step, we will use a state-of-the art sequencing technique recently implemented in our lab to selectively capture and sequence all the NB-LRRs in a plant genome. This will allow us to rapidly and cheaply compare wildtype with mutants to identify and clone resistance genes.The outputs of this research will be three-fold: (i) using known controls we will implement our generic strategy to isolate R genes from complex genomes, (ii) we will apply this strategy to identify novel R genes from potato and wheat (against late blight and wheat rusts respectively), and (iii) we will test our key wheat rust R genes in Indian and UK environments.We envisage that not only will our strategy significantly accelerate R gene cloning, it could also be used to pursue R genes not amenable to standard genetics, e.g. in low- or non-recombinogenic regions of the genome including centromeres, alien introgressed segments, and translocations. In wheat, this would allow accessing a plethora of useful R genes currently unusable due to linkage to deleterious yield-depressing alleles.