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

植物病害对农业造成重大的产量损失。小麦和土豆是世界上最重要的两种作物，包括印度和英国。最具破坏性的小麦病害是锈病。条状锈病在作物生长的任何地方都会发生，在一些地区造成年均产量损失高达10%。在绿色革命之前，茎锈病一直与农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。马铃薯晚疫病是导致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，但它们价格昂贵，与农业的可持续集约化不一致，在发展中国家和自给自足的农民根本负担不起。驯化作物的野生近缘种含有许多有用的抗病基因。引入这种自然抗性是控制疾病的一种优雅方式。然而，传统的引入R基因的方法通常涉及较长的育种轨迹，以避免连锁阻力，即同时引入有害性状。此外，如果一次部署一个R基因，往往会在几个季节内被病原体克服。我们的长期战略是通过分子克隆分离出尽可能多的新的R基因，并通过转基因方法将它们组合在一起。分子克隆使把几个新基因放在基因组的同一位置成为可能，实际上是直截了当的，这样育种者就可以把它们当作一个“单一”基因来处理，避免连锁效应。此外，从基本原理来看，具有不同特异性的R基因金字塔应该更持久。然而，传统的基于图谱的R基因克隆仍然具有挑战性。首先，由于缺乏重组，基于图谱的遗传学无法获取大量的植物基因组。其次，大多数R基因属于一个被称为NB-LRRs的基因结构类别，它倾向于驻留在复杂的集群中，并且数百个NB-LRRs组成一个典型的植物基因组。因此，科学家经常划定包含多个nb - lrr的地图区间，并必须找出哪一个具有兴趣阻力。一种已成功用于将候选名单缩小到单个NB-LRR的方法是诱变和筛选易感突变体。这就产生了离散的变异，通过简单地比较突变型和野生型就可以识别R基因。我们提出了一个策略，将显著提高R基因的鉴定率。在我们工作流程的第一步，我们将筛选大量诱变植物的易感突变体。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型，以鉴定和克隆抗性基因。这项研究的产出将有三个方面：(i)使用已知的控制，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）我们将应用这一策略，从马铃薯和小麦中识别新的R基因（分别针对晚疫病和小麦锈病）；（iii）我们将在印度和英国的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基因的克隆，它也可以用于追求不符合标准遗传学的R基因，例如在基因组的低或非重组区域，包括着丝粒、外来渗入片段和易位。在小麦中，这将允许获得大量有用的R基因，目前由于与有害的抑制产量的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.