Controlling important diseases in potato by cloning functional NB-LRR-type resistance genes

马铃薯是世界上最重要的粮食作物之一，其生产受到几种害虫和病原体的威胁，严重降低了作物的产量和质量。这些病原体包括导致臭名昭著的爱尔兰马铃薯饥荒的晚疫病、攻击马铃薯根部的居住在土壤中的囊肿线虫，以及一些细菌和病毒。在世界大多数地区，目前对许多这类病原体的控制方法主要基于使用化学喷雾剂，这既对环境有害，又往往无效。此外，其中一些化学品，例如杀线虫剂，正在被欧盟立法禁止使用，这严重限制了种植者控制囊肿线虫的能力。模型显示，如果减少产量的疾病能够得到控制，全球马铃薯产量可能增加多达三分之一。人们普遍认为，防治作物病虫害最有希望和最环保的途径是利用自然产生的植物抗性基因。在包括马铃薯在内的植物中，抗病性通常由抗性基因控制，即具有可识别的DNA“结构域”结构的“R基因”。在植物先天免疫系统中，由R基因编码的R蛋白通过识别病原体产生的“效应”分子，然后激活各种防御过程来提供抗性。因此，R基因是植物抗病的关键角色，它们对所有主要类型的各种病原体（卵菌、真菌、病毒、线虫和细菌）都有效。我们最近分析了已发表的马铃薯基因组，并使用我们开发的一种新技术从马铃薯品种或野生物种中快速分离出所有R基因，鉴定出约760个R基因。这种新方法被称为RenSeq （R基因富集测序），它利用了基因组测序和从复杂混合物中“捕获”目标DNA序列的最新发展。利用这项技术，我们现在可以对R基因进行测序，R基因仅占马铃薯基因组DNA的0.24%，可以从任何马铃薯或密切相关的植物中高效且经济地进行测序。此外，只要提供适当的序列信息，该方法可以很容易地应用于其他作物品种。该项目利用了野生马铃薯和其他茄科植物中存在的广泛多样性的R基因。本项目的主要目的是将RenSeq方法与最新的测序技术相结合，以确定选定的茄属植物的R基因互补体，这些茄属植物对一种或多种马铃薯病原体具有有效的功能抗性。我们将使用生成的序列数据，使用有效的遗传池方法来识别功能性抗性的标记和候选基因。这将利用抗性和易感植物之间的现有杂交来绘制植物基因组中染色体位置的功能性抗性。RenSeq将用于对杂交亲本以及具有抗性和易感的后代植物的所有R基因进行测序。利用新的生物信息学方法对这些序列数据进行比较，我们将开发出位于抗性基因内部或紧邻抗性基因两侧的遗传标记。标记将通过传统的植物育种来选择抗性，候选基因将进行功能研究，以备未来的生物技术应用。该项目的一个关键方面是JHI， UoD和TSL之间的合作伙伴关系，TGAC的植物基因组测序专业知识以及与Simplot的联系，Simplot将在美国进行抗性基因的田间试验。

Potato is one of the world's most important food crops, and production is threatened by several pests and pathogens that severely reduce crop yield and quality. These pathogens include late blight, the organism that caused the infamous Irish potato famine, soil-dwelling cyst nematodes that attack the roots, and a number of bacteria and viruses. Current control methods for many of these pathogens in most parts of the world are based mainly on the use of chemical sprays which are both environmentally hazardous and often ineffective. Moreover, some of these chemicals, for example nematicides, are being removed from use by EU legislation, severely limiting the ability of growers to control cyst nematodes. Modelling suggests that global potato production could increase by up to a third if the diseases that reduce yields could be controlled. It is widely thought that the most promising and environmentally benign route for combating crop pests and diseases is by deployment of naturally-occurring plant resistance genes. In plants, including potato, disease resistance is typically controlled by resistance genes, known as 'R genes' that have a recognizable DNA 'domain' structure. In the plant innate immune system, the R proteins encoded by R genes provide resistance by recognising 'effector' molecules produced by the pathogen and then activating various defence processes. Hence R genes are key players in plant disease resistance, and they are effective towards diverse pathogens of all major types (oomycetes, fungi, viruses, nematodes and bacteria). We recently analysed the published potato genome and identified ~760 R genes using a novel technique we have developed for rapid isolation of all R genes from potato varieties or wild species. This new method, called RenSeq (R gene enrichment sequencing), takes advantage of recent developments in genome sequencing and in 'capturing' targeted DNA sequences from complex mixtures. Using this technique we can now sequence the R genes, which represent only about 0.24% of the DNA in the potato genome, from any potato or closely related plant in an efficient and cost effective manner. Moreover, given the appropriate sequence information, this method can be easily applied to other crop species.This project makes use of the wide diversity of R genes present in wild potatoes and other Solanaceae. The main aims of this project are to combine the RenSeq method with the latest sequencing technologies to determine the R gene complements of selected Solanum species harbouring functionally effective resistances against one or more potato pathogen. We will use the sequence data generated to identify markers and gene candidates for functional resistances using an efficient genetic pooling approach. This will make use of existing crosses between resistant and susceptible plants to map the functional resistances to a chromosomal position in the plant genome. RenSeq will be deployed to sequence all R genes in the parents of crosses as well as pools of offspring plants that are resistant and susceptible. Comparing these sequence data using new bioinformatics approaches we will develop genetic markers that are within or immediately flanking the genes conferring resistance. Markers will be deployed via conventional plant breeding to select for resistance and candidate genes will be subjected to functional studies for potential future biotechnological deployment. A key aspect of this project is the partnership between the JHI, UoD and TSL, the plant genome sequencing expertise of TGAC as well as the link with Simplot, who will test resistance genes in field trials in the USA.