A genetic system to study resistance to the soil-borne pathogen Verticillium dahliae in strawberry

草莓是英国的一种重要作物，2005年的零售额为3.47亿英镑，在过去五年中，超市的销售额迅速增长。英国种植者从5月到10月供应市场，他们是创新和进步的，但面临着疾病问题的挑战。草莓最广泛的土壤传播疾病是由大丽黄萎病引起的，直到最近，这种疾病还通过甲基溴熏蒸土壤来控制。在欧盟，自2005年以来，这种化学物质已被禁止用于这种用途，而替代土壤消毒剂的效果较差。最好的选择是种植抗大丽花的品种，但这些品种数量有限，许多品种要么不适应英国，要么没有市场所需的果实特征。对大丽花弧菌的抗性是EMR草莓育种的优先事项，但与世界各地的其他规划一样，所使用的策略是经验性的，依赖于基于田间或盆栽试验中症状表达的筛选。该项目将采用现代分子生物学技术，为育种者提供更高的精度和有效性，以开发抗性草莓品种。栽培草莓对大丽花病菌的抗性表现出数量遗传，但单纯根据症状表现进行定量分析比较困难。该项目将采用实时PCR来测量植物内病原体定殖的程度，这将与表达的症状相关。这种方法将是有价值的，不仅因为提高了精度，而且它将区分不同类型的电阻。可能有些草莓品种在受到大丽花弧菌侵袭时只表现出轻微的症状，但它们能够抵抗病原体的入侵，而其他品种则可能耐受广泛的病原体定植。使用实时PCR来区分这两种类型的抗性对育种者来说是有价值的，并提供了将它们组合成单一基因型的机会。栽培草莓Fragaria x ananassa是八倍体，因此很难进行遗传研究。野生二倍体草莓与栽培型草莓有十多种亲缘关系，它们是遗传学和基因组学研究的理想模型。在BBSRC和Defra资助的早期工作之后，EMR基于两个二倍体物种之间的杂交开发了一个遗传图谱，该图谱已被采用为Fragaria的国际参考图谱。在此基础上，将筛选一系列二倍体物种对大丽花的抗性，并进行适当的杂交，以产生一个或多个抗性分离的二倍体群体。抗性位点的定位将与二倍体参考图谱相关，以便鉴定出紧密相连的可转移分子标记。同时，利用栽培的草莓杂交将产生对大丽花病菌抗性的后代，这将使用AFLP标记进行定位。然后将在二倍体图谱上鉴定的抗性标记转移到八倍体图谱上，以确定它们在草莓育种中的标记辅助选择（MAS）的有效性。除了鉴定与抗性位点密切相关的标记外，还将鉴定和测序候选抗性基因。当草莓植物受到大丽花弧菌的攻击时，基因的表达将通过差异展示进行研究，并与已知的茄属物种（如番茄）的抗性基因进行比较，以及其他蔷薇属水果作物和拟南芥。有希望的候选基因序列数据将用于绘制其在二倍体和八倍体Fragaria连锁图谱上的位置，并确定它们与大丽花弧菌抗性相关位点的相关性。这些方法的结合将为植物育种者提供分子工具，以提高对大丽花弧菌的抗性草莓的育种效率。

Strawberries are an important UK crop with retail sales of £347M in 2005 and rapid growth in supermarket sales over the last five years. UK growers supply the market from May to October and they are innovative and progressive but face challenges from disease problems. The most widespread soil borne disease of strawberries is caused by Verticillium dahliae and until recently this was controlled by fumigating the soil with methyl bromide. In the EU this chemical has been prohibited for this use since 2005 and alternative soil sterilants are less effective. The best option would be to grow cultivars resistant to V. dahliae but these are limited in number and many are either not adapted in the UK or do not have the fruit characteristics required by the market. Resistance to V. dahliae is a priority for strawberry breeding at EMR but, as with other programmes around the world, the strategy used is empirical and relies on screening based on symptom expression in field or pot tests. This project would employ modern molecular biology techniques to provide breeders with improved precision and effectiveness in developing resistant strawberry cultivars. In the cultivated strawberry resistance to V. dahliae shows quantitative inheritance but quantification is difficult based purely on symptom expression. This project will employ real time PCR to measure the extent of pathogen colonisation within the plant and this will be correlated to symptoms expressed. This approach will be valuable not only because of improved precision but also it will distinguish between different types of resistance. It is likely that some strawberry cultivars that show only mild symptoms when challenged with V. dahliae are able to resist invasion by the pathogen, whereas others may be tolerant of extensive pathogen colonisation. Using real time PCR to distinguish these two types of resistance would be valuable to breeders and provide the opportunity to combine them in a single genotype. The cultivated strawberry Fragaria x ananassa is octoploid and thus difficult for genetic studies. There are over ten wild diploid strawberries closely related to the cultivated type and these make an ideal model for genetic and genomic research. Following earlier work funded by BBSRC, and subsequently Defra, EMR has developed a genetic map, based on a cross between two diploid species, which has been adopted as the international reference map for Fragaria. Building on this, a collection of diploid species accessions will be screened for resistance to V. dahliae and the appropriate crosses made to produce one or more diploid populations segregating for resistance. The resistance loci will be mapped and their position related to the diploid reference map, so that closely linked transferable molecular markers can be identified. Concurrently, a cross using the cultivated strawberry will produce a progeny segregating for resistance to V. dahliae and this will be mapped using AFLP markers. Markers for resistance identified on the diploid maps will then be transferred to the octoploid map to determine their usefulness for marker assisted selection (MAS) in strawberry breeding. In addition to identifying markers closely linked to resistance loci, candidate resistance genes will be identified and sequenced. Genes that are expressed when strawberry plants are challenged with V. dahliae will be investigated using differential display and compared to known resistance genes from Solanaceous species, such as tomato, along with other Rosaceous fruit crops and Arabidopsis thaliana. Sequence data for promising candidate genes will then be used to map their position on diploid and octoploid Fragaria linkage maps and determine their correlation with loci involved in resistance to V. dahliae. The combination of these approaches will result in molecular tools for plant breeders to improve the efficiency of breeding strawberries resistant to V. dahliae.