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

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.

The hypothesis is that diploid Fragaria species can be used as a model to study the genetics of resistance to strawberry wilt (Verticillium dahliae), and develop markers for resistance to be used by breeders of the cultivated octoploid strawberry. Real time PCR will be used to study the colonisation of strawberry plants by V. dahliae and this will be related to the expression of symptoms, i.e. are plants without visual symptoms are resistant to colonisation or merely tolerant to extensive colonisation by the pathogen? This will distinguish between strawberry genotypes having different types of genetic resistance and the information will be used to identify parents to produce diploid and octoploid mapping progenies of c. 400 individuals segregating for resistance. The progenies will be challenged with V. dahliae and phenotypic data generated for resistance/susceptibility. The EMR diploid Fragaria reference map will be used to develop a reduced microsatellite genetic linkage map for the segregating diploid population and microsatellites closely linked to resistance loci will be identified. Concurrently a map of the segregating octoploid population will be generated using AFLPs and the phenotypic data will be used to position resistance loci, including QTL, on the map. Microsatellites co-segregating with resistance loci from the diploid map will be transferred to the octoploid map to establish linkage with resistance loci. Resistance to V. dahliae has been characterised in tomato and homologues of the key genes will be isolated from Fragaria along with other resistance gene analogues. Differential display will be used to study the expression of genes involved in the defence response to V. dahliae infection. Potential resistance genes identified from these approaches will be cloned from F. x ananassa. Markers will be produced and located on the diploid and octoploid linkage maps to determine correlation with loci involved in resistance to V. dahliae.