3.4.35
TOWARDS THE POSITIONAL CLONING OF Ry, AN EXTREME RESISTANCE GENE AGAINST PVY IN POTATO

G BRIGNETI1, J GARCIA-MAS2, M QUERCI3 and DC BAULCOMBE1

1The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich NR4 7UH, UK; 2IRTA, Departamento de Genetica Vegetal, Carretera Cabrils s/n, 08348 Cabrils, Barcelona, Spain; 3International Potato Center, Apartado 1558, Lima-100, Peru

Background and objectives
Potato virus Y (PVY) is one of the most important viral pathogens in potato crops around the world. The virus, which is actively transmitted by several aphid species, can cause up to an 80% crop yield reduction in susceptible potato cultivars. One of the best ways to control the spread of the disease is the use of resistant varieties. Extreme resistance or immunity to PVY was first reported by Cockerham in 1943 in Solanum stoloniferum and has since been introgressed into some commercial cultivars by conventional breeding. This resistance is controlled by the single dominant gene Rysto and is effective against all strains of PVY [1]. Our first objective was to map Rysto to a chromosomal position in the potato genome. Our second objective was to generate a high resolution map of the Rysto region by identification of linked markers using AFLP analysis on bulked segregant pools derived from an Fl tetraploid potato population segregating for the gene. This genetical map will be used in the positional cloning of Rysto. We have also started studies aiming to the identification of the corresponding avirulence determinant of the resistance interaction in the PVY genome using a biolistic approach.

Results and conclusions
Two F1 segregating populations for Rystowere used in these studies. The first one, consisting of 360 progeny was used for the assignment the gene to a potato linkage group and for the construction of a low resolution map in the vicinity of Rysto. A bulked segregant approach in combination with AFLP technology was used to obtain markers linked to Rysto. The AFLP marker M39b, linked to Rysto in potato, was mapped in a tomato F2 mapping population derived from the interspecific cross Lycopersicon esculentum x L. pennellii [2]. Results from the analysis indicated that M39b is located in the tomato linkage group Xi. This position allowed us to select shared RFLP markers (GP163 and GP259) between the tomato and potato maps [3] and to confirm the position of the gene in the potato linkage group Xi. In order to saturate the Rysto region with more markers, extensive AFLP analysis was carried out on the bulked segregants. The second F1 segregating population, consisting of 1779 progeny, was used to map several of the identified linked markers and to generate the high resolution map of the Rysto region. The current map positions Rysto flanked by the AFLP marker's M5-M45 and M16 at 0.16 and 0.5 cM, respectively. A dihaploid potato plant derived from the resistant parent of the crosses used in this study will be used for the construction of an Rysto potato genomic library. An estimation of the physical distance between flanking markers by PFGE will allow us to determine if a cosmid or a BAC library will be needed to achieve the cloning of the gene by chromosome landing.

We are also interested in the identification of the viral elicitor of the Rystomediated resistance. For this purpose, we are currently generating 35S promoter-based constructs carrying PVY sequences and the GUS reporter gene. These constructs will be inoculated on resistant and susceptible potato plants using the particle bombardment technique. We expect that monitoring of the GUS activity in the inoculated material will help us to identify which viral factor or factors are involved the resistance interaction.

References
1. Cockerham G, 1970. Heredity 25, 309-348.
2. Paul E, Goto M, Tanksley SD,1 994. Euphytica 79, 181-186.
3. Gebhardt C, Ritter E, Barone A et al., 1991. Theoretical and Applied Genetics 83, 49-57.