Laboratory of Potato Breeding, Hokkaido National Agricultural Experiment Station, Shinsei, Memuro, Hokkaido, 082-0071, Japan

Background and objectives
The effects of viruses on potato growth and yield are various depending on the virus species and potato cultivars. Potato virus Y (PVY) is a major causal pathogen of potato virus diseases. It spreads easily, evokes severe symptoms in potato plants, and causes 80% of potential yield loss. It is generally accepted that in contrast with PVY, potato virus S (PVS) usually causes slight symptoms and does not cause significant economic loss by itself. Potato virus X (PVX) occurs worldwide and reduces yields by 15% or more. It is known that mixed infection with PVX, PVS and PVY causes severe diseases that sometimes destroy the crop, and the severity of the symptoms depends on potato cultivars. Due to the lack of effective pesticide, use of resistant cultivars has been known to be the best method for disease control. In this study, 196 potato lines derived from interspecific hybrids were evaluated the susceptibility to PVY, PVX and PVS in order to identify genetic material for exploitation in breeding programs for virus resistance. Furthermore, in order to facilitate potato breeding programs, we tried to perform cloning of virus resistance genes.

Materials and methods
Potato interspecific hybrids with different genetic background (196 lines) were vegetatively propagated for several generations in the field. Crude sap of potato leaves was transferred mechanically to several indicator plants. Nicotiana tabacum cv. Ky57, Gomphrena globosa and Chenopodium quinoa were used as indicator plants to detect PVY, PVX and PVS infection, respectively. In order to detect the virus resistance genes, genomin DNA was extracted from mature tubers and used as templates for PCR. As primers for PCR, we used degenerate oligonucleotides that corresponding to amino acid sequences, which were conserved between resistance genes N, RPS2, RPM1, PRF, L6 and I2C. PCR products were cloned and sequenced.

Results and conclusions
Inoculation tests showed that 120 interspecific hybrid lines out of 196 were infected with PVY during their vegetative propagation in the field. Similarly, 75 and 109 lines were infected with PVX and PVS, respectively, and one-third of the potato interspecific hybrid lines were infected with all three virus species. Forty-eight lines were not infected with each of all three virus species. It was expected that potato plants which were susceptible to virus should be infected with virus during their vegetative propagation in the field; therefore it was supposed that these 48 lines were resistance to three virus species. Furthermore, it appeared that most of them were derived from Solanum tuberosum ssp. andigena, S. chacoense, S. stoloniferum, S. phureja, S. vernei, and US breeding line 96-56 in these pedigrees. These results suggest that these cultivars and breeding lines can be used as a source of useful genes required for virus resistance. In order to detect and identify the genes for virus resistance, diploid interspecific hybrids were used. Each of these lines showed different susceptibility to three virus species. Genomic DNA prepared from diploid plants which were not infected with three virus species was used for PCR. Several PCR products were obtained. By cloning and sequencing these DNA fragments, sequence similarities were found to plant resistance genes. We are now investigating a correlation between these DNA fragments and virus resistance genes.