Cloning of the PVY resistance gene derived from S. stoloniferum.

To clone Rysto gene, we developed a diploid potato (2n = 2x = 24) mapping population by a cross between PVY-resistant heterozygous dihaploid clone dH Alicja, and susceptible clone 83-3121. The extreme resistance (ER) to PVY in dH Alicja was conferred by the gene Rysto which was derived from clone MPI 55.957/54. MPI 55.957/54 had in its pedigree Solanum stoloniferum from wild Solanum spp. collection of Max Planck Institute (Ross H. (1958) Handbuch der Pflanzenzüchtung, 2nd, Vol. III:106-125). The mapping population consisted of 391 F1 individuals; the segregation ratio of resistant versus susceptible progeny in the mapping population deviated from the 1:1 ratio expected for the segregation of a single dominant gene and was distorted towards resistance (149 susceptible and 242 PVY-resistant F1 individuals). We hypothesized that the underlying PVY resistance genes encode a nucleotide binding-leucine rich repeat (NLR) protein. To predict candidate gene(s) for Rysto, we applied RenSeq combined with Illumina MiSeq 250 PE sequencing to 149 bulked susceptible plants from mapping population (BS), alongside with susceptible (S) and resistant (R) parent. To generate high confidence de novo assembly of R-parent NLRs, we also carried out SMRT RenSeq on gDNA derived from the resistant clone dH Alicja. Additionally we performed cDNA RenSeq to detect expressed NLRs.