Determining the S-genotypes of several sweet cherry cultivars based on PCR-RFLP analysis

Based on the cDNA sequences encoding sweet cherry self-incompatibility asso ciated ribonucleases (S-RNases), a PCR-based S-allele typing system for swe et cherry cultivars has been recently developed. Using this technique, we d etermined S-genotypes of the three newly released Japanese cvs Kouka-Nishik i, Beni-Sayaka and Beni-Shuho and one British cv Merton Glory that was clas sified as a Universal Donor, which is able to be used as a pollen donor for all cultivars in pollen incompatibility groups I to XIII. Furthermore, we also determined the partial sequences of the S-RNase genes of 'Rainier' ((S S4)-S-1)' and 'Sato-Nishiki ((SS6)-S-3)', which leads to the development of a more reliable S-allele identification method of PCR-RFLP for sweet cherr y cultivars. Total DNA isolated from leaves of the four cultivars along wit h those from ten cultivars with known S-genotypes were PCR amplified with t wo sets of primers that were designed from DNA sequences encoding the signa l peptide (Pru-T2) and two conserved domains (Pru-C2 and Pru-C4R) of sweet cherry S-RNases. By comparing the size of PCR products on agarose gel, the S-genotypes of 'Kouka-Nishiki', 'Beni-Sayaka', 'Beni-Shuho' and 'Merton Glo ry' were suggested to be (SS3)-S-1, (SS6)-S-1 (SS6)-S-4, and (SS6)-S-4, res pectively. Two of these three S-genotypes ((SS6)-S-1 and (SS6)-S-4) were fo und for the first time. DNA sequencing of PCR products from S-alleles of 'R ainier' and 'Sato-Nishiki' revealed that Ban II, Nru I, Apa LI and Ava I si tes, respectively, were unique in the S-1-, S-3-, S-4- and S-6- sequences f lanked by Pru-T2 and PN-C4R primers. RFLP analysis of the PCR products usin g these enzymes confirmed that S-1-, S-3-, S-4- and S-6-alleles of the four cultivars contained the respective restriction enzyme recognition sites.