Molecular characterization of new S-RNases ('S31' and 'S32') in apple (Malus x domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphic S ribonuclease (S-RNase) for the pistil-part S determinant. Information about S-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the S-genotypes of 'Charden' (S2S3S4), 'Winesap' (S1S28), 'York Imperial' (S2S31), 'Stark Earliblazel' (S1S28), and 'Burgundy' (S20S32), by S-RNase sequencing and S-allele-specific PCR analysis. Two new S-RNases, S-31 and S-32, were also identified from 'York imperial' and 'Burgundy', respectively. These new S-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S-31 showed high similarity to S-20 (94%), S-32 exhibited 58% (to S-24) to 76% (to S-25) similarity in the exon regions. We designed new S-allele-specific primers for amplifying S-31- and S-32-RNase-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those new S-RNases. They should be useful, along with an S-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.