Sequence of the S9-RNase cDNA and PCR-RFLP system for discriminating S1- to S9-allele in Japanese pear

A new S9-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S9-RNase, a stylar product of S9-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S9-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S9-RNase had a hypervariable (HV) region different from S1- to S8-RNase and shared higher similarity (95.2%) with apple S3-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S1- to S9-amplicon (product), but could not discriminate the S2 from the S9 of ca. 1.3 kb. The S2 and S9 were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S1 to S9, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S1- to S9-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S4S9 were determined to be cross incompatible.