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

A new S-9-allele was discovered in 6 Japanese pear cultivars, 'Shinkou', 'Shinsei', 'Niitaka', 'Amanogawa', 'Nangetsu' and 'Nansui'. cDNA encoding S-9-RNase, a stylar product of S-9-allele, was cloned from pistils of 'Shinkou' and 'Shinsei' by 3' and 5' RACE. The S-9-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S-9-RNase had a hypervariable (HV) region different from S-1- to S-8-RNase and shared higher similarity (95.2%) with apple S-3-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 S-1- to S-9-amplicon (product), but could not discriminate the S-2 from the S-9 of ca. 1.3 kb. The S-2 and S-9 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 S-1 to S-9, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S-1- to S-9-allele. 'Shinkou', 'Shinsei', 'Nangetsu' and 'Nansui' assigned as S4S9 were determined to be cross incompatible.