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To elucidate the function of S-allele and the possible molecular mechanism of gametophytic self-incompatibility (GSI) in Chinese white pear (Pyrus bretschneideri Rehd.), full length cDNA encoding S20-RNase was cloned by rapid amplification of cDNA ends (RACE) approach from the cultivar 'Donghuang' (S8S20), and analyzed by bioinformatics method. S20-RNase displayed typical sequence features of pear S20-RNases, i.e. five conserved regions and a hypervariable (HV) region. The predicted three-dimension (3D) structure of the S20-RNase was similar to that of 53-RNase of Japanese pear (P. pyrifolia), indicating that the S20-RNase gene behaves as RNase rejecting pollen tube growth with the same S-haplotype as S3-RNase does in GSI response. At the amino acid level, S20-RNase showed 31.5% to 90.8% similarities with other rosaceous S-RNases. Phylogenetic analysis revealed that rosaceous S-RNases occurred before divergence of species, but after divergence of subfamilies Maloideae and Amygdaloideae.