Establishment of identification method for self-incompatibility genotype in Japanese pears

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Stylar proteins associated with self-incompatibility alleles, S_1 to S_5, were identified by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE), and self-incompatibility genotypes of several cultivars including new ones were established by using the IEF-PAGE technique. The established S-genotypes were confirmed by pollination experiments in the orchard. Present study also suggests the usefulness of Concanavalin-A (Con-A) staining of S-protein bands for predicting S-genotypes of cultivars, because each S-protein was proved to be glycoprotein. The trial to make mono-clonal antibodies of respective S-proteins resulted in failure by the following reasons ; 1)two S-proteins in the cultivar were contaminated each other because of their very close pI points, 2)the recovery was trace level when each protein was purified.The S_4-gene was amplified by PCR techniques using 5'TTTACGCAGCAATATCAG3' and 5'G(C/T)GGGGGCA(A/G)T(T/C)TATGAA3'as primers. However, it was not amplified when genomic DNAs from young leaves of 'Shinsui'(S_4S_5) and 'Seigyoku'(S_3S_4) were used as templates. Therefore, probalble probe for S_4-gene could not be established in this study. The cDNAs were synthesized from stylar mRNA or rRNA by RT-PCR techniques, and PCR was conducted using the cDNAs as templates to select each S-gene. We could detect several candidates for each S-gene using RAPD primer, but probes for respective genes were not established yet.