GENE IDENTIFICATION BASED ON CONSERVED HOMOLOGY WITH A MODEL PLANT SPECIES: CLONING THE S-LOCUS IN PRUNUS AVIUM

Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

As with other self-incompatible species of the Rosaceae, sweet cherry (Prunus avium L.) exhibits the monofactorial gametophytic self-incompatibility system, which is controlled by a single locus, the S-locus, with multiple alleles. The molecular mechanisms of the monofactorial gametophytic self-incompatibility have been extensively studied in the solanaceous plant species and S-gene products in pistils of Solanaceae are shown to be S-RNases. Recently, S-RNases have also been shown to be involved in the self-incompatibility of rosaceous plant species. We have identified S-RNases of sweet cherry and cloned cDNAs encoding them based on conserved homology with other plant species. Stylar proteins of sweet cherry were surveyed by 2D-PAGE. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other S-RNases. These glycoproteins were present at highest concentration in upper segment of the mature style and shared the similarity in immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding them were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences all indicated that the cDNAs encode S-RNases and thus the S-proteins identified by 2D-PAGE are S-RNases. Although S₁ to S₆-alleles of sweet cherry cultivars could be distinguished each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.