Isolation of S-locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotype

This study characterises a series of 12 S-locus haplotype-specific F-box protein genes (SFB) in cherry (Prunus avium) that are likely candidates for the pollen component of gametophytic self-incompatibility in this species. Primers were designed to amplify 12 SFB alleles, including the introns present in the 5′ untranslated region; sequences representing the S-alleles S1, S2, S3, S4, S4′, S5, S6, S7, S10, S12, S13 and S16 were cloned and characterized. [The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: PaSFB1 (AY805048), PaSFB2 (AY805049), PaSFB3 (AY805057), PaSFB4 (AY649872), PaSFB4′ (AY649873), PaSFB5 (AY805050), PaSFB6 (AY805051), PaSFB7 (AY805052), PaSFB10 (AY805053), PaSFB12 (AY805054), PaSFB13 (AY805055), PaSFB16 (AY805056).] Though the coding regions of six of these alleles have been reported previously, the intron sequence has previously been reported only for S6. Analysis of the introns revealed sequence and length polymorphisms. A novel, PCR-based method to genotype cultivars and wild accessions was developed which combines fluorescently labelled primers amplifying the intron of SFB with similar primers for the first intron of S-RNase alleles. Intron length polymorphisms were then ascertained using a semi-automated sequencer. The convenience and reliability of this method for the determination of the self-incompatibility (SI) genotype was demonstrated both in sweet cherry cultivars representing alleles S1 to S16 and in individuals from a wild population encompassing S-alleles S17 to S22. This method will greatly expedite SI characterisation in sweet cherry and also facilitate large-scale studies of self-incompatibility in wild cherry and other Prunus populations.