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 S-1, S-2 , S-3, S-4, S-4', S-5, S-6, S-7, S-10, S-12, S-13 and S-16 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 S (6) . 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 S-1 to S-16 and in individuals from a wild population encompassing S-alleles S-17 to S-22 . 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.