Isolation and identification of the pollen-expressed polymorphic F-box genes linked to the S-locus in apple (Malus × domestica)

Using degenerate primers designed based on the conserved regions of the reported SLF/SFB genes, more than a dozen of different cDNA clones were isolated from pollen of three different apple S-genotypes by reverse transcription (RT)-PCR. The 5′- and 3′-rapid amplification of cDNA end in combination with RT-PCR allowed isolation of two full-length cDNAs, whose corresponding genes (RJ28-2 and Mg15, later renamed SLF1 and SLF2) were specifically expressed in pollen in an S-haplotype-specific manner. PCR analysis of seven different cultivars further showed the linkage between SLF1 and S1-RNase and between SLF2 and S2-RNase. The predicted ORFs of the two genes encode two F-box proteins of 393 amino acids in length with 70% amino acid identity. These features are consistent with the supposition that SLF1 and SLF2 are good candidates for pollen S-genes. Phylogenetic trees for the SLF/SFB and S-RNase proteins reported in the Solanaceae, Scrophulariaceae and Rosaceae (including apple) were constructed. It was found that the SLFs and S-RNases from apple were more closely related to those from Petunia (a genus of the Solanaceae) and Antirrhinum (a genus of the Scrophulariaceae) than to those from Prunus (a genus of the Rosaceae), implying a potential co-evolution between SLF/SFB and S-RNase. Furthermore, six SLF-like genes that shared a high level of sequence similarity (amino acid identity from 68 to 72%) to SLFs were also isolated. Among them, RJ28, Jt24 and Jt24-4 were confirmed to be expressed specifically in pollen in an S-haplotype-unspecific manner. These results are discussed in relation to the possible evolution of the SLF/SFB in S-RNase-based self-incompatible species.