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Petuniais controlled by the polymorphic S‐locus, which contains S‐RNaseencoding the pistil determinant and 16–20 S‐locus F‐box( SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2‐haplotype of the S‐locus in Petunia inflatausing bacterial artificial chromosome clones collectively containing all 17 SLFgenes, SLFLike1, and S‐RNase. Two SLFpseudogenes and 28 potential protein‐coding genes were identified, 20 of which were also found at the S‐loci of both the S6a‐haplotype of P. inflataand the SN‐haplotype of self‐compatible Petunia axillaris, but not in the S‐locus remnants of self‐compatible potato ( Solanum tuberosum) and tomato ( Solanum lycopersicum). Comparative analyses of S‐locus sequences of these three S‐haplotypes revealed potential genetic exchange in the flanking regions of SLFgenes, resulting in highly similar flanking regions between different types of SLFand between alleles of the same type of SLFof different S‐haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S‐loci of all three S‐haplotypes and in the flanking regions of all S‐locus genes examined. We also found evidence of the association of transposable elements with SLFpseudogenes. Based on the hypothesis that SLFgenes were derived by retrotransposition, we identified 10 F‐boxgenes as putative SLFparent genes. Our results shed light on the importance of non‐coding sequences in the evolution of the S‐locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLFgenes.
The collaborative non‐self‐recognition model for S‐
RNase‐based self‐incompatibility predicts that multiple S‐locus F‐box proteins ( SLFs) produced by pollen of a given S‐haplotype collectively mediate ubiquitination and degradation of all non‐self S‐ RNases, but not self S‐ RNases, in the pollen tube, thereby resulting in cross‐compatible pollination but self‐incompatible pollination. We had previously used pollen extracts containing GFP‐fused S2‐ SLF1 ( SLF1 with an S2‐haplotype) of Petunia inflatafor co‐immunoprecipitation (Co‐ IP) and mass spectrometry ( MS), and identified Pi CUL1‐P (a pollen‐specific Cullin1), Pi SSK1 (a pollen‐specific Skp1‐like protein) and Pi RBX1 (a conventional Rbx1) as components of the SCFS2– SLF1complex. Using pollen extracts containing Pi SSK1: FLAG: GFPfor Co‐ IP/ MS, we identified two additional SLFs ( SLF4 and SLF13) that were assembled into SCFSLFcomplexes. As 17 genes ( SLF to SLF1 ) have been identified in SLF17 S2and S3pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co‐ IP/ MSprocedure, including the addition of style extracts from four different S‐genotypes to pollen extracts containing Pi SSK1: FLAG: GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF‐like protein, SLFLike1 (encoded by an S‐locus‐linked gene), co‐immunoprecipitated with Pi SSK1: FLAG: GFP. Moreover, of the 179 other F‐box proteins predicted by S2and S3pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co‐immunoprecipitated with Pi SSK1: FLAG: GFP. These results suggest that SCFSLFcomplexes have evolved specifically to function in self‐incompatibility.