Duplication of the S-locus F-box gene is associated with breakdown of pollen function in an S-haplotype identified in a natural population of self-incompatible Petunia axillaris

We previously identified both self-incompatible and self-compatible plants in a natural population of self-incompatible Petunia axillaris subsp. axillaris, and found that all the self-compatible plants studied carried either S_C1- or S_C2-haplotype. Genetic crosses showed that S_C2 was identical to S₁₇ identified from another natural population of P. axillaris, except that its pollen function was defective, and that the pollen-part mutation in S_C2 was tightly linked to the S-locus. Recent identification of the S-locus F-box gene (SLF) as the gene that controls pollen specificity in S-RNase-based self-incompatibility has prompted us to examine the molecular basis of this pollen-part mutation. We cloned and sequenced the S₁₇-allele of SLF of P.axillaris, named PaSLF₁₇, and found that S_C2 S_C2 plants contained extra restriction fragments that hybridized to PaSLF₁₇ in addition to all of those observed in S₁₇ S₁₇ plants. Moreover, these additional fragments co-segregated with S_C2. We used the S_C2-specific restriction fragments as templates to clone an allele of PaSLF by PCR. To determine the identity of this allele, named PaSLF_x, primers based on its sequence were used to amplify PaSLFalleles from genomic DNA of 40 S-homozygotes of P. axillaris, S₁ S₁ through S₄₀ S₄₀. Sequence comparison revealed that PaSLF_x was completely identical with PaSLF₁₉ obtained from S₁₉ S₁₉. We conclude that the S-locus of S_C2 contained both S₁₇-allele and the duplicated S₁₉-allele of PaSLF. S_C2 is the first naturally occurring pollen-part mutation of a solanaceous species that was shown to be associated with duplication of the pollen S. This finding lends support to the proposal, based on studies of irradiation-generated pollen-part mutants of solanaceous species, that duplication, but not deletion, of the pollen S, causes breakdown of pollen function.