No abstract

SummaryMany Prunus species, including sweet cherry and Japanese apricot, of the Rosaceae, display an S-RNase-based gametophytic self-incompatibility (GSI). The specificity of this outcrossing mechanism is determined by a minimum of two genes that are located in a multigene complex, termed the S locus, which controls the pistil and pollen specificities. SFB, a gene located in the S locus region, encodes an F-box protein that has appropriate S haplotype-specific variation to be the pollen determinant in the self-incompatibility reaction. This study characterizes SFBs of two self-compatible (SC) haplotypes, S 4¢ and S f , of Prunus. S 4¢ of sweet cherry is a pollen-part mutant (PPM) that was produced by X-ray irradiation, while S f of Japanese apricot is a naturally occurring SC haplotype that is considered to be a PPM. DNA sequence analysis revealed defects in both SFB 4¢and SFB f . A 4 bp deletion upstream from the HVa coding region of SFB 4¢ causes a frame-shift that produces transcripts of a defective SFB lacking the two hypervariable regions, HVa and HVb. Similarly, the presence of a 6.8 kbp insertion in the middle of the SFB f coding region leads to transcripts for a defective SFB lacking the C-terminal half that contains HVa and HVb. As all reported SFBs of functional S haplotypes encode intact SFB, the fact that the partial loss-of-function mutations in SFB are present in SC mutant haplotypes of Prunus provides additional evidence that SFB is the pollen S gene in GSI in Prunus.

The transition from self-incompatibility (SI) to self-compatibility (SC) is regarded as one of the most prevalent transitions in Angiosperm evolution, having profound impacts on the genetic structure of populations. Yet, the identity and function of mutations that result in the breakdown of SI in nature are not well understood. This work provides the first detailed genetic description of the breakdown of S-RNase-mediated gametophytic self-incompatibility (GSI) in a polyploid species that exhibits genotypedependent loss of SI. Genetic analyses of six natural sour cherry (Rosaceae, Prunus cerasus) selections identified seven independent, nonfunctional S-haplotypes with disrupted pistil component (stylar-S) and/ or pollen component (pollen-S) function. A genetic model demonstrating that the breakdown of SI in sour cherry is due to the accumulation of a minimum of two nonfunctional S-haplotypes within a single individual is developed and validated. Our finding that sour cherry is SI when only one nonfunctional S-haplotype is present has significant evolutionary implications since nonfunctional S-haplotypes would be maintained in the population without causing an abrupt shift to SC. Furthermore, we demonstrate that heteroallelic sour cherry pollen is self-incompatible, which is counter to the well-documented phenomenon in the Solanaceae where SC accompanying polyploidization is frequently due to the SC of heteroallelic pollen.

The gene SFB encodes an F-box protein that has appropriate S-haplotype-specific variation to be the pollen determinant in the S-RNase-based gametophytic self-incompatibility (GSI) reaction in Prunus (Rosaceae). To further characterize Prunus SFB, we cloned and sequenced four additional alleles from sweet cherry (P. avium), SFB 1 , SFB 2 , SFB 4 , and SFB 5 . These four alleles showed haplotype-specific sequence diversity similar to the other nine SFB alleles that have been cloned. In an amino acid alignment of Prunus SFBs, including the four newly cloned alleles, 121 out of the 384 sites were conserved and an additional 65 sites had only conservative replacements. Amino acid identity among the SFBs ranged from 66.0% to 82.5%. Based on normed variability indices (NVI), 34 of the non-conserved sites were considered to be highly variable. Most of the variable sites were located at the C-terminal region. A windowaveraged plot of NVI indicated that there were two variable and two hypervariable regions. These variable and hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the GSI reaction. Evidence of positive selection was detected using maximum likelihood methods with sites under positive selection concentrated in the variable and hypervariable regions.

Tetraploid sour cherry (Prunus cerasus) exhibits a genotype-dependent loss of gametophytic self-incompatibility that is caused by the accumulation of non-functional S-haplotypes with disrupted pistil component (stylar-S) and/or pollen component (pollen-S) function. Genetic studies using diverse sour cherry germplasm identified non-functional S-haplotypes for which an equivalent wild-type S-haplotype was present in sweet cherry (Prunus avium), a diploid progenitor of sour cherry. In all cases, the non-functional S-haplotype resulted from mutations affecting the stylar component S-RNase or Prunus pollen component S-haplotype-specific F-box protein (SFB). This study determines the molecular bases of three of these S-haplotypes that confer unilateral incompatibility, two stylar-part mutants (S(6m2) and S(13m)) and one pollen-part mutant (S(13)'). Compared to their wild-type alleles, S(6m2)-RNase has a 1 bp deletion, S(13m) -RNase has a 23 bp deletion and SFB(13)' has a 1 bp substitution that lead to premature stop codons. Transcripts were identified for these three alleles, S(6m2)-RNase, S(13m)-RNase, and SFB(13)', however, these transcripts presumably result in altered proteins with a resulting loss of activity. Our characterization of natural pollen-part and stylar-part mutants in sour cherry along with other natural S-haplotype mutants identified in Prunus supports the view that loss of pollen specificity and stylar rejection evolve independently and are caused by structural alterations affecting the S-haplotype. The prevalence of non-functional S-haplotypes in sour cherry but not in sweet cherry (a diploid) suggests that polyploidization and gene duplication were indirectly responsible for the dysfunction of some S-haplotypes and the emergence of self-compatibility in sour cherry. This resembles the specific mode of evolution in yeast where accelerated evolution occurred to one member of the duplicated gene pair.