Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene

Abstract

Recently, an S haplotype-specific F-box (SFB) gene has been proposed as a candidate for the pollen-S specificity gene of RNase-mediated gametophytic self-incompatibility in Prunus (Rosaceae). We have examined two pollen-part mutant haplotypes of sweet cherry (Prunus avium). Both were found to retain the S-RNase, which determines stylar specificity, but one (S3' in JI 2434) has a deletion including the haplotype-specific SFB gene, and the other (S4' in JI 2420) has a frame-shift mutation of the haplotype-specific SFB gene, causing amino acid substitutions and premature termination of the protein. The loss or significant alteration of this highly polymorphic gene and the concomitant loss of pollen self-incompatibility function provides compelling evidence that the SFB gene encodes the pollen specificity component of self-incompatibility in Prunus. These loss-of-function mutations are inconsistent with SFB being the inactivator of non-self S-RNases and indicate the presence of a general inactivation mechanism, with SFB conferring specificity by protecting self S-RNases from inactivation.

Figure 1.

Segregation Analysis of S Alleles to Test Cosegregation of the JI 2434 Pollen-Part Mutation with the S₃ Allele. Genomic DNA of seedlings of Van (S₁S₃) × 9239-3 (S₁S₃′) was amplified by PCR using consensus primers revealing length polymorphism of the first intron of cherry S-RNases. Samples are as follows: ER, Early Rivers (S₁S₂); Victor (S₂S₃) (standards for S₁ and S₃, respectively); Van (S₁S₃); S₁S₃′ selection, 22 seedlings.

Figure 2.

DNA Gel Blot Analysis of the S₃′ and S₄′ Mutants Using an S₁-RNase cDNA Probe to Test for Rearrangements in the Regions Flanking the S₃- and S₄-RNase Genes, Respectively. (A) EcoRI digest of parents and pollen-part mutants. EF, Emperor Francis (S₃S₄); Nap, Napoleon (S₃S₄); 2420, JI 2420 (S₄S₄′); 2434, JI 2434 (S₃′S₄); Van (S₁S₃); ML, Merton Late (S₁S₄). (B) HindIII digest of parents and pollen-part mutants. EF, Emperor Francis (S₃S₄); Nap, Napoleon (S₃S₄); 2420, JI 2420 (S₄S₄′); 2434, JI 2434 (S₃′S₄); Van (S₁S₃); ML, Merton Late (S₁S₄). (C) EcoRI digest of the progeny Van (S₁S₃) × JI 2434 (S₃′S₄). Van (S₁S₃); 2434, JI 2434 (S₃′S₄); S₃S₃′ selection; S₃S₄ selection; S₁S₃′ selection; S₁S₄ selection; ML, Merton Late (S₁S₄). (D) SstI digest of parents and S₃′ mutant. EF, Emperor Francis (S₃S₄); Nap, Napoleon (S₃S₄); 2434, JI 2434 (S₃′S₄); S₃S₃′ selection; Van (S₁S₃); ML, Merton Late (S₁S₄). Low stringency post-hybridization washes allowed the detection of cross-hybridization of the S₁-RNase cDNA probe to both the S₃ and S₄ alleles.

Figure 3.

Alignment of the Predicted Amino Acid Sequence of the S₄-Haplotype–Specific SFB with Other Prunus SFB/SLF Sequences. Seven SFB sequences of Prunus (Pa, P. avium; Pd, P. dulcis; Pm, P. mume) aligned using ClustalW. Residues highlighted in gray denote divergence from the observed consensus, and dashes represent gaps. The F-box region and two variable regions Va and Vb (as indicated in Ushijima et al., 2003) are boxed.

Figure 4.

Segregation Analysis of the Putative S₄-Haplotype–Specific SFB Sequence to Test Cosegregation with the S₄-RNase Allele. PCR amplification with specific primers for the putative S₄-specific SFB sequence is shown for genomic DNA of parents and a representative sample of seedlings of the cross Bradbourne Black (S₃S₅) × Merton Late (S₁S₄): BB, Bradbourne Black; ML, Merton Late; 12 seedlings. Presence (+) or absence (−) of the S₄-RNase allele is indicated underneath the lanes. The bands amplified by the internal control primers included in each PCR are indicated with PAL.

Figure 5.

Genomic PCR Amplification of Parents and Pollen-Part Mutants with Specific Primers for the SFB and S-RNase Genes of the S₃- and S₄-Haplotypes. The primers used are specific for S₃-haplotype–specific SFB (A), S₃-RNase (B), S₄-haplotype–specific SFB (C), and S₄-RNase (D). Samples are as follows: EF, Emperor Francis (S₃S₄); Nap, Napoleon (S₃S₄); 2420, JI 2420 (S₄S₄′); S₄′S₄′ selection; 2434, JI 2434 (S₃′S₄); S₃S₃′ selection; S₃′S₃′ selection; Van (S₁S₃); ML, Merton Late (S₁S₄); water control. The bands amplified by the internal control primers that were included in each PCR are indicated with PAL.

Figure 6.

DNA Gel Blot Analysis of the S₃′ and S₄′ Mutants Using SFB Haplotype-Specific Probes. Genomic DNA was digested with DraI and probed with S₄-haplotype–specific SFB probe (A) and S₃-haplotype–specific SFB probe (B). The stringent washing conditions resulted in an allele-specific hybridization signal with the S₃-SFB probe; the S₄-SFB probe also cross-hybridized weakly to the S₁-SFB sequence. Samples are as follows: EF, Emperor Francis (S₃S₄); Nap, Napoleon (S₃S₄); 2420, JI 2420 (S₄S₄′); S₄′S₄′ selection; 2434, JI 2434 (S₃′S₄); S₃S₃′ selection; S₃′S₃′ selection; Van (S₁S₃); ML, Merton Late (S₁S₄).

Figure 7.

Intergenic PCR for the S₃-Haplotype Using S₃-RNase and S₃-SFB Specific Primers in Four Possible Combinations to Determine Distance between and Transcriptional Orientation of the Genes. The primers used on genomic DNA are S₃-RNase forward with S₃-haplotype–specific SFB forward (A), S₃-RNase forward with S₃-haplotype–specific SFB reverse (B), S₃-RNase reverse with S₃-haplotype–specific SFB forward (C), and S₃-RNase reverse with S₃-haplotype–specific SFB reverse (D). Samples are as follows: ER, Early Rivers (S₁S₂); Col, Colney (S₅S₆); EF, Emperor Francis (S₃S₄); Nap, Napoleon (S₃S₄); 2434, JI 2434 (S₃′S₄); S₃S₃′ selection; water control. Only combination (A) shows specific amplification for accessions with the S₃ allele.

Figure 8.

Preliminary Restriction Maps of the S₃- and S₃′-Haplotypes of Cherry of Napoleon (S₃S₄) and JI 2434 (S₃′S₄). Restriction enzyme positions and distances were inferred from restriction fragment size estimates on DNA gel blots (Napoleon [S₃S₄] and JI 2434 [S₃′S₄]) (Table 5), except for the regions of known sequence. Genes are represented by black boxes, with white boxes for introns. Arrows underneath gene boxes indicate the direction of transcription as deduced from the intergenic PCR (Figure 7). The breakpoint of the rearrangement in the S₃′-haplotype was deduced to be ∼3.5 kb downstream of the S₃-RNase; sequence downstream of the breakpoint (dotted line) is linked to S₃ in the progenitor. Restriction enzyme abbreviations are as follows: Ba, BamHI; E, EcoRI; K, KnpI; Nd, NdeI; Ps, PstI; Sc, ScaI; Sp, SspI; St, SstI.

Figure 9.

DNA Gel Blot Analysis of the S₃′ and S₄′ Mutants Using an SLFL2-Specific Probe. Genomic DNA was digested with SstI and probed with a cherry SLFL2 probe. Samples are as follows: EF, Emperor Francis (S₃S₄); Nap, Napoleon (S₃S₄); 2420, JI 2420 (S₄S₄′); S₄′S₄′ selection; 2434, JI 2434 (S₃′S₄); S₃S₃′ selection; S₃′S₃′ selection; Van (S₁S₃); ML, Merton Late (S₁S₄); Victor (S₂S₃); Vic (S₂S₄); ER, Early Rivers (S₁S₂); MH, Merton Heart (S₃S₆); MG, Merton Glory (S₄S₆); Mermat (S₁S₆).

Figure 10.

Alignment of Partial Genomic DNA Sequences and Deduced Amino Acid Sequences of the S₄- and S₄′-Haplotype–Specific SFB Genes. Nucleotide (nt) and predicted amino acid (aa) sequences representing P. avium SFB sequences for S₄ and S₄′. Putative intron sequence within the 5′ untranslated region is presented in lower case and indicated by asterisks. Twelve amino acids are missing at the 3′ end of the S₄ sequence. Residues highlighted in gray denote divergence from the original S₄-SFB sequence. The F-box region and the two variable regions Va and Vb as identified by Ushijima et al. (2003) are boxed. A 4-bp deletion in the S₄′-SFB sequence at position 742 to 745 in the first variable region leads to the deletion of a single Tyr residue (#) and a subsequent frame shift in translation. Premature stop codons present in the S₄′-SFB sequence are highlighted in black.