Although recent findings suggest that the F-box genes SFB/SLF control pollen-part S specificity in the S-RNase-based gametophytic self-incompatibility (GSI) system, how these genes operate in the system is unknown, and functional variation of pollen S genes in different species has been reported. Here, we analyzed the S locus of two species of Maloideae: apple (Malus domestica) and Japanese pear (Pyrus pyrifolia). The sequencing of a 317-kb region of the apple S 9 haplotype revealed two similar F-box genes. Homologous sequences were isolated from different haplotypes of apple and Japanese pear, and they were found to be polymorphic genes derived from the S locus. Since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes are specifically expressed in pollen, and variable regions of the SFBB genes are under positive selection. In a style-specific mutant S haplotype of Japanese pear, the SFBB genes are retained. Apart from their multiplicity, SFBB genes meet the expected characteristics of pollen S. The unique multiplicity of SFBB genes as the pollen S candidate is discussed in the context of mechanistic variation in the S-RNase-based GSI system.

SUMMARYThe S-RNase-based gametophytic self-incompatibility (SI) of Rosaceae, Solanaceae, and Plantaginaceae is controlled by at least two tightly linked genes located at the complex S locus; the highly polymorphic S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen. Self-incompatibility in Prunus (Rosaceae) is considered to represent a 'self recognition by a single factor' system, because loss-of-function of SFB is associated with self-compatibility, and allelic divergence of SFB is high and comparable to that of S-RNase. In contrast, Petunia (Solanaceae) exhibits 'non-self recognition by multiple factors'. However, the distribution of 'self recognition' and 'non-self recognition' SI systems in different taxa is not clear. In addition, in 'non-self recognition' systems, a loss-of-function phenotype of pollen S is unknown. Here we analyze the divergence of SFBB genes, the multiple pollen S candidates, of a rosaceous plant Japanese pear (Pyrus pyrifolia) and show that intrahaplotypic divergence is high and comparable to the allelic diversity of S-RNase while interhaplotypic divergence is very low. Next, we analyzed loss-of-function of the SFBB1 type gene. Genetic analysis showed that pollen with the mutant haplotype S 4sm lacking SFBB1-S 4 is rejected by pistils with an otherwise compatible S 1 while it is accepted by other non-self pistils. We found that the S 5 haplotype encodes a truncated SFBB1 protein, even though S 5 pollen is accepted normally by pistils with S 1 and other non-self haplotypes. These findings suggest that Japanese pear has a 'non-self recognition by multiple factors' SI system, although it is a species of Rosaceae to which Prunus also belongs.

Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a complex S locus that encodes separate proteins for pistil and pollen specificities, extracellular ribonucleases (S-RNases) and F-box proteins SFB/SLF, respectively. SFB/SLFs of Prunus (subfamily Prunoideae of Rosaceae), Solanaceae and Plantaginaceae are single copy in each S haplotype, while recently identified pollen S candidates SFBBs of subfamily Maloideae of Rosaceae, apple and Japanese pear, are multiple; two and three related SFBBs were isolated from each S haplotype of apple and Japanese pear, respectively. Here, we show that apple (Malus x domestica) SFBBs constitute a gene family that is much larger than initially thought. Twenty additional SFBB-like genes/alleles were isolated by screening of a BAC library derived from S (3) S (9) genotype, and tentatively named MdFBX1-20. All but one MdFBX showed S haplotype-specific polymorphisms. All the polymorphic MdFBXs were completely linked to S-RNase in 239 segregants. In addition, FISH revealed that the monomorphic gene MdFBX11 is also located near S-RNase, and the S locus is located in a subtelomeric region of a chromosome and is not close to the centromere. All MdFBXs were specifically expressed in pollen, except for a pseudogene MdFBX4 that showed no expression in any organs analyzed. Phylogenetic analysis revealed that the closest relatives of most MdFBXs were from a different S haplotype, suggesting that proliferation of MdSFBB/FBXs predates diversification of the S haplotypes.

The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.

Many species of Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI) in which pistil-part specificity is controlled by S locus-encoded ribonuclease (S-RNase). Although recent findings revealed that S locus-encoded F-box protein, SLF/SFB, determines pollen-part specificity, how these pistil- and pollen-part S locus products interact in vivo and elicit the SI reaction is largely unclear. Furthermore, genetic studies suggested that pollen S function can differ among species. In Solanaceae and the rosaceous subfamily Maloideae (e.g., apple and pear), the coexistence of two different pollen S alleles in a pollen breaks down SI of the pollen, a phenomenon known as competitive interaction. However, competitive interaction seems not to occur in the subfamily Prunoideae (e.g., cherry and almond) of Rosaceae. Furthermore, the effect of the deletion of pollen S seems to vary among taxa. This review focuses on the potential differences in pollen-part function between subfamilies of Rosaceae, Maloideae, and Prunoideae, and discusses implications for the mechanistic divergence of the S-RNase-based SI.

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The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum).

Many flowering plants exhibit self-incompatibility (SI) to prevent inbreeding and promote outcrossing. This self/non-self discrimination mechanism is controlled by the S locus, which contains separate genes for pistil and pollen specificities. In the gametophytic SI (GSI) of Rosaceae, Solanaceae and Plantaginaceae, the pistil S determinant, S-RNase, encodes extracellular ribonuclease which is thought to act as a cytotoxin to the self pollen tube, while the pollen S determinant is the F-box gene called SLF/SFB/SFBB. In Petunia (Solanaceae), SLF is reported to be a component of the noncanonical E3 ubiquitin ligase complex with S-RNase binding protein1 (SBP1) and Cullin1 (CUL1), and interact with non-self S-RNases to ubiquitinate them for degradation. Here, we isolated an apple (Malus×domestica) homolog of SBP1 (MdSBP1) from pollen RNA by RT-PCR. MdSBP1 included a RING-HC domain required for E3 ubiquitin ligase activity, and showed 64.0-68.2% amino acid identities with solanaceous SBP1 proteins. Expression analysis showed that MdSBP1 was expressed in all the organs analyzed. We detected an interaction between recombinant MdSBP1 protein and S-RNase of apple using a pull-down assay.Key words: Self-incompatibility, S-RNase binding protein, pollen, apple, Rosaceae.Self-incompatibility (SI) is a mechanism adopted by many flowering plants to prevent inbreeding and promote outcrossing. The S-RNase-based gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a single multiallelic S locus which contains separate genes for pistil and pollen specificities. When the S haplotype of a pollen matches one of the two S haplotypes of the diploid pistil, the pollen is recognized as self and rejected (de et al. 1986;McClure et al. 1989;Sassa et al. 1996Sassa et al. , 1997Tao et al. 1997;Xue et al. 1996) which is thought to be taken up by pollen tubes and act as a cytotoxin to self pollen (Goldraij et al. 2006;Luu et al. 2000). In Petunia of Solanaceae and species of Rosaceae tribe Pyreae, i.e., apples (Malus×domestica) and pears (Pyrus spp.), multiple F-box genes SLFs/SFBBs are implicated in pollen-part specificity (De Franceschi et al. 2011;Kubo et al. 2010;Kakui et al. 2011;Minamikawa et al. 2010;Saito et al. 2012;Sassa et al. 2007).SLF has been predicted to act as a component of the E3 ubiquitin ligase complex and interact with non-self S-RNases to ubiquitinate them for degradation (Huang et al. 2006;Qiao et al. 2004aQiao et al. , 2004bSijacic et al. 2004). Canonical E3 complex comprises Skp1, Cullin1, F-box protein and Rbx1 (Cardozo and Pagano 2004). In Petunia inflata, however, SLF-containing E3 ubiquitin ligase is reported to be a noncanonical SCF-like complex which includes S-RNase binding protein1 ( Abbreviations: GSI, Gametophytic self-incompatibility; RNase, ribonuclease; SBP1, S-RNase binding protein1; MBP, maltose binding protein.† These authors contributed equally to this work. This article can be found at