Hiroyuki Kakui

S Locus F-Box Brothers: Multiple and Pollen-Specific F-Box Genes With S Haplotype-Specific Polymorphisms in Apple and Japanese Pear

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 S9 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.

Pollen-expressed F-box gene family and mechanism of S-RNase-based gametophytic self-incompatibility (GSI) in Rosaceae.

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.

Polymorphism of SFBB −γ and its use for S genotyping in Japanese pear (Pyrus pyrifolia)

Japanese pear (Pyrus pyrifolia) exhibits the S-RNase-based gametophytic self-incompatibility where the pollen-part determinant, pollen S, had long remained elusive. Recent identification of S locus F-box brothers (SFBB) in Japanese pear and apple suggested that the multiple F-box genes are the pollen S candidates as they exhibited pollen specific expression, S haplotype-specific polymorphisms and linkage to the S locus. In Japanese pear, three SFBBs were identified from a single S haplotype, and they were more homologous to other haplotype genes of the same group (i.e., α-, β- and γ-groups). In this study, we isolated new seven PpSFBB−γ genes from different S genotypes of Japanese pear. These genes showed S haplotype-specific polymorphisms, however, sequence similarities among them were very high. Based on the sequence polymorphisms of the PpSFBB−γ genes, we developed a CAPS/dCAPS system for S genotyping of the Japanese pear cultivars. This new S genotyping system was found to not only be able to discriminate the S1–S9, but also be suitable for identification of the mutant S4sm haplotype for the breeding of self-compatible cultivars, and detection of new S haplotypes such as Sk.

Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.).

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.

Interhaplotypic heterogeneity and heterochromatic features may contribute to recombination suppression at the S locus in apple (Malus×domestica)

Gametophytic self-incompatibility (GSI) is controlled by a complex S locus containing the pistil determinant S-RNase and pollen determinant SFB/SLF. Tight linkage of the pistil and pollen determinants is necessary to guarantee the self-incompatibility (SI) function. However, multiple probable pollen determinants of apple and Japanese pear, SFBBs (S locus F-box brothers), exist in each S haplotype, and how these multiple genes maintain the SI function remains unclear. It is shown here by high-resolution fluorescence in situ hybridization (FISH) that SFBB genes of the apple S 9 haplotype are physically linked to the S 9 -RNase gene, and the S locus is located in the subtelomeric region. FISH analyses also determined the relative order of SFBB genes and S-RNase in the S 9 haplotype, and showed that gene order differs between the S 9 and S 3 haplotypes. Furthermore, it is shown that the apple S locus is located in a knob-like large heterochromatin block where DNA is highly methylated. It is proposed that interhaplotypic heterogeneity and the heterochromatic nature of the S locus help to suppress recombination at the S locus in apple.