Interhaplotypic heterogeneity and heterochromatic features may contribute to recombination suppression at the S locus in apple (Malusxdomestica).

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 S9 haplotype are physically linked to the S9-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 S9 haplotype, and showed that gene order differs between the S9 and S3 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.

Introduction

Self-incompatibility (SI) is a mechanism to prevent self-fertilization and to promote outcrossing in plants (Franklin-Tong and Franklin, 2003). S-RNase-based gametophytic self-incompatibility (GSI) is the most widespread SI system, and has been found in the families Solanaceae, Rosaceae, and Plantaginaceae. In GSI systems, the S haplotype contains at least two tightly linked S-determinant genes, pistil S and pollen S. The pistil S gene encodes an extracellular ribonuclease called S-RNase, which is thought to act as a cytotoxin to inhibit growth of the self pollen tube via degradation of pollen RNAs (Anderson ; McClure et al., 1989, 1990; Lee et al., 1994; Murfett ; Broothaerts et al., 1995; Xue ; Sassa , 1997). For pollen S, F-box genes, SLF (S locus F-box)/SFB 
(S haplotype-specific F-box), were identified as candidates (Lai et al., 2002; Entani ; Ushijima , 2004; Yamane ; Sijacic et al., 2004; Sonneveld et al., 2005; Hauck ). Recently, in Petunia (Solanaceae), a ‘collaborative non-self recognition model’ was proposed where at least three related SLF genes in an S haplotype encode pollen S determinants, and these factors work together to suppress non-self S-RNases (Kubo ). Multiple F-box genes have also been identified as pollen S candidates in Japanese pear and apple of the tribe Pyreae (Rosaceae) (Sassa ; Minamikawa et al., 2010; Kakui ). Minamikawa et al. (2010) reported that the S locus of apple contains more than ten SFBB genes. Recent findings on the SFBBs of Japanese pear suggested that GSI in Pyreae is, like in petunia, a ‘non-self recognition by multiple factors’ system in which numerous SFBB genes in an S haplotype are components of the pollen determinant, and each SFBB targets a subset of non-self S-RNases (Kakui et al., 2011).

Tight linkage of the pollen S and pistil S genes is necessary for GSI to function. Recombination at the S locus would cause the breakdown of SI by generating differences in pollen and pistil specificity within a single S haplotype (Sijacic et al., 2004). As a result of recombination at the S locus, otherwise incompatible ‘self’ pollen would exhibit specificity different from the pistil and be accepted by the pistil. The centromeric localization of the S locus of the Solanaceae is considered important to suppress recombination at the locus and to maintain the SI system (Brewbaker and Natarajan, 1960; Pandy, 1965; Bernatzky, 1993; Bernacchi and Tanksley, 1997; ten Hoopen et al., 1998; Entani et al., 1999; Golz et al., 2001). Although the S locus is located at the distal end of the chromosome in Antirrhinum and Ipomoea, heterochromatinization at the locus is assumed to have epigenetic roles in the suppression of recombination (Suzuki et al., 2004; Yang et al., 2007). The S locus in apple was found to be located in the subtelomeric region by fluorescence in situ hybridization (FISH) (Minamikawa et al., 2010). However, the order and precise chromosomal location of the multiple SFBB genes remain unclear. In this study, a BAC-FISH analysis was conducted on the apple pachytene chromosome to determine the precise location and relative order of SFBB and S-RNase genes. The relative order of these genes differed between the S and S haplotypes, indicating structural heterogeneity at the S locus among S haplotypes. In addition, the BAC-FISH and immunostaining of 5-methylcytosine (5mC) revealed that the apple S locus shows heterochromatinization and DNA methylation compared with surrounding regions. The interhaplotypic heterogeneity and heterochromatic and methylated nature of the apple S locus may contribute to the recombination suppression and maintenance of the SI function.

Materials and methods

Plant materials

Immature flower buds of Malus × domestica ‘Florina’ (S S ) were collected from the Nagano Fruit Tree Experiment Station (Nagano prefecture, Japan) and fixed in Farmer’s solution [3:1(v/v) ethanol:acetic acid] for more than 24h. The fixed materials were transferred to 70% ethanol and stored at 4 ºC.

FISH

Pachytene chromosomes were prepared using squashes of anthers from the fixed flower buds. Chromosome slides were prepared following established protocols (Minamikawa ). Interphase nuclei were also prepared from fixed anthers to check the specificity of BAC probes.

BAC DNA was extracted by a standard alkaline lysis method and purified using PEG 8000 (Minamikawa et al., 2010). DNA probes were labelled by nick translation using the DIG Nick Translation Mix (Roche) or Biotin Nick Translation Mix (Roche). For triple-colour FISH, the third probe was labelled with a combination of 50% DIG-11-dUTP and 50% Biotin-16-dUTP, which would result in the detection of the intermediate.

Pretreatment, post-fixation, and FISH were conducted according to the procedures of Minamikawa et al. (2010) with some modifications. Briefly, the slides were incubated in 100ng µl–1 of RNase at 37 °C for 60min followed by 5ng µl–1 of pepsin at 37 °C for 20min, and post-fixed in 3:1 (v/v) ethanol:acetic acid for 10min. Ten microlitres of hybridization solution (50% formamide, 10% dextran sulphate, 2× SSC, 20ng of each probe, and 3 µg of apple genomic blocking DNA) was applied to each slide. Chromosomal DNA and probe DNA were denatured for 5min at 80 °C. The slide was incubated at 37 °C for 2 d. After washing in distilled water, 125 µl of the 1st antibody cocktail [1% BSA, 4× SSC, 1 µg of FITC streptavidin conjugates (Molecular Probes), and 0.1 µg of mouse anti-digoxigenin (Roche)] was applied for 30min at 37 °C. After washing, 125 µl of the 2nd antibody cocktail [1% BSA, 4× SSC, and 0.5 µg of biotinylated anti-streptavidin (Vector Laboratories)] was then applied and incubated. Finally, after washing, 125 µl of the 3rd antibody cocktail [1% BSA, 4× SSC, 1 µg of FITC streptavidin conjugates (Molecular Probes), and 1 µg of Alexa fluor 568-conjugated rabbit anti-mouse IgG (H+L) (Molecular Probes)] was applied. Chromosomes were counterstained with VECTASHIELD (Vector Laboratories) containing 5 µg ml–1 of 4, 6 diamidino-2- phenylindole (DAPI).

FISH signals were captured with a Leica DM RXA2 fluorescence microscope equipped with a CCD camera (Leica DC350F) and processed by Leica CW4000 and Adobe Photoshop v5.1. At least ten FISH images were analysed to determine the location on the chromosomes.

Immunodetection of 5mC

The chromosome slides for the immunofluorescence assay of 5mC were prepared according to the above FISH protocol. To detect 5mC, the slides were denatured in 70% formamide in 2× SSC, at 80 °C for 2min, and then dehydrated in a series of cold (–20 °C) ethanol solutions (70%, 95%, and 100%) for 5min each. 100 µl of blocking solution (1% BSA, 1× PBS) was dropped onto each slide and the slides were then incubated at 37 °C for 30min in a wet chamber. After washes in 1× PBS, the slides were incubated with 125 µl of blocking solution containing mouse monoclonal antibody against 5-methylcytosine (1:500; Eurogentec) overnight at 4 °C. The mouse antibodies were detected using Alexa Fluor 568 rabbit anti-mouse IgG (1:1000; Invitrogen). Chromosomes were counterstained with 5 µg ml–1 of DAPI in VECTASHIELD.

For the dual detection of 5mC and FISH signals, the slides were first processed for the detection of 5mC, dehydrated in an ethanol series, and air-dried. FISH was then conducted as mentioned above.

Results

SFBB-S genes, which show genetic linkage to S -RNase, cluster in the small subtelomeric and haplotype-specific region of an apple chromosome

Previous genetic analysis showed that SFBB-S genes are closely linked to S -RNase (Minamikawa et al., 2010). To verify whether they are physically linked to S -RNase, BAC-FISH was conducted on pachytene chromosomes of ‘Florina’ (S S ) with S -haplotype BAC clones as probes. Out of 27 S -haplotype BAC clones which constructed seven contigs (Minamikawa et al., 2010), 12 BAC clones covering all the contigs which contain S -RNase and SFBB-S genes were used in this study (Table 1). All 12 BACs produced distinct signals on pachytene chromosomes and the signals completely colocalized to the subtelomeric region with S -RNase (Fig. 1). Although BAC 9P16, BAC 69A4, and BAC90A15 also gave signals in subtelomeric regions of other chromosomes probably because of repetitive sequences, simultaneous detection of the three BACs provided distinct locations and the order of S -RNase and SFBB-S genes on pachytene chromosome as mentioned below. The subtelomeric locations of FISH signals are consistent with genetic linkage analyses mapping the apple S locus to the bottom of linkage group 17 (Igarashi et al., 2008).

Table 1. The S -haplotype BAC clones used

Contig	BAC	Gene(s) c
1	45M19 b	SFBB5-S 9 (SFBB 9-α )
	78B14	SFBB5-S 9 (SFBB 9-α ),S 9 -RNase
	34G16	S 9 -RNase
	96N6	S 9 -RNase, SFBB7-S 9 (SFBB 9-β)
	14P21
	90A15	SFBB7-S 9 (SFBB 9-β )
2	72L2
	52B7
	25J12	SFBB3-S 9 (FBX17), FBX4
3	24C22
	21O2	SFBB6-S 9 (FBX15)
4	9P16	FBX12
	21M5	FBX12, SFBB1-S 9 (FBX16)
	10D16	FBX6, SFBB1-S 9 (FBX16)
	27P17	FBX6, SFBB1-S 9 (FBX16)
	6N9	FBX6
	42J3	FBX19
	70J19	FBX6,19
	69A4	FBX19
5	9J18	SFBB8-S 9 (FBX10)
	22C6
	68N5
6	33E2	FBX13
	89I13
	92P4
	20J13
7 a	9L6	FBX11

FBX11is a monomorphic gene and exists in both the S - and 
S -haplotypes (Minamikawa ).

BAC clones and genes used in this study are indicated in bold.

New naming system for SFBB genes was adopted, i.e. SFBBx-S for the type-x SFBB from the S haplotype (Kakui et al., 2011). Previous gene names (Minamikawa et al., 2010) are in parentheses. Genes that were not classified into the eight SFBB types (Kakui et al., 2011) are shown with their old names (Minamikawa et al., 2010).

Fig. 1.

FISH mapping of BACs containing S-RNase and SFBB genes on pachytene chromosomes of apple. BAC names (red, green, and yellow) are indicated for each signal (arrowheads). Yellow signals are a mix of red and green signals. Bar = 10 µm. (A, B, C, D) Rough mapping of contigs 1–5 and 7. Signals of BACs 25J12 (contig 2 including SFBB3-S ), 21O2 (contig 3 including SFBB6-S ), and 69A4 (contig 4 including FBX19) were located between signals of 45M19 (contig 1 including SFBB5-S ) and 9L6 (contig 7 including FBX11). The signal of 45M19 was located between that of 22C6 (contig 5 including SFBB8-S ) and 9L6. (E) 25J12 (contig 2 including SFBB3-S ) was mapped between 33E2 (contig 6 including FBX13) and 9L6. (F) Localization of contig 4 (69A4) between contigs 7 (9L6) and 5 (22C6) (Fig. 1C, D) was confirmed by using another contig 5 probe 9J18. (G) Relative order of 22C6, 45M19, and 33E2 was determined. The results (E, G) show the following order; 22C6 (contig 5), 45M19 (contig 1), 33E2 (contig 6), 25J12 (contig 2), and 9L6 (contig 7). (H, I, J) The relative order of contigs 2, 3, 4, and 6 that were localized between contigs 1 (45M19) and 7 (9L6). The order was determined to be as follows; contig 1 (45M19), contig 6 (89I3)/contig 3 (21O2), contig 4 (9P16), contig 2 (25J12), and contig 7 (9L6). Relative order of contigs 3 and 6 were not determined. (K, L) Determination of the relative order of SFBBs and S -RNase included in contigs 1 and 4. Two contig 1 BACs 45M19, which includes SFBB5-S , and 90A15, which includes SFBB7-S (Sassa ; Table 1), and 9L6 of contig 7 were localized by FISH to determine the relative order of SFBB genes in contig 1 (K). Two contig 4 BACs 9P16, which includes FBX12, and 69A4, which includes FBX19 (Minamikawa et al., 2010; Table 1), and 45M19 of contig 1 were localized by FISH to determine the relative order of SFBB genes included in contig 4 (L). The results are summarized in Fig. 2.

Fig. 2.

Gene order of the S haplotype and its comparison to the S haplotype. S haplotype genes (left) were placed based on the BAC-FISH signals in Fig. 1. Previous gene names are in parentheses (Minamikawa ). Numbers in black boxes denotes contigs of the S haplotype. Solid lines indicates pairs of SFBBs with high homology (>90% amino acid identity; Minamikawa ). The dashed line denotes alleles of S-RNase. The order of S haplotype contigs (bars) was not determined (Minamikawa ). Bars in the S haplotype (right) are not to scale.

Although interhaplotypic homology of the same types of SFBB genes is generally very high (Minamikawa et al., 2010; Kakui ), each of the 12 BAC probes detected single signal on a chromosome except for probable repetitive sequence-derived signals for some BACs (Fig. 1, see above). For some BACs, S -specificities were further confirmed by FISH on nuclei and chromosomes of somatic cells (see Supplementary Figs S1 and S2 at JXB online). Lack of cross-hybridization of S -BAC probes on the S chromosome may be because the flanking regions of the SFBB genes are rich in S-haplotype-specific sequences.

High-resolution mapping and the relative order of S -RNase and SFBB-S genes on pachytene chromosome

FISH mapping on pachytene chromosomes is a powerful approach for high-resolution mapping because pachytene chromosomes are, on average, 10–50 times longer than somatic chromosomes (de Jong ; Cheng ). Previous BAC-FISH analysis showed that S -RNase and SFBB5-S (SFBB ) genes, which are separated by 42kb from each other (Sassa et al., 2007), are located in a subtelomeric region of a somatic chromosome (Minamikawa et al., 2010). However, their relative orientation could not be determined by FISH using somatic chromosomes. Therefore, high-resolution FISH mapping was conducted on pachytene chromosomes to determine the location and order of all SFBB-S  genes.

First, rough mapping of the seven contigs were conducted by using eight BAC clones as probes. FISH analysis using the eight BAC probes showed that five contigs, contigs 1 (45M19), 2 (25J12), 3 (21O2), 4 (69A4), and 6 (33E2), were located between contig 7 (BAC 9L6 including FBX11) and contig 5 (BAC 9J18/BAC 22C6 including SFBB8-S ) (Fig. 1A–G). The signals of BAC 9L6 (contig 7) and BAC 22C6 (contig 5) were localized to the side of the centromere and telomere, respectively (Fig. 1D). Next, the relative order of the five contigs located between contigs 5 and 7 was analysed. BAC 9P16 (contig 4) was located between BAC 9L6 (contig 7) and BAC 89I13 (contig 6) (Fig. 1H), i.e. contig 4 was between contig 7 and contig 6. Similarly, BAC 21O2 (contig 3) was located 
between BAC 9P16 (contig 4) and BAC 45M19 (contig 1) 
(Fig. 1I), and BAC 9P16 (contig 4) between BAC 25J12 (contig 2) and BAC 45M19 (contig 1) (Fig. 1J). The signals of BAC 33E2/BAC 89I13 (contig 6) and BAC 21O2 (contig 3) overlapped even on pachytene chromosome (data not shown), thus the relative order of contigs 3 and 6 among the seven contigs could not be determined. Together, the relative order of the seven contigs were determined to be as follows; contigs 5, 1, 3/6, 4, 2, and 7 (Fig. 2).

Because contig 1 and contig 4 contain more than one SFBB gene, FISH mapping was conducted to determine the orientation of SFBB1-S , and FBX6, 12 and 19 in contig 4 and S -RNase, SFBB5-S and SFBB7-S in contig 1. Two BAC clones of each contig and BAC 9L6 (contig 7) or 45M19 (contig 1) were used as FISH probes. For contig 1, BAC 90A15 (SFBB7-S ) was closer to BAC 9L6 (contig 7) than BAC 45M19 (SFBB5-S ) (Fig. 1K). For contig 4, BAC 69A4 (FBX19) was closer to BAC 45M19 (contig 1) than 9P16 (FBX12) (Fig. 1L). Based on the results of FISH mapping, therefore, an ideogram was constructed of the order of the S -RNase and SFBB-S genes from the telomere to centromere, including SFBB8-S , SFBB5-S , S -RNase, SFBB7-S , FBX13/SFBB6-S , FBX19, FBX6, SFBB1-S , FBX12, SFBB3-S, and FBX11 (Fig. 2). Because the BAC 33E2/BAC 89I13 (FBX13) and BAC 21O2 (SFBB6-S ) signals overlapped (see above), the two genes were placed at the same position in the ideogram.

Previously, it was reported that gene order is not conserved between S and S haplotypes of apple (Minamikawa ). Comparison of the gene order of S haplotype determined by high-resolution FISH with previously reported S contigs showed additional structural difference between the two S haplotypes. Although the two proximal SFBBs of S -RNase are SFBB and SFBB (Minamikawa ), their probable alleles, FBX19 and FBX6, respectively, were shown to be the 4th and 5th SFBB genes from S -RNase, respectively (Fig. 2).

S-RNase and SFBB genes were located on heterochromatic and heavily methylated DNA sequence

Pachytene chromosomes provide high-resolution cytogenetic information on euchromatin and heterochromatin. To elucidate the cytological basis of recombination suppression of the region of S -RNase and SFBB genes, the chromatin structure and DNA methylation were examined at the S locus in apple. FISH and black-and-white images of DAPI-stained chromosomes showed that the chromosomal region containing the S -RNase and SFBB genes was embedded in a large knob-like heterochromatin block (Fig. 3). There were nine heterochromatin blocks on the chromosome, and the S locus was located in the third heterochromatic domain from the nearest telomere end.

Fig. 3.

Distribution of heterochromatin and euchromatin regions in the apple chromosome with the S locus. (A, C): Apple chromosome probed with BAC 45M19 (red) and 34G16 (green), or 45M19 (red) 9L6 (green). (B, D): The DAPI-stained chromosomes were converted to black-white images of (A) and (C). The arrowheads point to the S locus. Numbers represent each heterochromatin region. Bars = 5 µm.

Cytosine DNA methylation is a conserved epigenetic silencing mechanism in higher eukaryotes, and plays an important role in many biological and biochemical processes including heterochromatinization (Zhang et al., 2008) and DNA recombination suppression (Meyers et al., 2002). To investigate whether the methylation is associated with the apple S locus, an immunofluorescence assay was conducted on relaxed chromosomes of early pachytene of ‘Florina’ apple using the anti-5mC antibody. The S locus showed much brighter fluorescence than the surrounding regions (Fig. 4). This pattern is similar to the distribution of heterochromatin along the chromosomes, but there were several spots where fluorescence signals were missing (Fig. 4).

Fig. 4.

Immunodetection of 5mC on early pachytene chromosomes of apple. (A) DAPI-stained apple chromosome at the pachytene stage. Bar = 5 µm. (B) Detection of BAC 45M19 (red) and 9L6 (green) on the same chromosome. (C) Detection of 5mC (red). Note; Arrowhead indicates a large block of 5mC signals that is occupied by BACs of the S locus. Domains missing the 5mC signal are indicated by arrows.

Discussion

SFBB and S-RNase genes are physically linked

In a previous study, more than ten SFBB genes were identified by screening a BAC library five times the size of the apple genome and it was characterized as having pollen-specific expression, S haplotype-specific polymorphisms, and genetic linkage to S-RNase (Minamikawa et al., 2010). On the other hand, the physical linkage of multiple genes and the size of the apple S locus are still unknown. It has been demonstrated here that the S -haplotype SFBBs and S -RNase genes clustered in the subtelomeric region on the apple chromosome. Among SFBBs, FBX11 was considered to be a monomorphic gene and existing in the S and S haplotypes (Minamikawa et al., 2010). Although two chromosomes form FISH signals of BAC 9L6 (including FBX11), the signal on the chromosome with the signal from the S haplotype was much stronger than that on the other chromosome (S Wang, unpublished data). The difference in intensity suggests that the former signal was specific and the latter unspecific. Therefore, it is considered that, although FBX11 is present in both the S and S haplotypes (Minamikawa et al., 2010), BAC 9L6 was derived from the S haplotype and is rich in S haplotype-specific sequences.

The apple S locus has been mapped to the bottom of linkage group 17 (Igarashi ), and physically located in a subtelomeric region of a somatic chromosome using FISH (Minamikawa ). The subtelomeric location of the apple S locus was further confirmed by high-resolution FISH on pachytene chromosomes in this study. To maintain SI, pollen S and pistil S must be tightly linked. Otherwise, intergenic recombination would cause the breakdown of SI by generating differences in pollen and pistil specificity within a single S-haplotype (Sijacic ). In addition, the entire apple S locus containing SFBB-S and S -RNase genes is estimated to be ~1Mb or more, because contig 1 contains the sequenced 312kb region (Sassa et al., 2007) and extends the region, and contigs 2–7 are located outside contig 1. Consistent with this, in Japanese pear (Pyrus pyrifolia), Okada et al. (2011) found six and ten SFBB genes in 649kb around S -RNase and 378kb around S -RNase, respectively, and PpSFBB and PpSFBB (Sassa ) were located outside the sequenced regions. Thus, to protect the broad apple S locus from meiotic recombination, mechanisms of suppression are necessary.

Structural and genetic features of the apple S locus might help to suppress recombination in this region

The suppression of recombination around the S locus has been widely observed in different plants, such as Brassica, Petunia inflata, Ipomoea trifida, Antirrhinum, and Hordeum bulbosum (Casselman ; Wang ; Rahman et al., 2007; Yang ; Kakeda ). It was also observed in apple, for example, no recombination occurred between SFBB and S-RNase genes in 239 segregants (Minamikawa ). Sequence heterogeneity among S haplotypes has been suggested to be one of the genetic factors responsible for the suppression of recombination in Brassica and Ipomoea (Iwano ; Casselman ; Suzuki ; Rahman ). In apple, the FISH results showed that the relative order of probable alleles of SFBB genes was not conserved between the S and S haplotypes (Fig. 2; Minamikawa et al., 2010). The order of SFBB genes in the S haplotype determined in this study provides further evidence of interhaplotypic heterogeneity. Although SFBB and SFBB are located next to 
S -RNase (Minamikawa ), their probable alleles FBX19 and FBX6 were shown to be the 4th and 5th SFBB genes from S -RNase, respectively. In Japanese pear S and S haplotypes (Okada et al., 2011), sequence heterogeneity was also found. The FISH results of this study provide further evidence of sequence heterogeneity of the apple S locus region. The 12 BAC probes containing SFBB-S genes detected S -specific signals, although the haplotypic homology of SFBB genes are very high (Sassa et al., 2007; Minamikawa ; Kakui ). Previous study has also shown that two BAC probes 
containing SFBB-S detected S -specific signals on a somatic chromosome of a heterozygous (S S ) apple seedling (Minamikawa et al., 2010). This is probably because SFBB genes are embedded in S haplotype-specific sequences. Hence, sequence heterogeneity might help to inhibit homologous recombination at the apple S locus and guarantee inheritance of SFBBs and S-RNase as a unit.

Heterochromatin is concentrated in pericentromeric and tel omeric regions in most eukaryotes, is abundant in repetitive sequences and transposable elements, and has a relatively low gene density (Peng and Karpen, 2008). The DNA sequences in heterochromatic chromosomal domains are often hypermethylated (Zhang ). Heterochromatinization is considered to be an important factor inhibiting genetic recombination at involved loci in different genetic systems (Haber ; Meyers ; Wei ; Walsh ; Zhang ). In this research, it has been shown that the apple S locus is located in the third heterochomatin knob of the chromosome and the DNA elements in the heterochomatin were highly methylated, which might also contribute to the suppression of recombination at the apple S locus. Recent findings on Japanese pear, a close relative of apple, suggested that multiple SFBB genes are involved in determining pollen S specificity (Kakui ), suggesting that many, if not all, apple SFBB genes located in the large S locus region would also be the components of pollen determinants. Structural heterogeneity and the heterochromatic nature of the apple S locus would be necessary to guarantee stable inheritance of GSI by suppressing recombination in the large region.