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

Abstract

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.

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 ⁹), 21O2 (contig 3 including SFBB6-S ⁹), and 69A4 (contig 4 including FBX19) were located between signals of 45M19 (contig 1 including SFBB5-S ⁹) and 9L6 (contig 7 including FBX11). The signal of 45M19 was located between that of 22C6 (contig 5 including SFBB8-S ⁹) and 9L6. (E) 25J12 (contig 2 including SFBB3-S ⁹) 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 ⁹-RNase included in contigs 1 and 4. Two contig 1 BACs 45M19, which includes SFBB5-S ⁹, and 90A15, which includes SFBB7-S ⁹ (Sassa et al., 2007; 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 ⁹ haplotype and its comparison to the S ³ haplotype. S ⁹ haplotype genes (left) were placed based on the BAC-FISH signals in Fig. 1. Previous gene names are in parentheses (Minamikawa et al., 2010). Numbers in black boxes denotes contigs of the S ⁹ haplotype. Solid lines indicates pairs of SFBBs with high homology (>90% amino acid identity; Minamikawa et al., 2010). The dashed line denotes alleles of S-RNase. The order of S ³ haplotype contigs (bars) was not determined (Minamikawa et al., 2010). Bars in the S ³ haplotype (right) are not to scale.

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.

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.