Chromosomes carrying meiotic avoidance loci in three apomictic eudicot Hieracium subgenus Pilosella species share structural features with two monocot apomicts

Abstract

The LOSS OF APOMEIOSIS (LOA) locus is one of two dominant loci known to control apomixis in the eudicot Hieracium praealtum. LOA stimulates the differentiation of somatic aposporous initial cells after the initiation of meiosis in ovules. Aposporous initial cells undergo nuclear proliferation close to sexual megaspores, forming unreduced aposporous embryo sacs, and the sexual program ceases. LOA-linked genetic markers were used to isolate 1.2 Mb of LOA-associated DNAs from H. praealtum. Physical mapping defined the genomic region essential for LOA function between two markers, flanking 400 kb of identified sequence and central unknown sequences. Cytogenetic and sequence analyses revealed that the LOA locus is located on a single chromosome near the tip of the long arm and surrounded by extensive, abundant complex repeat and transposon sequences. Chromosomal features and LOA-linked markers are conserved in aposporous Hieracium caespitosum and Hieracium piloselloides but absent in sexual Hieracium pilosella. Their absence in apomictic Hieracium aurantiacum suggests that meiotic avoidance may have evolved independently in aposporous subgenus Pilosella species. The structure of the hemizygous chromosomal region containing the LOA locus in the three Hieracium subgenus Pilosella species resembles that of the hemizygous apospory-specific genomic regions in monocot Pennisetum squamulatum and Cenchrus ciliaris. Analyses of partial DNA sequences at these loci show no obvious conservation, indicating that they are unlikely to share a common ancestral origin. This suggests convergent evolution of repeat-rich hemizygous chromosomal regions containing apospory loci in these monocot and eudicot species, which may be required for the function and maintenance of the trait.

Figure 1.

A physical map of the region containing the LOA locus in H. praealtum. The SCAR markers linked to the LOA locus are indicated in order along the solid line representing the chromosome. Boxed markers indicate those initially used for the identification of BACs from the H. praealtum BAC library. Unboxed markers are SCARs developed in this study. The BAC clones identified and assembled into three contigs are shown in gray boxes. The term “gap” indicates sequences yet to be identified to complete the BAC contig. The genomic region essential for LOA function between 14-T7 and 9-HR required for AI cell formation, aposporous embryo sac formation, and sexual suppression is indicated. BAC clones pooled from contig A and contig B for 454 pyrosequencing are indicated with asterisks.

Figure 2.

Physical location of the LOA locus on Hieracium chromosomes. A, BAC clones and specific DNA probes (A-mix and B-mix) used for FISH analysis and their locations in contigs A and B. B, Hybridization of the LOA300.4 BAC probe (green) to a single chromosome in H. praealtum R35. C, Hybridization of the LOA275.12 BAC probe (red) to the same metaphase chromosome spread as in B. D, Merge of images in B and C. E, Phase-contrast image of metaphase chromosomes of H. praealtum R35. The elongated chromosome and an adjacent chromosome magnified in subsequent images is boxed. F, Chromosome spread in E, showing hybridization of the LOA267.14 probe (red) and 18S-5.8S-26S rDNA probe (green). G, Magnified phase-contrast image of the boxed region in E. H, Magnified fluorescent image of the chromosomes in F. I, Hybridization of three probes to the metaphase chromosomes of H. praealtum R35: LOA267.14 BAC (green) and specific DNA probes A-mix (red) and B-mix (yellow). J, Magnification of the long chromosome in I showing the location of contig A (red arrowheads) and contig B (yellow arrowheads) and surrounding repeats (green and bracket). K, Another chromosome showing the subtelomeric location of contigs A and B in R35. L, Schematic representation of the positions of contigs A and B on the elongated chromosome in R35. M to O, FISH analyses using LOA267.14 BAC (green and brackets) and A-mix (red arrowheads) and B-mix (yellow arrowheads) specific DNA probes in three other apomictic Hieracium accessions: H. caespitosum C36 (M), H. piloselloides D36 (N), and H. piloselloides D18 (O). P, Size of the region hybridized by the LOA267.14 BAC in H. praealtum R35, H. caespitosum C36, and H. piloselloides D36 and D18. The repetitive region hybridized by the LOA267.14 BAC is significantly smaller in D36 and D18 (asterisk; t test; P < 0.01). Bars = 10 μm (B–F and I) and 2 μm (G, H, J, K, and M–O).

Figure 3.

FISH karyotypes of different Hieracium accessions hybridized with probes to detect LOA-associated repeat sequences and rDNA loci, and DNA methylation status in H. caespitosum C36. A, H. praealtum R35, H. caespitosum C36, and H. piloselloides D36 and D18 were hybridized with LOA267.14 BAC (yellow), 5S rDNA (red), and 18S-5.8S-26S rDNA (green) probes. Yellow arrows indicate hybridization of the LOA267.14 BAC probe. White asterisks indicate chromosomes containing rDNA that do not have a corresponding hybridization pattern match to another chromosome. B, H. aurantiacum A35 and A36 and sexual H. pilosella P36 chromosomes were hybridized with rDNA probes as in A. The LOA267.14 probe does not hybridize to chromosomes in these species (Supplemental Fig. S3). It was not used as a probe in this image to reduce background noise. C to E, Analysis of the methylation status of H. caespitosum chromosomes using anti-5-methylcytosine (Anti-5mC) antibody. Shown are 4′,6-diamidino-2-phenylindole (DAPI) staining in red (C), DNA methylation in green (D), and the merge of DAPI and DNA methylation images (E). The long chromosome is indicated by the white dotted lines. The arrowheads indicate the position of the LOA locus. Bar = 10 μm.

Figure 4.

Analysis of the segregation of LOA-linked markers and AI cell formation in F1 progeny derived from a cross between sexual P36 (female) and apomict R35 (male). A, Presence (white) or absence (black) of SCAR markers in individual F1 plants and the parent plants used in the cross. Plants fall into classes containing markers that cross-reference with those shown in Table IV. The presence (+) or absence (−) of AI cells in the ovule is indicated. B to M, Microscopy images of cleared ovules of the plants in A. The plant identifier number is shown at the bottom right of each panel. ai, AI cell; dms, degenerating megaspores; fm, functional meiotic megaspore. Bar = 20 μm.

Figure 5.

Functions of the LOA locus and models for LOA action to enable AI cell formation. A, The events of meiosis leading to megaspore tetrad formation in ovules are required to activate the function of the LOA locus. LOA stimulates the formation of somatic AI cells, and their nuclei undergo mitosis to form unreduced embryo sacs. During this process, the sexual reproductive pathway is suppressed. Factors that might activate LOA are discussed in the text. B, The LOA locus resides in a subtelomeric position on the long arm of a single hemizygous chromosomal region in three subgenus Pilosella species. In H. praealtum, it is flanked by the indicated markers. Three possible scenarios for LOA induction of AI cell formation are discussed in the text.