The large-scale organization of the centromeric region in Beta species

Abstract

In higher eukaryotes, the DNA composition of centromeres displays a high degree of variation, even between chromosomes of a single species. However, the long-range organization of centromeric DNA apparently follows similar structural rules. In our study, a comparative analysis of the DNA at centromeric regions of Beta species, including cultivated and wild beets, was performed using a set of repetitive DNA sequences. Our results show that these regions in Beta genomes have a complex structure and consist of variable repetitive sequences, including satellite DNA, Ty3-gypsy-like retrotransposons, and microsatellites. Based on their molecular characterization and chromosomal distribution determined by fluorescent in situ hybridization (FISH), centromeric repeated DNA sequences were grouped into three classes. By high-resolution multicolor-FISH on pachytene chromosomes and extended DNA fibers we analyzed the long-range organization of centromeric DNA sequences, leading to a structural model of a centromeric region of the wild beet species Beta procumbens. The chromosomal mutants PRO1 and PAT2 contain a single wild beet minichromosome with centromere activity and provide, together with cloned centromeric DNA sequences, an experimental system toward the molecular isolation of individual plant centromeres. In particular, FISH to extended DNA fibers of the PRO1 minichromosome and pulsed-field gel electrophoresis of large restriction fragments enabled estimations of the array size, interspersion patterns, and higher order organization of these centromere-associated satellite families. Regarding the overall structure, Beta centromeric regions show similarities to their counterparts in the few animal and plant species in which centromeres have been analyzed in detail.

Figure 1

Homology of plant Ty3-gypsy-like retrotransposons. Predicted amino acid sequences of reverse transcriptase domains of Beta vulgaris (pBv26), B. procumbens (pBp10), Oryza sativa(EMBL accession no. AF111709), Sorghum bicolor (accession no. BAA95869), Arabidopsis thaliana (accession no. AAF67363). Homologous amino acids that are conserved in at least two sequences are shaded.

Figure 2

Physical mapping of centromere-associated, repetitive sequences on Beta chromosomes by fluorescent in situ hybridization (FISH). Blue fluorescence in panels shows the DNA stained with DAPI. (A) Hybridization of the Ty3-gypsy-like sequence pBv26 to mitotic metaphase chromosomes of B. vulgaris. Signals are largely confined to centromeric regions (green fluorescence). (B) Decondensed, paired chromosomes of B. procumbens at pachytene stage of meiosis. The Ty3-gypsy-like sequence pBp10 strongly hybridizes to the centromeric regions of five chromosome pairs (brightly stained with DAPI) and shows weak dispersion along chromosomes (red fluorescence). (C) Close-up image of the polymorphic pBp10 hybridization (red signal) to pachytene chromosomes of B. procumbens. The Ty3-gypsy-like sequence pBp10 shows confined amplification in the primary constriction (arrow) or clustering in the pericentromeric region with depletion in the constriction (arrowhead). (D) The satellite repeat pHC8 is detectable on three pairs of mitotic metaphase chromosomes of B. lomatogona (red fluorescence). Only one chromosome pair contains pHC8 arrays in the centromeric region (arrows) indicating polymorphisms of the centromeric heterochromatin between chromosomes in Beta species. (E) Pachytene chromosomes of B. vulgaris probed with the satellite family pBV1 (green signals). The satellite forms large arrays within the DAPI-positive heterochromatin of all centromeres. (F) FISH with the oligonucleotide (GATA)₄ to a mitotic metaphase plate of B. vulgaris shows that microsatellites are structural components amplified at Beta centromeres. (G) Rehybridization of B. procumbens pachytene chromosomes. After removal of the probe, chromosomes shown in (B) were hybridized with the satellite repeat pTS5 (strong green fluorescence). Note that the pTS5 satellite family colocalizes with DAPI-positive regions which also showed amplification of the Ty3-gypsy-like sequence pBp10. (H) Multicolor FISH of differently labeled satellite repeats to pachytene chromosomes of two cells of a heterozygous B. procumbens plant. The satellite family pTS5 (red signals) is amplified at most centromeres (middle left), the merged image (middle right) shows the morphology of decondensed pachytene chromosomes (gray) to indicate sites of hybridization. Examination with Filter 09 (right) shows that pTS4.1 satellite arrays (green signals) flank the prominent TS5 repeats (yellow). (I) Close examination of (H) showing the physical order of the pTS5 (yellow) and pTS4.1 (green) satellites at B. procumbens centromeres. (J) The chromosomal mutant PRO1 of B. vulgaris contains a B. procumbens chromosome fragment, which resembles a minichromosome. At mitotic metaphase, the PRO1 minichromosome is highly condensed, and both chromatides are visible (arrow). The minichromosome shows strong signals after FISH with the satellite repeat pTS5 (green fluorescence, arrow). (K) Close-up of FISH to pachytene chromosomes of PRO1. The arrow points to the minichromosome. Hybridization with the satellite pTS5 (red signal) and pTS4.1 (green signal) indicates that the centromeric region is close to the physical end of the minichromosome. (L) FISH to extended DNA fibers reveals the order of B. procumbens satellite repeats in the centromeric region of the PRO1 minichromosome. Stretches of red fluorescence indicate arrays of the satellite pTS5 (top panel). Note, that the pTS5 arrays are interrupted, detectable as gaps within the string of fluorescence signals. Double-target hybridization shows that the satellite pTS4.1 (green signals) forms an array adjacent to the longer pTS5 repeat block (red signals). Three examples are shown (lower panel). Yellow fluorescence indicates overlapping signals caused by interspersion of pTS4.1 repeats within gaps of the neighboring pTS5 array. (M) In the chromosomal mutant PAT2, the chromosome fragment from B. patellaris forms a minichromosome that is barely visible after DAPI staining (top, arrow) but is clearly detectable after FISH with the satellite repeat pTS5 (bottom, green fluorescence). Orange signals originate from simultaneous hybridization of the telomeric repeat (TTTAGGG).

Figure 3

Genomic organization and conservation of six centromere-associated repetitive sequences of the genus Beta. Representative species of the sections Beta (1–3), Corollinae (4–6), Nanae (7), and Procumbentes(8–9) were investigated. The following samples were loaded: (1) B. vulgaris (cultivar 'Rosamona'), (2) B. vulgaris cicla, (3) B. patula, (4) B. corolliflora, (5) B. lomatogona, (6) B. macrorhiza, (7) B. nana, (8) B. procumbens, and (9) B. patellaris. Spinacea oleracea(10) was included as an out-group species. Genomic DNA was digested with (A) BamHI, (B) HaeIII, (C,D) Sau3AI, and (E,F) RsaI, size-separated and transferred onto nylon membranes. Filters were hybridized with (A) pBV1, (B) pHC8, (C) pTS4.1, (D) pTS5, (E) pBv26, and (F) pBp10.

Figure 4

A model of the organization of repetitive DNA sequences at a Beta procumbens centromere. Major satellite families are represented by different colors. The region containing the presumable chromosomal breakpoint that results in the PRO1 minichromosome is indicated by a dotted line. Not included are microsatellite sequences, such as GATA.

Figure 5

Southern hybridization of high-molecular-weight DNA fragments of the minichromosome containing Beta vulgaris line PRO1. DNA was digested with HindIII (1), HaeIII (2), HpaII (3), XbaI (4), and restriction fragments were separated by pulsed-field gel electrophoresis. After transfer onto nylon membranes, filters were hybridized with radioactively labeled pTS4.1 (A) and pTS5 (B).

Figure 6

Alignment of sequences showing homology with the CENP-B motif. The following plant centromeric sequences were included: pTS5 fromBeta procumbens (this paper), CCS1 from Brachypodium sylvaticum, RCS1 from Oryza sativa, BCS1 from Hordeum vulgare, and MCS1A from Zea mays (all taken from Aragon-Aclaide et al. 1996). (A) Nucleotides matching the CENP-B motif are shaded. (B) Cladogram representing the homology of plant centromeric sequences containing a region with similarity to the CENP-B consensus motif and the CENP-B sequence of the human alphoid DNA (Choo 1997).