Heterochromatic pericentromeres and selectively shared DNA repeats

Fluorescence in situ hybridization (FISH) revealed the presence of the telomeric (TTTAGGG)n motif and 45S rDNA in (su)btelomeric and pericentromeric regions. In addition, a multiplicity of telomeric loci along the chromosomal arms and their positional variability were uncovered. The chromosomal position of FISH signals is shown in Figs. 2, 3a and b, 3e and h, and 4. Pericentromeric telomere sequence signals and signals of 45S rDNA could be seen evenly dispersed across the heterochromatin and as distinct peripheral bands (Figs. 2 and 3a). The latter clearly correspond to the previously revealed pericentromeric chromomycin A3 bands (Golczyk et al. 2010). Whether they reflect a trend for a regional modification of the amount of repetitive DNAs around breakpoint sites (Eichler 1999; Bulazel et al. 2007), remains to be explored.

Fig. 2

FISH-mapping of intrachromosomal telomeric sites; 1–12, individual chromosomes. DAPI staining in blue color. Telomeric sites in orange. Bars = 10 µm. Dispersed pericentromeric signals on all chromosomes except chromosome 1 (asterisk) tended to fade in b. Arrowheads, interstitial (TTTAGGG)n loci; double arrows, (TTTAGGG)n loci duplications; arrowheads, (TTTAGGG)n cluster concentrations (bands) on the heterochromatin periphery. a ND-FISH with the synthetic (TTTAGGG)3 probe. The asterisks in the right panel indicate 2–3 (TTTAGGG)n distinct loci within distal chromosome regions that could be resolved in this metaphase plate. b Standard FISH with the mixture of ca. 100 bp dsDNA fragments of the (TTTAGGG)n array used as a probe on individual chromosomes

Fig. 3

FISH-mapping of the repetitive DNAs. a All pericentromeric regions display the presence of 45S rDNA at standard stringency. Arrowheads, 45S rDNA clusters concentrated on the heterochromatin periphery as bands. Asterisks, centromeric regions. b TSrepI sequence arrangement under long (no. 1), moderate (no. 2), and short (no. 3) camera exposure. The FISH-exposure patterns indicate that the pericentromeric regions are heavily but rather uniformly loaded with the TSrepI sequence, but much more abundantly than in the subtelomeric and interstitial chromosomal sites. c Pericentromeric regions positive for DAPI (left panel) and for strong TSrepI-FISH signals (right panel) typically cluster into collective chromocenters at one pole at interphase. The remaining TSrepI sites are located some distance apart towards the opposite nuclear hemisphere (right panel). The asterisk marks the centromere pole. d The (AC)10 oligonucleotide (red) is generally absent or very rare in pericentromeric heterochromatin. e EC-FISH with the mixture of ca. 100 bp dsDNA fragments of the (TTTAGGG)n array. Chromosome arms may possess from two big (arm 11F – right panel) to twelve smaller (arm 8d – left panel, with graphical interpretation) punctate (TTAGGG)n foci at their subtermini, depending on the degree of contraction (compare pictures of chromosome 8 in the two panels). (f) Standard FISH with TSrepI (green) and 5SrDNA (red) probes. The arrowheads mark interstitial 5S rDNA sites, and the asterisks point to subterminal sites where both repeats are adjacent. The duplicated 5S rDNA loci on chromosomal arms 8E-9E tend to fuse, forming bigger joint clusters. g Standard FISH with TSrepI (red) and the 100 bp dsDNA telomeric arrays (green) as probes. The arrowheads or arrows point to interstitial (TTTAGGG)n or TSrepI sites, respectively. The asterisks mark subtelomeric sites where the two repeats are adjacent. h EC-FISH with 5S rDNA (red) and 63-mer telomeric oligonucleotide (green) as probes. The arrowheads or arrows point to interstitial (TTAGGG)n or 5S rDNA or sites, respectively. The asterisks mark subtelomeric sites where the two repeats are adjacent

Fig. 4

Repetitive DNA sites on chromosomes at meiosis. The intercalary sections are dot-outlined. They could be distinguished when at least two well distanced interstitial loci could be detected on one or on both conjoining arms. The distances between intercalary sections and subtelomeric regions mark distal sections

The third type of repeated DNA that is shared by subtelomeres and pericentromeric heterochromatin is short-degenerated semi-tandemly arranged motifs, as shown for the newly cloned sequence designated TSrepI, which contains these motifs (“Methods,” Fig. 3b and c, Data Set S1 and Fig. S1). However, pericentromeres also differ from the subtelomeric domains and from the rest of chromosome regions in that they lack detectable 5S rDNA (Fig. 3f) and CA/GT dinucleotide repeat sequence clusters (Fig. 3d). This may reflect an independent molecular evolution of the subtelomeric and pericentromeric region via sequence elimination/degeneration, counterbalancing the subtelomere-pericentromere homogenization.

The repetitive sequence landscape along chromosomal arms can now be revealed

Mapping of the numerous (TTTAGGG)n, TSrepI, and rDNA loci (Fig. 3f–h as examples) has shown that repetitive sequences tend to occupy different niches along the chromosomal arms (Fig. 4). Notably, making copies of clusters manifests as interstitial duplicated (TTAGGG)n loci on arms 1a, 2a, 7D, and 12A (Figs. 2 and 4), as well as the 5S rDNA locus duplication on chromosome arms 8E and 9E (Figs. 3f and 4, also see Golczyk et al. 2010). Eight pairs of intercalary chromosomal sections were distinguished (Fig. 4) whose size is 22% to 61% of the arm length (Table S1), thus representing a substantial fraction of euchromatin. A high degree of structural heterozygosity was revealed, since within each of such five pairs (localized on arms 4C–5C, 7d–8d, 9e–10e, 10F–11F, and 12A–1A), sections differ in their structure (Fig. 4). However, some of the conjoining arms express structural homology or its remnants. Arms 1a and 2a appear to have their Sects. (43–51% of arm length) perfectly homologous in terms of the (TTTAGGG)n sequence arrangement and arms 3b–4b (39–43%) show incomplete structural homology of their sections. The most striking is that the distance between the distal and interstitial 5S rDNA locus (ca. 40% of arm length) in both “b” arms is the same. Another feature of the PTH karyotype indicative of structural homologies is that, within each of the five arm pairs 1a–2a, 3b–4b, 4C–5C, 6D–7D, and 10F–11F, the most proximal (TTTAGGG)n loci are equally or similarly distanced from the chromosome termini (Fig. 4).

TSrepI repeat invasion into non-pericentromeric sites is related with the size of chromosomal arms

As previously shown (Golczyk et al. 2010), fifteen long (1A, 1a, 2a, 3b, 4b, 4C, 5C, 6D, 7d, 8d, 9e, 10e, 10F, 11F, 12A) and nine short (2B, 3B, 5c, 6c, 7D, 8E, 9E, 11f, 12f) chromosome arms can be distinguished in the PTH karyotype of the oyster plant (Fig. 4). It is then striking that all the eight TSrepI distal loci occupy exclusively long arms (arms: 3b, 4b, 7d, 8d, 9e, 10e, 10F, 11F). Even on chromosome 4, whose long arms are somewhat unequal in length (Golczyk et al. 2010), the TSrepI distal locus resides on the longer arm (Fig. 4). On the other hand, the long arms of chromosome 10 are strictly equal in length (Golczyk et al. 2010) and each is equipped with the TSrepI distal locus (Fig. 4). Thus, if a chromosome has arms even slightly different in length, the distal TSrepI locus (if present) is located on the longer arm. It has already been statistically established by Golczyk et al. (2010) that mismatches in length between conjoining chromosome arms in the ring exist for six arm positions, the rest of the conjoining arms being equal in length. These length differences can be expressed in a gradient, starting from the highest value: 1.79% of karyotype length for 6D–7D > 1.67% for 4C–5C > 0.86% for 1a–2a > 0.61% for 12A–1A > 0.56% for 9e–10e > 0.55% for 7d–8d (Golczyk et al. 2010). Thus, the lack of distal TSrepI loci on the remaining long arms (Fig. 4) is inherent for those arm pairs whose members do not exactly fit in length (1a–2a, 12A–1A) or are vastly uneven in length (6D–7D, 4C–5C).

Subtelomeres that conjoin in the meiotic ring are complex and homologous at the sequence level

Distal chromosome regions of metaphase chromosomes display 2–12 punctate (TTTAGGG)n fluorescence foci, which means that approximately up to six distinct telomeric loci can be present (Figs. 2a right 3e). Such duplications of (TTAGGG)n subtelomeric loci were also found in the closely related Tradescantia virginiana, which also forms terminal chasmata at metaphase I of meiosis (Golczyk 2011a). Notably, if a chromosomal arm has (sub)terminally located rDNA (5S and/or 45S) and/or TSrepI clusters, the adjacent arm of the neighboring chromosome in the meiotic ring has always the same composition of the relevant region (arm pairs: 2B–3B, 3b–4b, 5c–6c, 7d–8d, 8E–9E, 9e–10e, 10F–11F, 11f–12f (Fig. 4)). Furthermore, there are five unique sequence combinations specific for a given pair of arms: 3b–4b (TSrepI + 5S rDNA + telomeric motif), 7d–8d (TSrepI + telomeric motif), 9e–10e (TSrepI + 5S rDNA + 45S rDNA + telomeric motif), 10F–11F (TSrepI + 45S rDNA + telomeric motif), 11f–12f (5S rDNA + telomeric motif).