Telomere sequence variability in genotypes from natural plant populations: unusual block-organized double-monomer terminal telomeric arrays

Abstract

Background: Telomeres are the nucleoprotein complexes that physically cap the ends of eukaryotic chromosomes. Most plants possess Arabidopsis-type telomere sequences (TSs). In addition to terminal TSs, more diverse interstitial TSs exists in plants. Although telomeres have been sufficiently studied, the actual diversity of TSs in land plants is underestimated.

Results: We investigate genotypes from seven natural populations with contrasting environments of four Chenopodium species to reveal the variability in TSs by analyzing Oxford Nanopore reads. Fluorescent in situ hybridization was used to localize telomeric repeats on chromosomes. We identified a number of derivative monomers that arise in part of both terminal and interstitial telomeric arrays of a single genotype. The former presents a case of block-organized double-monomer telomers, where blocks of Arabidopsis-type TTTAGGG motifs were interspersed with blocks of derivative TTTAAAA motifs. The latter is an integral part of the satellitome with transformations specific to the inactive genome fraction.

Conclusions: We suggested two alternative models for the possible formation of derivative monomers from telomeric heptamer motifs of Arabidopsis-type. It was assumed that derivatization of TSs is a ubiquitous process in the plant genome but occurrence and frequencies of derivatives may be genotype-specific. We also propose that the formation of non-canonical arrays of TSs, especially at chromosomal termini, may be a source for genomic variability in nature.

Keywords: Evolution; Oxford nanopore sequencing; Plant; Population; Species; Telomere.

Fig. 1

Analysis of the ON reads with telomeric sequences and their derivatives in the genomes of Chenopodium species. Self-to-self comparisons of the ON reads displayed as dot plots (YASS program output) (Supplementary information), where parallel lines indicate tandem repeats (the distance between the diagonals equals the lengths of the motifs; green lines are forward, red lines are reverse) at the top. Arrays of telomeric sequences in ON reads detected by the “search for motifs” command of Geneious Prime software based on of telomeric monomers at the bottom. Yellow – (TTTAGGG)₃, red – (TTTAAAA)₃ and green – ((C)CCTGGG + (C)CCTAGG)₃. (A) Array of the Arabidopsis-type monomer TTTAGGG at the edge of the ON read (C. pamiricum, accession 830-3С, read #21 644 of 19 572 bp, see also Data S1). (B) Block-organized double-monomer terminal telomeric arrays where clusters of TTTAGGG monomers interchange with clusters of TTTAAAA monomers at the edge of the ON read (C. acuminatum, accession 429-3, read #5413 of 20 152 bp, see also Data S2). (C) Array of the Arabidopsis-type monomer TTTAGGG in the middle of the ON read, ITR array (C. acuminatum, accession 429-3, read #1954 of 24 378 bp, see also Data S3). (D) Colocation of ITR and DTR arrays (C. pamiricum, accession 830-3С, read #36 779 of14 352 bp, see also Data S4). (E) ITR array where elongated monomers are forming (C. pamiricum, accession 177, read #10 528of 11 922 bp, see also Data S6). (F) DTR array where elongated monomers are forming (C. acuminatum, accession 429-3, read #8544 of 18 185 bp, see also Data S7)

Fig. 2

Multi-FISH with telomeric sequences of the Arabidopsis-type labelled with Cy3 (red signal) and derivative sequences labelled with biotin (green signal) in the somatic chromosomes of Chenopodium species. (A) Somatic chromosomes of C. acuminatum. (1) Non-canonical TTTAAAA probe (green channel). (2) Canonical TTTAGGG telomeric probe (red channel). (3) Combination of non-canonical and canonical telomere probes (red-green-blue channels). (B, C) An enlargement of the two chromosomes from the metaphase plate (shown with white arrows on A) showing chromosomal constitution. Green arrows indicate the position of combined TTTAGGG + TTTAAAA cluster-organized sequence arrays, red arrows indicate the positions of terminal telomeric arrays of the Arabidopsis-type, yellow arrows indicate the positions of the ITRs, and white square brackets show pericentromeric heterochromatin. (D) Somatic chromosomes of C. iljinii (433-9). Green arrows indicate the position of combined TTTAGGG + TTTAAAA cluster-organized sequence arrays. (E) Somatic chromosomes of C. iljinii (441-6) hybridized with sequences of the Arabidopsis-type (red signal) and DTR CCTGGG (green signal). All chromosomes were counterstained with DAPI (blue signal). Bars represent 5 μm. (F) Fiber-FISH conformation of ON data on the interchange of monomer blocks within the telomeric array on DNA strands of C. pamiricum (830-3 C). (1) Pure TTTAGGG array (red signal); (2, 3) two examples of combined TTTAGGG (red signal) + TTTAAAA (green signal) arrays

Fig. 3

Putative recombination between G- and C-rich strands. (A) Scheme of the alleged exchange between DNA strands with the formation of derivative monomers that physically exist in the genomes of the studied species. (B) ITR-DTR junction (C. iljinii 433-9 read #13,986, see also Data S5). ITRs: TTTAGGG – yellow, TTTAGG and TTAGGG (small percentage, approximately 2–5%) – blue; DTRs: CCTGGG – green, CCCTGGG and CCTAGG – grey. (C) FISH on somatic chromosomes of C. iljinii (441-6) of the synthetic probe mimicking junction of TTTAGGG and TTTAAAA blocks

Fig. 4

Probes for in situ experiments. PCR products obtained with forward (TTTAGGG)₃ and reverse (CCCTAAA)₃, and (TTTAAAA)₃ primers (left). Hybridization of labelled PCR products on the interphase nucleus of C. acuminatum (central and right). Bar represents 5 μm

Fig. 5

Hybridization of synthetic probe mimicking junction of TTTAGGG and TTTAAAA blocks together with telomeric probe on chromosomes of C. iljinii (A-C). (A) Synthetic probe (TTTAGGG)₅ + (TTTAAAA)₅ (red signal). (B) Telomeric probe (green signal). (C) Combination of synthetic and telomere probes. Control experiment for determination of the efficiency of synthetic probe hybridization on chromosomes of C. acuminatum (D-F). Simultaneous FISH of synthetic probe and 45 S rDNA (pTa71) was performed. (D) Synthetic probe (TTTAGGG)₅ + (TTTAAAA)₅ (red signal). (E) 45 S rDNA (green signal). (F) Combination of synthetic and 45 S rDNA probes. Bar represents 5 μm