Genome-wide analysis of tandem repeats in Tribolium castaneum genome reveals abundant and highly dynamic tandem repeat families with satellite DNA features in euchromatic chromosomal arms

Abstract

Although satellite DNAs are well-explored components of heterochromatin and centromeres, little is known about emergence, dispersal and possible impact of comparably structured tandem repeats (TRs) on the genome-wide scale. Our bioinformatics analysis of assembled Tribolium castaneum genome disclosed significant contribution of TRs in euchromatic chromosomal arms and clear predominance of satellite DNA-typical 170 bp monomers in arrays of ≥5 repeats. By applying different experimental approaches, we revealed that the nine most prominent TR families Cast1-Cast9 extracted from the assembly comprise ∼4.3% of the entire genome and reside almost exclusively in euchromatic regions. Among them, seven families that build ∼3.9% of the genome are based on ∼170 and ∼340 bp long monomers. Results of phylogenetic analyses of 2500 monomers originating from these families show high-sequence dynamics, evident by extensive exchanges between arrays on non-homologous chromosomes. In addition, our analysis shows that concerted evolution acts more efficiently on longer than on shorter arrays. Efficient genome-wide distribution of nine TR families implies the role of transposition only in expansion of the most dispersed family, and involvement of other mechanisms is anticipated. Despite similarities in sequence features, FISH experiments indicate high-level compartmentalization of centromeric and euchromatic tandem repeats.

Keywords: Tandem repeats; Tribolium castaneum; euchromatic regions; evolutionary trends; transposition.

Figure 1.

Genomic distribution of arrays of TRs on T. castaneum assembled chromosomes (CH1–CH10). Vertical bars represent short arrays (<5 monomers/array, upper line) and long arrays (≥5 monomers/array, lower line). The actual number of arrays per Mb for short and long arrays is indicated above each chromosome. Red dots correspond to centromeric TCAST satDNA found in the assembled genome. Horizontal bar represents putative euchromatin (white) and heterochromatin (HighA domain, grey) regions as identified in Wang et al. Locations of the 300 kb placeholders were included to define uncaptured gaps (yellow). Red triangles indicate assumed position of the centromere and large blocks of centromeric heterochromatin based on our FISH analyses (Fig. 4), chromosome banding and HighA domain (putative heterochromatin) defined in Wang et al.

Figure 2.

Correlation of monomer number in extracted TRF arrays and monomer length. Plotted is number of arrays as a function of monomer length for arrays with ≤2 monomers (A) 3–4 monomers (B) and ≥5 monomers (C). This figure is available in black and white in print and in colour at DNA Research online.

Figure 3.

Alignment of consensus monomer of five TCAST satDNA subfamilies from unassembled reads of T. castaneum genome. Consensus sequences of subfamilies are derived according to the majority principle in alignment of variants from unassembled reads. Monomer length variation in the central region is highlighted in grey.

Figure 4.

Fluorescence in situ hybridization of centromeric TCAST satDNA and TR families determined in this work by TRF analysis. Chromosomes are counter-stained with DAPI. FISH showing centromeric TCAST satDNA (TCAST subf3 as a probe; red signals) on T. castaneum chromosomes in meiotic pro metaphase (A). Arrows point to chromosomes CH2, CH3, CH4 and Yp. Chromosomes were named according to the karyotype analysis provided by Stuart and Mocelin. Two-coloured FISH performed on chromosomes in mitotic pro metaphase show localization of new TR families (green): Cast1 (B), Cast2 (C), Cast3 (D), Cast4 (E), Cast5 (F), Cast6 (G), Cast7 (H), Cast8 (I) and Cast9 (J) with respect to centromeric regions marked with TCAST satDNA (red). The bar represents 1 μm. Aside to chromosome spreads are shown Southern blot analyses of genomic DNA digested with REs and hybridized with Cast1 (B), Cast2 (C), Cast4 (E), Cast 5 (F) and Cast6 (G). Only TR families with >0.5% of genomic DNA are presented.

Figure 5.

Phylogenetic relationships of monomers of Cast1–Cast6 families presented in ML trees (A–F). Terminal branches are coloured according to the chromosome of origin. Monomers originating from a single array and grouped in a single branch of the tree are not specifically marked. Dominant groups which display putative recent exchange events are indicated with arches. Black arches mark monomers that group together in the tree, although they originate from arrays located on non-homologous chromosomes. Grey arches represent monomers in chromosome-specific arrays distant one from the other (>20 kb). Blank arches indicate monomers located in dominant chromosome-specific arrays positioned <20 kb apart. Symbols indicate monomers originating from the same array but dispersed in the tree. The branch support values are indicated at major branch points. (G) FISH of Cast6 family on T. castaneum chromosomes. Arrow points to the CH3 chromosome.

Figure 6.

Schematic presentation of flanking regions composed of R66-like and R140-like sequences associated with arrays of Cast5 (A). Two-colour FISH of R66-like (green) and Cast5 (red) probes hybridized on T. castaneum chromosomes (B).