Characterization and repeat analysis of the compact genome of the freshwater pufferfish Tetraodon nigroviridis

Abstract

Tetraodon nigroviridis is a freshwater pufferfish 20-30 million years distant from Fugu rubripes. The genome of both tetraodontiforms is compact, mostly because intergenic and intronic sequences are reduced in size compared to other vertebrate genomes. The previously uncharacterized Tetraodon genome is described here together with a detailed analysis of its repeat content and organization. We report the sequencing of 46 megabases of bacterial artificial chromosome (BAC) end sequences, which represents a random DNA sample equivalent to 13% of the genome. The sequence and location of rRNA gene clusters, centromeric and subtelocentric satellite sequences have been determined. Minisatellites and microsatellites have been cataloged and notable differences were observed in comparison with microsatellites from Fugu. The genome contains homologies to all known families of transposable elements, including Ty3-gypsy, Ty1-copia, Line retrotransposons, DNA transposons, and retroviruses, although their overall abundance is <1%. This structural analysis is an important prerequisite to sequencing the Tetraodon genome.

Figure 1

Schematic representation of the human (top) and Tetraodon (bottom) rRNA gene organization. In humans, a 30-kb nontranscribed spacer (open boxes, partially represented) separates a tandem repetition of the 18S, 5.8S, and 28S genes (black boxes) interspersed by intergenic transcribed spacers and external transcribed spacers (ITSs and ETSs, respectively; horizontal lines). In the 8303-bp Tetraodon sequence, the positions of the first and last base of each gene are indicated based on their homology with the human sequence. The sequence and position of the Tetraodon nontranscribed spacer is unknown. A multiple alignment of the Tetraodon sequence is available under EMBL accession number DS42722.

Figure 2

Fluorescence in situ hybridization of repetitive probes on Tetraodon nigroviridis metaphase chromosome. (A) A cloned 180-bp fragment of the 118-bp satellite hybridizes uniformly to all centromeres. Fluorescein isothiocyanate signals are in green, and chromosomes are counterstained with DAPI. (B) A synthetic probe that includes 4 consecutive monomers of the 10 bp satellite labels specifically the short arms of 10 pairs of subtelocentric chromosomes. Fluorescein isothiocyanate signals are in yellow, and chromosomes are counterstained with DAPI and PI. Arrows indicate the 11th pair of subtelocentric chromosomes that carries the 18S-5.8S-28S rRNA gene clusters and which is strongly stained with propidium iodide.

Figure 3

(A) Alignment of 22 different 118 bp monomers of the centromeric satellite sequence. The first 4 monomers with names starting with 118 are cloned PCR products; the first clone was used as in situ probe in Figure 1C. The last 8 monomers, with names identical two by two except for the last letter, are extracted from the two ends of the same BAC clones. (B) Smith-Waterman alignment between the Tetraodon and the Fugu 118 bp repeat unit. The optimal alignment was obtained by comparing in forward and reverse orientation the Tetraodon sequence to a database of 118 versions of the Fugu monomer obtained by shifting the starting position by one base. (C) Alignment of 25 consecutive subtelocentric satellite monomers, together with the resulting consensus sequence. The only nonvariable base is the thymidine in fifth position.

Figure 4

Distribution of the percentage of DNA contributed by tandemly repeated sequences in the Tetraodon genome according to their period size (7 to 300 bases).

Figure 5

Distribution of microsatellite relative frequencies in Tetraodon (top) and Fugu (bottom).