Enriched samples containing repetitive DNA sequences from S. insignis and S. taeniurus were constructed based on the renaturation kinetics of Cot-1 DNA (DNA enriched for highly and moderately repetitive DNA sequences) according to the protocol described by Zwick et al. 2010) and recently adapted by Ferreira and Martins (2008). DNA samples (50 μl of 100-500 ng/μl of DNA in 0.3 M NaCl) were autoclaved (121 °C) for 5 minutes (min) to obtain fragments ranging from 100 to 2000 base pairs. Next, the DNA was denatured at 95 °C for 10 min, placed on ice for 10 seconds (s) and subsequently placed at 65 °C for 1 min for re-annealing. The samples were incubated at 37 °C for 8 min with 1 U of S1 nuclease to permit the digestion of single-stranded DNA. The repetitive portion of this DNA was recovered by freezing in liquid nitrogen, and the DNA was extracted using phenol-chloroform. The resulting DNA fragments were used as probes for fluorescence in situ hybridization, cloned and sequenced.

The repetitive S. taeniurus and S. insignis sequence probes isolated using Cot-1 DNA were labeled with digoxigenin-11-dUTP and biotin-16-dUTP (Dig-Nick Translation mix and Biotin-Nick Translation mix; Roche), respectively, by nick translation reactions following the manufacturer’s instructions. Two antibodies, namely, anti-digoxigenin-rhodamine and streptavidin (Life Technologies), were used for signal detection. Fluorescence in situ hybridization (FISH) was performed on mitotic chromosome spreads (Pinkel et al. 1986). Homologous and heterologous in situ fluorescent hybridizations were performed using 77% stringency (2.5 ng/µl of DNA, 50% deionized formamide, 10% dextran sulfate and 2 × SSC at 37 °C for 18 hours). The chromosomes were counterstained with DAPI (2 µg/ml) in Vectashield mounting medium (Vector).

Hybridized chromosomes were analyzed using an Olympus BX51 epifluorescence microscope, and the images were captured with a digital camera (Olympus DP71) using the Image-Pro MC 6.3 software.

One microgram of the Cot-1 DNA products was cloned using a pMOS Blunt-ended PCR Cloning Kit (GE Healthcare), purified using the GFX PCR Purification Kit (GE Healthcare) and sequenced using the Big Dye Kit (Applied Biosystems) in an ABI 3130 genetic analyzer. Sequence alignment was performed using Clustal W (Thompson et al. 1994), which is included in the BioEdit 7.0 software program (Hall 1999). Each clone was used as a query in BLASTN (Basic Local Alignment Search Tool nucleotide) searches against the NCBI nucleotide collection (http://www.ncbi.nlm.nih.gov) and in searches against the Repbase database (Jurka et al. 2005) at the Genetic Information Research Institute (Giri) (http://www.girinst.org/repbase/) using CENSOR software (Kohany et al. 2006).

Recent studies have indicated that repetitive sequences have definitely influenced genome evolution by controlling gene activity and by their involvement in chromosomal rearrangements (Valente et al. 2011). The cloning and sequencing the repetitive genome elements obtained from S. insignis and S. taeniurus Cot-1 DNA displayed high similarities to repetitive sequences deposited in public DNA databases and classified as microsatellites, transposons, and retrotransposons.

Although some repetitive sequences are shared between the two Semaprochilodus species analyzed here, DNA sequencing indicated that the genomes of S. insignis and S. taeniurus were composed of different classes of repetitive sequences. Most of the clones displayed high similarity to microsatellites known from fish species in the order Characiformes (Colossoma macropomum Cuvier, 1816) and the family Prochilodontidae (Prochilodus mariae Eigenmann, 1922). We believed that the microsatellites were more abundant in this analysis because the method used to obtain the repetitive fraction of the genome (renaturation kinetics) generates short fragments of DNA (200−300bp) enabling the identification of microsatellites with full homology.

Microsatellites have been observed in a wide range of organisms and are common and widespread in both prokaryote and eukaryote genomes. Among the functions assigned to microsatellites are their participation in chromatin organization, DNA replication, recombination, and the regulation of gene activities (Martins et al. 2011, Li et al. 2011). In fish species such as Steindachneridion scripta (Miranda Ribeiro, 1918) Rineloricaria latirostris (Boulenger, 1900), and Danio rerio (Hamilton, 1822) these repetitive sequences tend to be clustered in the centromeric and telomeric regions (Shimoda et al. 1999, Vanzela et al. 2002). Future studies aimed at mapping microsatellites within the chromosomes of Prochilodontidae will further our understanding of the roles of those sequences in chromosomal evolution in that group.

A certain proportion of these DNA fragments displayed high degrees of similarity to transposable elements (i.e., both transposons and retrotransposons) (Tables 1 and 2) found in the genomes of fish species such as Xiphophorus maculates Gunther, 1866, Leporinus elongatus Valenciennes, 1850 and Oryzias hubbsi Roberts, 1998 (Volff et al. 2000, Bohne et al. 2012, Marreta et al. 2012, Takehana et al. 2012).

The sequences described in the present study may play an evolutionary role in the genomes of S. insignis and S. taeniurus as one of the sequences identified in the genome of S. taeniurus (Ste17) displayed 82% homology with a retrotransposon called SINE3 identified by (Kapitonov and Jurka 2003) as originating from 5S rRNA. As other species of the Prochilodontidae family have only one pair of chromosomes carrying these ribosomal sites, this information strengthens the hypothesis that the multiple 5S rDNA sites observed in S. insignis and S. taeniurus are pseudogenes (or repetitive sequences) derived from 5S rDNA (Terencio et al. 2012a). In Gymnotus paraguensis (Albert & Crampton, 2003) the multiplication of 5S rDNA gene clusters might has be caused by the involvement of transposable elements because the NTS has high identity (90%) with a Tc1-like transposon (Silva et al. 2011).

We were also able to identify sequences in S. insignis that exhibited high similarity with the transposable element Helitron. In maize, this TE seems to continually produce new non autonomous elements responsible for the duplicative insertion of gene segments at new locations and for the unprecedented genomic diversity of this species (Morgante et al. 2005). Intact Helitron elements were identified in the sex-determining region of the sex chromosomes of the platyfish X. maculatus, suggesting that TE are still active in the genome of platyfish and related species – where they may have roles in the evolution of sex chromosomes and other genomic regions (Zhou et al. 2003).

Tc1/mariner (isolated from the genomes of S. insignis and S. taeniurus) are the most widespread superfamily of DNA transposons and can be found in fungi, plants, ciliates, and animals (including nematodes, arthropods, fish, frogs, and humans). Most of the transposon copies isolated from vertebrates are clearly inactive remnants of once active transposons that were inactivated by mutations, but only after successfully colonizing their genomes (Plasterk et al. 2009, Ivics et al. 2006).

The retroelement Rex1 was also detected in the repetitive fraction of the genomes of S. insignis and S. taeniurus. The Rex family has been widely studied in fish, and a number of different lineages have been described in this group (Volff et al. 2000) where they are known to be scattered or grouped into conspicuous clusters in the chromosomes of Neotropical cichlids (Mazzuchelli and Martins 2009, Gross et al. 2009, Teixeira et al. 2009, Oliveira et al. 1999). These elements display compartmentalized distributions in some autosomes and show clear signals along the full lengths of W chromosomes in S. taeniurus (Terencio et al. 2012).

The Line2 element was detected only in the repetitive fraction of the S. taeniurus genome. This repetitive sequence may be present in the S. insignis genome but simply not sampled in our study, or alternatively, it may have been eliminated from the genome of this species. FISH showed that Line2 sequences are organized in small clusters dispersed over all of the chromosomes of Oreochromis niloticus (Linnaeus, 1758), but with higher concentrations near chromosome ends (Oliveira et al. 1999). Line elements in mammals appear clustered in the G-banding regions of the chromosomes, and on the sex chromosomes in some cases (Wichman et al. 1992, Fishcher et al. 2004).

Repetitive DNA sequences comprising mostly the heterochromatic portions of the genome were observed using the C-banding technique. Previous studies (Feldberg et al. 1987, Vicari et al. 2006, Terencio et al. 2012b) revealed that this technique revealed that repetitive DNA sequence fractions in the genomes of fish of the family Prochilodontidae are not abundant and are located mainly in the centromeric regions of the chromosomes and, less frequently, in the terminal regions of the long arms of some chromosome pairs. Large heterochromatic blocks can be observed, however, in the supernumerary chromosomes (i.e., the B chromosomes) of Prochilodus spp. (Vicari et al. 2006, Voltolin et al. 2011) and in the W sex chromosome of S. taeniurus (Feldberg et al. 1987, Terencio et al. 2012a). Fluorescence in situ hybridization using species-specific probes of the repetitive fractions of the genomes partially confirmed the heterochromatic pattern demonstrated with the C-banding technique in both S. insignis and S. taeniurus – and positive signals were detected in the centromeric regions of all of their chromosomes. Markers were also observed in the terminal regions of some chromosomes, confirming that repetitive DNAs are also present in this area, although heterochromatin was not observed. Repetitive sequences located outside of heterochromatic regions are believed to significantly influence genome evolution, particularly by controlling and regulating gene activities, and genome sequencing has frequently revealed short and truncated copies of repetitive sequences in euchromatic genomic regions (Fischer et al. 2002, Biémont and Vieira 2006, Timberlake 1978, Yuan and Wessler 2011, Torres et al. 2011). This observation does not necessarily indicate that these repetitive sequences are constitutively expressed, however, since they tend to be silenced and undergo subsequent molecular deterioration. In other words, these sequences becomes inactive and progressively accumulate mutations, insertions, and deletions at neutral rates until completely losing their identities or become lost in the host genome (Fernández-Medina et al. 2012). The presence of repetitive DNAs in euchromatic regions has been observed in many groups, such as insects (Cabral-de-Mello et al. 2011), fish (Teixeira et al. 2009, Valente et al. 2011), and lizards (Pokorná et al. 2011), and these TEs have acquired structural/regulatory functions so that their accumulation in euchromatic regions may lend advantages to the host genome.

Cross-hybridizations of S. insignis and S. taeniurus showed patterns similar to those observed in homologous hybridization – which suggests that this portion of the genome has been conserved throughout evolution, perhaps due to a functional role. However, revealed that these species have species-specific centromeric and terminal sites not identified by heterologous hybridization.

Cross-FISH using S. insignis and S. taeniurus Cot-1 DNA probes revealed hybridization signals in the subterminal regions of P. lineatus, in contrast to the heterochromatic pattern revealed by the C-banding technique with heterochromatin blocks being primarily observed in the centromeric region (Pauls and Bertollo 1990, Venere et al. 1990, Cavallaro et al. 2000, Artoni et al. 2006, Vicari et al. 2006, Voltolin et al. 2011). These data indicate that the repetitive fraction of centromeric heterochromatin of P. lineatus is different from the other two species examined (S. insignis and S. taeniurus) and that shared repetitive sequences are located on the subtelomeric portions of their chromosomes. This same pattern was observed in three species of Prochilodus using (AATTT)n microsatellite (Hatanaka et al. 2002) and W-specific probes (Terencio et al. 2012b).

A common karyotypic feature of species belonging to the genus Prochilodus is the presence of supernumerary chromosomes (B chromosomes). Many studies of B chromosomes have indicated that these supernumerary chromosomes are rich in repetitive sequences and, in certain cases, may contain a number of functional genes (Camacho 2005, Ruiz-Estévez et al. 2012). The P. lineatus population analyzed in the present study displayed two B chromosomes with distinct hybridization sites. The S. insignis Cot-1 DNA probe did not hybridize to the B chromosomes, possibly because the repetitive fraction of the S. insignis genome is not shared with the B chromosomes of P. lineatus. One possible hypothesis explaining this result would be that these repetitive sequences have undergone rapid differentiation and evolution in the genome of S. insignis, resulting in a loss of homology with sequences on the B chromosomes of P. lineatus. Another explanation could be due to the fact it is different genera, and therefore the Bs found in each species could have different origins. The S. taeniurus Cot-1 DNA probe was positive, revealing sequence sharing with P. lineatus B chromosomes. A number of studies have suggested that B chromosomes can influence sex determination in fish (Noleto et al. 2012, Yoshida et al. 2012), although no relationship between the occurrence of B chromosomes and sex determination has been observed in P. lineatus. The B chromosomes detected in P. lineatus were recently shown to demonstrate positive signals when hybridized with the W-specific probe, indicating the sharing of repetitive DNA families between these two species (Terencio et al. 2012a).