Asian seabass (Lates calcarifer), also known as barramundi, belongs to the family Latidae in the order Perciformes, which represents the largest order of vertebrates. Most members of the Latidae family are endemic fishes in Africa and the Indian and Pacific Oceans (Luna, 2008). The Asian seabass is a fast-growing, popular food fish found in tropical and sub-tropical fresh and salt waters. Their white flaky meat and the low number of Y-bones have made them a highly rated table fish. This species is a protandrous hermaphrodite: individuals typically mature as males and later reverse their sex to become females (Moore, 1979; Guiguen et al., 1993). The availability of classical aquaculture technologies made the Asian seabass potentially suitable for marker-assisted selection (MAS). Over the past 10 years, together with our two partners in Singapore, we have been working on a MAS project of the Asian seabass. Information encoded in the genes and in the whole genome is required to increase the resolution of selection from MAS to genomic selection in order to make the approach suitable to identify minor-effect genes that control only a small proportion of a trait (Liu and Cordes, 2004; Gjedrem and Baranski, 2009; Gjedrem, 2010).

Recently, we have started the Asian seabass Genome Project in order to develop new platforms for the eventual improvement of the selection process. A substantial part of nuclear genomes of most eukaryotes is occupied by various types of repetitive DNA sequences (Britten and Kohne, 1968) that make the assembly of genomic sequences difficult (Sutton et al., 1995). On the other hand, repeated DNA elements have numerous advantages for genomic studies. They have been extensively applied as physical chromosome markers in comparative studies for the identification of chromosomal rearrangements, the identification of sex chromosomes, chromosome evolution analysis and applied genetics (Ferreira and Martins, 2008).

Repetitive DNA sequences are classified based on genomic organization of repetitive units as dispersed and tandem. Dispersed repeats, represented by various classes of transposable elements encode for proteins which facilitate their replication and integration into nuclear genome (Devine et al., 1997). Tandem repeats are organized in tandem arrays that may be large and consist of thousands or even millions of repetitive units arranged in head-to-tail orientation (Willard, 1991). Chromosome loci rich in polymorphic satellite DNA usually show specific banding patterns and this makes them potentially useful as cytogenetic markers to discriminate individual chromosomes (Lee et al., 2005; Han et al., 2008). The presence of repeat-derived chromosome landmarks enables the identification of individual chromosomes and is a prerequisite to study structural changes accompanying evolution and speciation and to follow chromosome behavior and transmission in interspecific hybrids (Lee et al., 2005).

The processes by which satellites arise and evolve are not well understood; unequal crossing over, gene conversion, transposition and formation of extra chromosomal circular DNA were all implicated (Carone et al., 2009; Enukashvily and Ponomartsev, 2013). The Nucleus Organizer Regions (NOR) are described as highly repetitive genome sites related to the rRNA synthesis. These regions present small, active transcription sites (highly conserved during evolution) and non-transcribed spacing segments (highly variable), organized as two distinct multigene families 47S and 5S rRNA genes. Tandemly arrayed rDNA repeats are composed of hundreds to thousands copies, disturbed by dispersed elements and usually belong to separate chromosomes (Cioffi et al., 2011).

Repeat regions can cause serious problems during genome assemblies, especially for those that involve short reads produced by next generation sequencing (NGS) technologies. This is due to the fact that a fragment from a repeat region can have false overlaps with fragments from other repeat regions, resulting in the merging of unrelated regions and incorrect final assemblies. The fragments from repeat regions are identified by the number of potential overlaps each fragment has based on pairwise comparisons (Sutton et al., 1995).

In this study, we focus on the primary analysis of repeat elements of the Asian seabass transcriptome and genome using approaches based on classical cytogenetics, molecular biology and next generation sequencing technology. Through this work we have demonstrated that the transcriptome assembly and cDNA data contains repeats and it is suitable for the identification of repeat DNAs from an unknown genome and for comparative investigation of conserved repeat elements between species. Altogether thirteen repeat elements, including 5S rDNA and 18s rDNA, have been identified from the Asian seabass genome, together they constitute 8–14% of the genome. All of them produce short transcripts. Some repeats are enriched on specific chromosomes, while others are generally distributed on the karyotype, except for B-chromosomes.

Detailed information about repetitive elements present in a particular genome can provide useful information that can be used to improve de novo genome assemblies. For this reason, we have initiated a repeat inventory of the Asian seabass, as part of our genome project. The transcriptome and genome of this teleost species were searched for repetitive elements with experimental and bioinformatic tools. Studies involving comparative genomics have revealed that most vertebrate lineages contain different populations of dispersed elements, including retrotransposons and DNA transposons, and significant differences could be observed in their proportions among species of the same lineage. Highly repetitive satellite sequences and moderately repetitive transposable elements form constitutive heterochromatin, which is mostly located in the preTEL, CEN and periCEN regions of chromosomes (for reviews see Choo, 1997; Kawai et al., 2007; Enukashvily and Ponomartsev, 2013). Our data have demonstrated that transcriptomes show similarity to the non-coding regions of genomes as they also contain various dispersed and tandem elements in different teleost species (Figure 3A; Supplementary Tables S3–S6). The current size of the Asian seabass transcriptome assembly (169.6 Mb) is much larger than those of the other fish species (38–68 Mb; Table 1), presumably due to the following reasons: (i) it is partial and yet to be mapped onto the genome; (ii) while the other teleost transcriptomes were mostly generated with the aid of Sanger- and/or pyrosequencing of cDNAs, the Asian seabass transcriptome was produced by high throughput next generation sequencing resulting in a much larger amount of sequence data; (iii) Ensembl transcripts were obtained from RNA-seq-based gene models (Flicek et al., 2012), whereas the current version of the Asian seabass transcriptome wasn't; and (iv) there is a possibility of “inflation” due to unique transcripts that have retained introns (i.e., sequencing of immature mRNAs). Although our “in progress” Asian seabass transcriptome is derived from over a dozen different adult tissues and samples collected at several developmental stages, we cannot exclude the possibility that we have missed repeat-derived transcripts that were active, but not expressed in the samples used for sequencing.

Repeat-derived transcript sequences form only ca. 6–13% of the Asian seabass transcriptome, whereas most of the teleost genomes assembled so far contain more than 14% of various repeat types (Table 2). This indicates that the large differences in the repeat content between species are due to the repeat DNAs being present not only in the non-coding regions of genomes (Kramerov and Vassetzky, 2005), but also in the transcriptomes (Table 2, Supplementary Tables S3–S6). Studies involving comparative genomics have revealed that most vertebrate lineages contain different populations of retrotransposable elements and DNA transposons with significant differences frequently observed among species of the same lineage (Ferreira et al., 2011). The repeat-derived transcripts of teleosts are generally short, their average length is 50–400 bp; the largest transcript length is no longer than 3.6 kb for Asian seabass compared to 6 kb for human transcriptome (Supplementary Table S3). The longest repeat-derived transcripts in human and mouse generally belong to the non-LTR retrotransposon L1, ERV, or Mariner/TC1 groups (Supplementary Table S3), while those in fish transcriptomes are part of a partially overlapping set of repeat types, excluding ERV. The longest repeat transcript for Asian seabass is MAUI_SB with 3575 bp (Supplementary Table S3). Some dispersed, transcript-derived repeats from retrotransposons, such as MAUI (Poulter et al., 1999), Gypsy, Rex1 (Volff et al., 2000, 2003), Bell, and TART (Casacuberta and Pardue, 2003) are longer than 1 kb and show similarity with each other across different fish genomes (Supplementary Table S3). The maintenance of D. melanogaster telomeric DNA is accomplished by repeated retrotransposition of the non-LTR retrotransposon HeT-A/TART specifically to telomeres, in contrast to the maintenance of tandem arrays of species-specific simple DNA repeats at telomeres by telomerase in many eukaryotic organisms (Maxwell, 2004). The result of transcript analysis was consistent with TART producing an array of heterogeneous sense and antisense transcripts (Maxwell, 2004). TART, a non-LTR retrotransposon, was also found in the Asian seabass transcriptome (Supplementary Tables S3, S6). This repeat belongs to the Jockey group (Casacuberta and Pardue, 2003) and its transcript size is longer in teleosts than in mammals (Figure 7). The DNA transposon-based transcript content in human and mouse is lower than those of fishes, but that of the endogenous retrovirus group is higher in mammals (Figure 3). We demonstrated a fish-specific transcriptome-based repeat profile with a few group of repeats, where the proportion of groups Rex1, SINE, R4, Gypsy, and Jockey is higher in fishes then in human and mouse (Figure 7), while that of ERV3 and L1 is lower (Figure 7; Supplementary Table S6). The relative amount of primate-specific SINE1/7SL or Alu transcripts is about three magnitudes higher in human than in fishes. The same tendency has been observed in the distribution of R4/Rex6, Rex1, and Gypsy retrotransposons based on comparative analysis of whole genome shotgun sequences of pufferfishes (Japanese fugu and green-spotted pufferfish), zebrafish, human and mouse genomes (Volff et al., 2003; Shirak et al., 2009). The short sequence length of most repeats from different fish genomes and transcriptomes, including satDNA (Volff et al., 2003; Shirak et al., 2009), indicates that a large part of fish-specific repeat sequences are not characterized.

Moreover, although transcriptomes tested so far tend to have a unique satDNA distribution profile, all investigated fish transcriptomes have similar sets of satellite DNA (Supplementary Table S5). For example, the major satDNA transcript in Asian seabass is OnSat_SB, which has shown similarity with Nile tilapia genome, and is present in the other fish transcriptome datasets, albeit with lower level of similarity. On the other hand, the two main satDNA sequences of the Nile tilapia genome, Satellite A (SATA) and Satellite B (SATB) (Shirak et al., 2009), have not been found in the partial transcriptome data of Asian seabass so far (Supplementary Table S5). The phylogeny based on mitochondrial and nuclear markers suggests independent amplification from the “library.” The existence of a “library of satDNA” has been previously demonstrated experimentally (Mestrović et al., 1998; Bruvo et al., 2003; Pons et al., 2004) and suggested a common ancestor whose genome harbored all or most of the major satDNA families present in the living species at low copy numbers (Pons et al., 2004). Thus, we can confirm that satDNA is shared by a group of related organisms at variable copy number (Mravinac and Plohl, 2010). The existence of short RNA fragments originating from satDNA has been shown experimentally (Rizzi et al., 2004; Valgardsdottir et al., 2005; Enukashvily et al., 2009; Ting et al., 2011; Kuznetzova et al., 2012). Sequencing of such transcripts confirmed that they consisted of satellites only. They are polyadenylated (Rizzi et al., 2004; Enukashvily et al., 2009) and exhibit mostly intranuclear spot-like localization (Valgardsdottir et al., 2005; Kuznetzova et al., 2012). The reported length of satDNA transcripts in mammals varied from 20 bp to 5 kb (Valgardsdottir et al., 2005; Ting et al., 2011) probably because the transcripts were revealed at different stages of their processing. In the various cell types and at different stages of development or cell cycle, the transcription of satellites is asymmetrical: either the sense or the antisense strand is transcribed (Rizzi et al., 2004; Valgardsdottir et al., 2005; Enukashvily et al., 2009; Ting et al., 2011; Kuznetzova et al., 2012). Our experiments have demonstrated a different expression level of satDNA (MoSat_SB) and Cr1 non-LTR retrotransposon Rex1_SB, among the ovary and testis—but not between the male and female brain—of adult Asian seabass (Table 3).

The distribution pattern of constitutive heterochromatin is a good chromosome marker for some teleosts (Kantek et al., 2009; Vicari et al., 2010). Different strategies have been utilized for isolating repetitive DNA sequences: (1) traditional method genomic DNA restriction (Beridze, 1986); (2) re-association kinetics based on C0t–1 DNA (Devine et al., 1997; Langmead et al., 2009); (3) the microdissection of chromosomes submitted to C-banding and subsequent amplification of heterochromatic sequences using DOP-PCR or WGS amplification primers (Cioffi et al., 2013); and (4) bioinformatic analysis of WGS and NGS sequences (Komissarov et al., 2011) and transcriptome data (Jiang et al., 2012). Some of these approaches have been used to investigate the repeats in this study.

Six new repeats belonging to tandem and dispersed groups of repetitive DNA have been identified, they constitute ~8–14% of the Asian seabass genome. The MoSat_SB and YRep_SB repeats are enriched on individual chromosomes, while the other four—Rex1_SB, MAUI_SB, OnSat_SB, and GGSat_SB—are generally distributed on the autosomes throughout the karyotype (Figures 4, 8). The analysis of the chromosomal location of repeated elements demonstrated that they are, in most cases, compartmentalized in heterochromatic regions and the periCEN regions of chromosomes tend to show a unique repeat distribution as has been observed in several other vertebrates (Beridze, 1986; Enukashvily and Ponomartsev, 2013). Our results have demonstrated that the compartmentalization of repeat elements is mostly restricted to heterochromatic AT-rich segments, whereas most of the chromosomes have large GC-rich blocks within their centromeric regions (Figures 1, 8). The AT-rich B-chromosomes of the Asian seabass contain chromatin which differs from heterochromatin identified in periCEN regions of the autosomal chromosomes (Figure 8). The GC-rich B-chromosomes, as chromosomes with potentially higher recombination rate could be useful for future characterization Lates populations throughout the Indian, South-East Asian and Australian regions.

Transposable elements (TEs) can be organized in clusters or dispersed throughout the genome. CR1 elements regulate gene activity and located within genes, as MAUI from the fugu genome (Poulter et al., 1999). Among the CR1 clade of LINE element family of non-LTR retrotransposons, Rex was characterized for the first time in the genome of the Xiphophorus and it was found to be widely present in different fish genomes (Volff et al., 2000). Phylogenetic analysis revealed that Rex1 retrotransposons were frequently active during fish evolution. They formed multiple ancient lineages, which underwent several independent and recent bursts of retrotransposition and invaded fish genomes with variable success rate (Volff et al., 2000; Ferreira et al., 2011). The divergence between two cloned sequences of Rex1 in the Asian seabass was ~10%, at the same time about 20% of divergence was found among Rex1 elements isolated from three different teleost species (Volff et al., 2000, 2003). The physical mapping of different Rex elements showed that they were primarily compartmentalized in the periCEN heterochromatic regions, although dispersed or clustered signals in euchromatic regions were also observed (Figure 4A, Valente et al., 2011). The presence of TEs in heterochromatin can be correlated with their role in structure and organization of heterochromatic areas (such as centromeres) or with the lower selective pressure that act on these gene-poor regions. Rex elements were also concentrated in the largest chromosome pair of the Nile tilapia, Oreochromis niloticus. This chromosome pair is supposed to have originated by fusions, demonstrating the possible involvement of TEs with chromosome rearrangements (Valente et al., 2011). Rex1, 3, and 6 are non-LTR elements that have been active during the evolution of different fish lineages. In the family of Cichlidae (Perciformes), Rex elements are organized in clusters within the genome of the majority of the species (Martins et al., 2004). Rex elements showed a wide distribution among fishes and could be observed both in the periCEN region and euchromatic regions of chromosomes. However, at present, due to the lack of an assembled genome, we are unable to resolve their exact location in relation to the periCEN region. Rex elements can also be associated with genes involved with sex determination (Volff et al., 2000). In this study, we have demonstrated the existence of Rex1-derived transcripts in Asian seabass; moreover, their expression levels were significantly lower in the adult ovary than in the testis (Table 3).

The Nucleolus Organizer Regions (NORs) are highly repetitive genome sites related to rRNA synthesis (Preuss and Pikaard, 2007; Pinhal et al., 2008). NORs present small, active transcription sites (evolutionally conserved 120 bp for 5S rDNA and 300–2800 bp for 18S rDNA) and highly variable non-transcribed spacing segments with their own structural dynamics, in which the presence of transposons located close to the genome regions has been identified (Martins et al., 2004). rDNA sequences and their chromosomal location have proven to be valuable as genetic markers to distinguish closely related species and also in the understanding of the dynamic of repetitive sequences in the genomes. In this study we have shown that the chromosomal position of 5S rDNA and 18S rDNA probes in Asian seabass is similar to those observed in different fish species (Martins et al., 2004; Mantovani et al., 2005; Cioffi et al., 2010; Merlo et al., 2010; Nakajima et al., 2012). At the cytogenetic level, the 5S rDNA probes hybridized mostly near to the centromere region; while the 18S rRNA gene probe has identified variable positions across different fish species (Martins et al., 2004; Mantovani et al., 2005; Cioffi et al., 2010; Merlo et al., 2010; Nakajima et al., 2012).

In order to improve our understanding of their organization, rDNA sequences were analyzed in the Asian seabass genome. Two clones were identified through hybridization with 18SrDNA-derived probes from a BAC library; they formed a single contig (A6N6). They were sequenced with two NGS platforms (Illumina HiSeq and Ion Torrent) and assembled. The A6N6 contig is extremely rich in repeats, containing conserved (Supplementary Table S7) and novel sequences. Some of them are chromosome-specific, whereas others are dispersed throughout the chromosomes (Figure 5D). Due to the shortness of reads, the assembled repeats tend to be highly fragmented. In teleosts, the spacer portion of the major rDNA is generally GC-rich (Schmid and Guttenbach, 1988), whereas our A6N6 scaffold shows a high (59%) AT content. It was shown in the red wolf fish (Erythrinus erythrinus) that rDNA sequences were co-localized with the Rex3 retrotransposable element in the centromeric heterochromatin (Valente et al., 2011) and they have also shown strong association with Tc1/Mariner and Rex elements (Schmid and Guttenbach, 1988; Cioffi et al., 2010; Merlo et al., 2010; Nakajima et al., 2012). The presence of Tc1/Mariner and Rex1_SB repeats was observed in the A6N6 scaffold (Supplementary Table S7). Transcripts derived from the A6N6 scaffold showed sequence homology with a predicted gene (PDZ domain containing ring finger 3) from fish and amphibian genomes. The synteny of Asian seabass A6N6 scaffold with a European seabass linkage group (Figure 6) and their phylogenetic association (Figure 3B) strengthened the idea that the chromosomes have undergone rearrangements during evolution. These rearrangements were likely mediated by retrotransposon activity in which the insertion of the retrotransposable element into rDNA sequences created an rDNA-transposon complex that moved and dispersed in the karyotype (Cioffi et al., 2010; Nakajima et al., 2012).

The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. In summary, our data indicate that the sequence and structure of most Asian seabass repeat DNAs are likely to be unknown. Detailed analysis of the completed genome assembly will provide the final proof for this suggestion. The approach used for the analysis of repeats in this study has also yielded useful knowledge only about evolutionarily conserved repeats. All analyzed sequences, except Tel, belong to the periCEN part of chromosomes (Figure 8), they could form chromosome-specific periCEN patterns of compartmentalization and the transcripts of some show differential expression between the adult gonads of Asian seabass sex.