Karyological characterization and identification of four repetitive element groups (the 18S – 28S rRNA gene, telomeric sequences, microsatellite repeat motifs, Rex retroelements) of the Asian swamp eel (Monopterus albus)

Abstract Among teleost fishes, Asian swamp eel (Monopterus albus Zuiew, 1793) possesses the lowest chromosome number, 2n = 24. To characterize the chromosome constitution and investigate the genome organization of repetitive sequences in M. albus, karyotyping and chromosome mapping were performed with the 18S – 28S rRNA gene, telomeric repeats, microsatellite repeat motifs, and Rex retroelements. The 18S – 28S rRNA genes were observed to the pericentromeric region of chromosome 4 at the same position with large propidium iodide and C-positive bands, suggesting that the molecular structure of the pericentromeric regions of chromosome 4 has evolved in a concerted manner with amplification of the 18S – 28S rRNA genes. (TTAGGG)n sequences were found at the telomeric ends of all chromosomes. Eight of 19 microsatellite repeat motifs were dispersedly mapped on different chromosomes suggesting the independent amplification of microsatellite repeat motifs in M. albus. Monopterus albus Rex1 (MALRex1) was observed at interstitial sites of all chromosomes and in the pericentromeric regions of most chromosomes whereas MALRex3 was scattered and localized to all chromosomes and MALRex6 to several chromosomes. This suggests that these retroelements were independently amplified or lost in M. albus. Among MALRexs (MALRex1, MALRex3, and MALRex6), MALRex6 showed higher interspecific sequence divergences from other teleost species in comparison. This suggests that the divergence of Rex6 sequences of M. albus might have occurred a relatively long time ago.


Introduction
Teleost fishes possess high morphological and physiological variation with nearly 30,000 extant species (Nelson 2016). The Asian swamp eel (Monopterus albus Zuiew, 1793) is a commercially important, air-breathing fish (Synbranchidae, Synbranchiformes) which is a protogynous hermaphrodite native in freshwaters of East and Southeast Asia and invasive elsewhere in the world including North America (Liem 1963, Chan et al. 1972, Cheng et al. 2003. The diploid chromosome number of M. albus is 24, comprising 12 pairs of acrocentric chromosomes (Yu et al. 1989, Ji et al. 2003. This is considered to be the lowest chromosome number known in teleosts (genome sizes 0.6-0.8 pg), while common chromosome numbers of teleosts are 2n = 40-50 and genome sizes around 0.8-2 pg (Zhou et al. 2002). The Asian swamp eel is, therefore, a good model to investigate genome evolution and the developmental process in teleosts.
Synbranchids are freshwater eel-like fishes which include four genera (Macrotrema Cantor, 1849, Monopterus Lacépède, 1800, Ophisternon McClelland, 1844, and Synbranchus Bloch, 1795 and Monopterus is phylogenetically located at the basal position except for the Macrotrema (Perdices et al. 2005, Betancur et al. 2013). This phylogenetic relationship suggests that the Asian swamp eel might retain the ancestral karyotype of Synbranchidae. When compared to other synbranchids, it has a unique karyotype with very few chromosomes. For example, the diploid chromosome numbers of Monopterus cuchia Hamilton, 1822, a closely related species, is 42 and those of Synbranchus and Ophisternon species are 42 and 46, respectively (Rishi and Haobam 1984, Foresti et al. 1992, Nirchio et al. 2011, Carvalho et al. 2012). An investigation of M. albus chromosome constitution to compare it with other synbranchid fishes could shed light evolutionary scenarios of chromosomal rearrangements and genome organization within Synbranchidae.
Vertebrate genomes are commonly characterized by a large copy number of repetitive sequences, belonging to two main classes: the site-specific type (such as satellite DNA, microsatellite repeats, ribosomal RNA genes and telomeric sequences), and the interspersed type (transposable elements, TEs) (Jelinek and Schmid 1982). Although most repetitive DNAs do not code for proteins, repetitive sequences can also play important role in the function, dynamics, and evolution of genomes (Csink andHenikoff 1998, Henikoff et al. 2001). Microsatellites, which are tandem repeats of small stretches of DNA motifs, are widespread in the genomes. Amplification of microsatellite repeat motifs has often been observed on sex chromosomes (Cioffi et al. 2011, Matsubara et al. 2015 or several autosomes (Schneider et al. 2015) of vertebrates. Microsatellite repeat motifs have been widely used as cytogenetic markers for chromosome identification, particularly for map-poor species (Srikulnath 2010). TEs are also thought to play an important role in genome evolution (Kidwell and Lisch 2000) acting as a substrate for homologous recombination resulting in chromosomal rearrangements. Additionally, TEs can be transmitted by both vertical and horizontal transfers being present in genomes of phylogenetically distant species (Tang et al. 2015). Retrotransposons (retroelements) are a class of TEs which have RNA as an intermediate, and the Rex retroelements (Rex1, Rex3, and Rex6) were active during teleost evolution (Volff et al. 1999(Volff et al. , 2000(Volff et al. , 2001. These retroelements are widely used as markers for molecular evolution and physical mapping, which allow to understand the role of repetitive elements in genome organization and evolution of teleosts , Schneider et al. 2013. In this study, karyotyping was performed with conventional Giemsa staining, 4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) fluorescent staining, C-banding, and fluorescence in situ hybridization (FISH) with four repetitive elements; namely, the 18S − 28S ribosomal RNA genes, telomeric (TTAGGG)n sequences, Rex retroelements and 19 microsatellite repeat motifs. Partial DNA fragments of Rex retroelements (Rex1, Rex3, and Rex6) were molecularly characterized and the evolutionary processes responsible for these retroelements in teleost genomes were discussed, together with the organization of synbranchid genomes.

Specimens and chromosome preparation
Ten specimens of the Asian swamp eel were purchased from an animal pet shop in Bangkok, Thailand. Animal care and all experimental procedures were approved by the Animal Experiment Committee, Kasetsart University, Thailand (approval no. ACKU00958), and conducted according to the Regulations on Animal Experiments at Kasetsart University, Thailand. Mitotic chromosomes were obtained from gill and kidney cells using the air drying method. Briefly, after intraperitoneal injection of 0.01% colchicine (Sigma, St. Louis, Missouri, USA) in the proportion of 0.7 ml per 100 g of fish weight for 2 h, fishes were anesthetized in ice-cold water, and the anterior portion of the gill and kidney were removed and used for mitotic chromosome preparation. After hypotonic treatment of gill and kidney in 0.075 M KCl for 50 min at room temperature, the organs were minced and placed in the first fixative solution (3:1 methanol/acetic acid) for 5 min and in the second fixative solution (2:1 methanol/ acetic acid) for 5 min on ice. The cells were collected by filtration using gauze, and then fixed with 3:1 methanol/acetic acid. The cells in suspension were dropped onto clean glass slides and air-dried. The slides were kept at -80°C until use. For karyotyping with conventional Giemsa staining, the chromosome slides were stained with 4% Giemsa solution (pH 7.2) for 10 min.

C-banding
To examine the chromosomal distribution of constitutive heterochromatin, C-banding was performed using the standard barium hydroxide/saline/Giemsa method (Sumner 1972) with slight modification as follows: chromosome slides were treated with 0.2 N HCl at room temperature for 60 min and then with 5% Ba(OH) 2 at 50°C for 15 s, followed by 2× SSC at 65°C for 60 min.

Polymerase chain reaction (PCR) amplification and molecular cloning
Genomic DNA was extracted from liver and muscle tissue following the standard salting-out protocol as described previously (Supikamolseni et al. 2015), and used as templates for polymerase chain reaction (PCR). Partial DNA fragments of the 18S − 28S rRNA genes, and Rex retroelements (Rex1, Rex3, and Rex6) were amplified using following PCR primers (see Suppl. material 1). PCR amplification was performed using 20 μl of 1× ExTaq buffer containing 1.5 mM MgCl 2 , 0.2 mM dNTPs, 5.0 μM the primers, and 0.25 U of TaKaRa Ex Taq (TaKaRa Bio, Otsu, Japan), and 25 ng of genomic DNA. PCR conditions were as follows: an initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 53-59°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 10 min. The PCR products were cloned using the pTG19-T vector (Vivantis Technologies Sdn Bhd, Selangor Darul Ehsan, Malaysia), and nucleotide sequences of the DNA fragments were determined using DNA sequencing service (First BASE Laboratories Sdn Bhd, Seri Kembangan, Selangor, Malaysia). Nucleotide sequences of three to five DNA clones, and their consensus sequences were searched for homologies with annotated sequences in the National Center for Biotechnology Information (NCBI) database to identify the amplified DNA fragments, using the BLASTx and BLASTn programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi). They were then deposited in the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/ index-e.html) (Suppl. material 2).

Sequence analysis
Multiple sequence alignments of the three data sets (Rex1, Rex3, and Rex6) were performed with those of other teleosts taken from the NCBI database (Suppl. material 2), using the default parameters of Molecular Evolutionary Genetics Analysis 6 (MEGA6) software (Center for Evolutionary Functional Genomics, The Biodesign Institute, Tempe, AZ, USA) (Tamura et al. 2013). Numbers of indels (insertions and deletions) for each data set of Rex retroelements were calculated using the multiallelic mode of DNAsp 5.0 (Librado and Rozas 2009). All unalignable and gap-containing sites were carefully removed from the data sets. Interspecific sequence divergence was estimated using uncorrected pairwise distances (p-distances), and for the Rex reverse transcriptase region, synonymous (K s ) and nonsynonymous (K a ) substitution rates (±standard error) were calculated using the Nei-Gojobori method (Nei and Gojobori 1986) with Jukes-Cantor correction (Jukes and Cantor 1969). Phylogenetic analyses were then performed using Bayesian Inference (BI) using MrBayes v3.0b4 (Huelsenbeck and Ronquist 2001) and the optimal model of DNA substitution was determined for each data set using Kaku-san4 (Tanabe 2011). The Markov Chain Monte Carlo (MCMC) process was set to run four chains simultaneously for one million generations. After the log-likelihood value plateaued, a sampling procedure was performed every 100 generations to obtain 10,000 trees, and subsequently to provide a majority-rule consensus tree with average branch lengths. All sample points were discarded as burn-in prior to reaching convergence, and the Bayesian posterior probability in the sampled tree population was obtained in percentage terms. All phylogenetic trees were midpoint-rooted due to the absence of suitable outgroup in Rex3 data set. However, additional phylogenetic tree based on Rex1 and Rex6 sequences were constructed with using outgroup method from other Rex sequences.

Karyotype of Monopterus albus
Over 10 Giemsa-stained metaphase spreads were examined for each M. albus individual. Diploid chromosome number is 24 (FN = 24) comprising twelve pairs of acrocentric chromosomes (Fig. 1a). The size difference of chromosome pairs was sequential, but most pairs were identified by size and banding pattern with DAPI and PI fluorescent staining. Large DAPI-positive bands were observed at the pericentromeric region of chromosome 9 (Fig. 1b), and large PI-positive bands were found at the pericentromeric region of chromosome 4 ( Fig. 1c) coincident with a large C-positive heterochromatin bands (Fig. 1d).

Chromosomal location of the 18S -28S rRNA genes and (TTAGGG) n sequences
Fluorescence hybridization signals for the 18S -28S rRNA genes were also detected at the pericentromeric region of chromosome 4 co-localizing with both PI-positive bands and large C-positive heterochromatin blocks (Fig. 2a,c,d,e). Hybridization signals of TTAGGG repeats were observed at telomeric ends of all chromosomes, but no interstitial signal was found (Fig. 2b, c).

Molecular evolutionary dynamics of Rex retroelements
The nucleotide sequence of a 533 bp-fragment of MALRex1 was used in multiple sequence alignment with 28 other teleosts, evidencing 32 indel sites. Sequence divergence among species varied from 0 to 50.13% with an average of 29.56±1.13% (Suppl. material 3). MALRex1 sequences in M. albus showed the minimum interspecific sequence divergence of 1.88% from nototheniids Dissostichus mawsoni Norman, 1937 and Notothenia coriiceps Richardson, 1844 (Perciformes) and the maximum divergence of 41.95% to Poeciliopsis gracilis Heckel, 1848 (Cyprinodontiformes); the average is 24.51±8.14%. The phylogenetic placement of Rex1 sequences showed that most species were grouped in their respective orders (Fig. 6  placement of Rex3 sequences showed a clade for each order except for Perciformes fishes (Fig. 7). The average K s /K a value of Rex3 sequences was 1.05±0.05 (  (Fig. 8, Suppl. material 7). The average K s /K a value of Rex6 sequences was 0.85±0.04 (Table 3).

OLA GAF PFO PGR XMA MAL AOC CLA CMO CRE GPR HBI MAU ONI PSC SDI RSO
Oryzias latipes  . The fundamental numbers of M. albus is reduced to 50% of norm in synbranchid fishes and teleosts, which suggests that the acrocentric chromosomes of M. albus may have been formed by repeated tandem fusion of the ancestral acrocentric chromosomes contained in the ancestral karyotype of Synbranchidae. However, the hybridization signal of (TTAGGG)n at interstitial telomeric sites (ITSs) that appears to be remnants of fusion or inversion (Srikulnath et al. 2009(Srikulnath et al. , 2011 was not found in any chromosomes of M. albus in this study (Fig. 2). Comparative chromosome mapping of Asian swamp eel with zebrafish (Danio rerio Hamilton, 1822) using human bacterial artificial chromosome (BAC) probes revealed the Asian swamp eel retains a number of gene copies found in tetrapods, while other teleosts underwent the third genome duplication (GD), leading to multiple copies of the genes (Yi et al. 2001, Zhou et al. 2002. This suggests that Asian swamp eel retained the genome composition before the event of the third GD that occurred in teleosts (Zhou et al. 2002). Molecular structure of the pericentromeric regions of chromosome 4 which were high GC-rich have evolved in a concerted manner with amplification of the 18S -28S rRNA genes. However, the chromosomal locations of the 18S -28S rRNA genes varied in M. albus individuals (Fig. 2d, e), a phenomanon also observed in Chinese population on pair of chromosome 3 and/or chromosome 7 (Ji et al. 2003). In other synbranchid fishes, the 18S -28S rRNA genes are generally located on a pair of chromosome 1 and on a pair of medium-sized acrocentric chromosomes in O. aenigmaticum (Nirchio et al. 2011), as well as on several other chromosome pairs in various pattern of S. marmoratus . These results suggest that chromosomal locations of the 18S -28S rRNA genes considerably differ in Synbranchidae. In this study, eight microsatellite repeat motifs [(CAG) 10 , (CAA) 10 , (CGG) 10 , (GAG) 10 , (AGAT) 8 , (ACGC) 8 , (AAAT) 8 , and (AAATC) 6 ] were dispersedly mapped on different chromosomes (Fig. 3). This suggests that the amplification of several microsatellite repeat motifs has occurred independently in the genome of M. albus. Interestingly, the dispersion of the microsatellite repeat motifs signals was co-localized to M. albus chromosomes with Rex retroelements. A similar case was found in cichlid species Cichla monoculus Agassiz, 1831, Pterophyllum scalare Schultze, 1823, and Symphysodon discus Heckel, 1840 (Schneider et al. 2015). This suggests that both Rex retroelements and microsatellite repeat motifs have co-amplified in the evolutionary process of the genome of M. albus.

Monopterus albus chromosomes
The diversity of chromosomal distribution for Rex retroelements (Rex1, Rex3, and Rex6) was found in teleosts (Table 4). Two major distinctive patterns were observed: (1) compartmentalization as found in pericentromeric, centromeric, or telomeric regions, and (2) uniform dispersion throughout the genome or along the chromosomes (Ozouf-Costaz et al. 2004). Chromosomal distribution of Rex1, Rex3, and Rex6 were generally located in the specific region together as compartmentalization within each family/order (Table 4). In this study, although MALRex1 was dispersed throughout the genome, this element was predominantly localized to pericentromeric regions of all chromosomes except for chromosomes 4 and 9. By contrast, strong hybridization signals of MALRex3 were dispersed on five chromosome pairs, with weak signals on seven chromosome pairs, which implies that MALRex3 were specifically amplified in chromosomal regions of M. albus.
The differences in the copy number and chromosomal distribution of MALRex1, MALRex3, and MALRex6 suggest that these retroelements were independently amplified or lost in the lineage of M. albus, where MALRex3 is prone to retain a copy number higher than MALRex1 and MALRex6. A similar case of copy number variation in Rex retroelements was also found in several Antarctic nototheniid species (Ozouf-Costaz et al. 2004).

Molecular diversity of Rex retroelements (Rex1, Rex3, and Rex6)
Three Rex retroelements were identified in the genome of M. albus, and the degree of sequence divergence for the three retroelements was high (14-67%) from other species in comparison. MALRex1 and MALRex3 showed high interspecific sequence divergences from Cyprinodontiformes and Characiformes, respectively, but low interspecific sequence divergences from Perciformes fishes for Rex1 and Escociformes for Rex3 (Suppl. materials 3 and 4). This suggests that M. albus and Perciformes or Escociformes shared relatively recent activity of Rex1 or Rex3, respectively. The average K s /K a value of Rex1 was higher than 1 between all compared species and between M. albus and other species (Table 1). These results suggest that Rex1 evolved under purifying selection and that retrotranspositions occurred during the evolution of teleosts. By contrast, the average K s / K a value of Rex3 was closer to 1, which suggests that after retrotransposition, Rex3 was influenced by pseudogene-like evolution (Table 2) (McAllister et al. 1997).
Only few data of Rex6 sequences were available because specific PCR primers were not feasibly effective to detect this element in the genome of teleosts (Volff et al. 2001, Ozouf-Costaz et al. 2004, Schneider et al. 2013. The absence of Rex6 was observed in several Antarctic nototheniid species, but Rex6 exists in some other species of the same order Perciformes (Volff et al. 2001, Ozouf-Costaz et al. 2004, Schneider et al. 2013). This suggests that Rex6 might have rapidly diverged in teleosts. MALRex6 showed high interspecific sequence divergences (approximately 60%) of M. albus from other teleosts (Suppl. material 5). This may indicate that the divergence of Rex6 sequences of M. albus (or Synbranchidae in general) and other teleosts was rather ancestral. The average K s /K a value of Rex6 was less than 1 (Table 3). This suggests that Rex6 has a more diverse function in teleosts.
The present results of chromosomal distribution and molecular diversity of four repetitive element groups (the 18S -28S rRNA gene, telomeric sequences, microsatellite repeat motifs, and Rex retroelements) revealed the chromosome constitution and genome organization of Asian swamp eels. This enabled us to learn more about the chromosome constitution in synbranchid fishes and teleosts as a whole. Further work is required to investigate and compare synbranchid fishes, including M. cuchia, to better understand the process of karyotype and genome evolution in this lineage.