Edited by Hidenori Tachida. Laureana Rebordinos: Corresponding author. E-mail: laureana.rebordinos@uca.es
Index
INTRODUCTION
MATERIALS AND METHODS
Sampling
Cloning and sequencing
Sequence analysis
Cytogenetic techniques
RESULTS AND DISCUSSION
5S rDNA
U2 snRNA
ITS-1
Cytogenetic analysis
References

INTRODUCTION

The Moronidae family of fishes, which were originally part of the Serranidae family, is composed of only 6 species from marine, anadromous and freshwater habitats (McGinnis, 2006). These six species belong to two genera, the genus Morone, which is exclusive to the Northwestern Atlantic (4 species); and the genus Dicentrarchus from the Northeastern Atlantic (2 species). The two Dicentrarchus species, Dicentrarchus labrax and Dicentrarchus punctatus, have similar morphologies in their early stages and the species cannot be distinguished from one another (Bonhomme et al., 2002). In their adult stages they can be easily differentiated, since D. labrax grows to a greater commercial length than D. punctatus, which has multiple black spots scattered over its body.

Since the two species are closely related, hybrid specimens between females of D. labrax and males of D. punctatus, have been obtained in captivity (IFAPA “El Toruño” Research Center, Junta de Andalucía, Puerto de Santa María, Cádiz, Spain).

D. labrax is distributed in the Atlantic Ocean from the coast of Norway as far south as Senegal, including the Mediterranean and Black Seas, Canary Islands and Iceland. Although both species have a high sympatric distribution, D. punctatus presents a more southern distribution (Bonhomme et al., 2002), from the English Channel (occasionally) to the Senegal coast, also including the Mediterranean Sea and Canary Islands.

D. labrax is one of the most commercially important species in Europe, for both fisheries and aquaculture, and is one of the species that is most well-established in aquaculture production. However, the aquaculture of D. punctatus is only in the experimental stage, but the species is very important in fisheries.

In humans, multigene families encoding the 18S-5.8S-28S rRNA (45S rDNA), 5S rRNA, U1 and U2 snRNA together account for approximately 2% of the total genome (Manchado et al., 2006b). The 5S rDNA comprises a conserved coding region of 120 bp and a non-transcribed spacer (NTS) which is variable between species in length and sequence. The 45S rDNA has a transcribed unit which comprises two external transcribed spacers (5’ ETS and 3’ ETS), the coding 18S, 5.8S and 28S rRNAs, and two internal transcribed spacers, which separate the 18S rRNA from the 5.8S rRNA (ITS-1) and the 5.8S rRNA from the 28S rRNA (ITS-2); the transcribed units are separated by the intergenic spacers (IGS). The U2 snRNA gene comprises a 188 bp coding region and an interspecies variable spacer. These families fundamentally evolve in a concerted fashion, in which the sequences of the units are homogenised by various mechanisms as gene conversion or unequal crossing-over. However, some authors have reported an alternative evolution model (birth-and-death model) of these multigene families in other species (Úbeda-Manzanaro et al., 2010b; Vierna et al., 2010).

Given the close relationship between D. labrax and D. punctatus, and the commercial interest in these two species, the objective of the genetic characterization procedures performed in this study is to contribute to assessing the variability existing between the species and the taxonomic status of the hybrid D. labrax (♀) × D. punctatus (♂). To this end, three sequences (5S rDNA, ITS-1 and U2 snRNA gene) have been characterized. Cytogenetic studies have also been carried out to locate the 5S rRNA, 18S rRNA and U2 snRNA genes by means of fluorescence in situ hybridization (FISH) techniques.


MATERIALS AND METHODS

Sampling

Molecular analysis of the multigene families was carried out using three adult individuals of the D. labrax, D. punctatus species and D. labrax (♀) × D. punctatus (♂) hybrid. Hybrid specimens were obtained by controlled crosses between D. labrax females and D. punctatus males. One or two days-old (post hatching) larvae were used for cytogenetic analysis. All samples were kindly supplied by the IFAPA “El Toruño” Research Center (Junta de Andalucía, Puerto de Santa María, Cádiz, Spain).

Cloning and sequencing

Genomic DNA was isolated from muscle tissue using FastDNA kit® (Q-Biogene). Extraction quality was validated by electrophoresis in agarose gel (1.5%) containing 0.5 μg ml–1 ethidium bromide. At least three individuals per species were used to amplify the three different multigene families. These PCR amplifications were made using the primers described in Table 1 (sets 1, 2 and 4). Reactions were carried out in a final volume of 50 μl containing 60–80 ng of genomic DNA, 3 mM Cl2Mg, 300 μM dNTP, 0.2 pmol of the forward and reverse primers and 3 U of Taq polymerase (Euroclone). The PCR amplification reactions were performed in a Gene Amp® PCR System 2700 (Applied Biosystems) thermal cycler. Cycles and PCR conditions were according to Cross et al. (2005).


View Details		Table 1
Description of primers used to amplify the multigene families


The PCR products were purified using a NucleoSpin® Extract II kit (Macherey-Nagel), and cloned into pGEM®-T Easy Vector (Promega). Plasmid DNA was extracted using NucleoSpin® Plasmid (Macherey-Nagel). DNA sequencing was performed with fluorescence-labeled terminator (BigDye Terminator 3.1 Cycle Sequencing Kit; Applied Biosystems) in an ABI3100 Genetic Analyzer.

Sequence analysis

Sequence data from this article have been deposited with the GenBank Data Library under Accession Numbers HM014362 to HM014399, HQ291485 to HQ291531 and HQ292261. Sequence alignments were performed using the ClustalX program (Thompson et al., 1997). Consensus sequences were obtained with the Bioedit software (Hall, 1999). A sequence-similarity search of the coding and spacers sequences from the different multigene families was performed in BLASTN (Altschul et al., 1990) to determine the similarities of the sequences obtained, with other sequences from the GenBank database. DnaSP version 5 program (Librado and Rozas, 2009) was used to obtain the nucleotide variability at two levels:

i) The nucleotide polymorphism within species (π), estimated as the average number of nucleotide differences per site between two sequences.

ii) The nucleotide divergence between species (K), estimated as the average number of nucleotide substitutions per site between two species.

In both cases the Jukes and Cantor correction was used. On the other hand, the nucleotide variation between types of units was calculated using the Kimura-2-parameter in relation to evolutionary distances (d). We also applied the complete deletion option, using the MEGA v4 program (Tamura et al., 2007).

Cytogenetic techniques

Chromosome preparations for the cytogenetic analysis of the three species analyzed in the present work were made from larvae, according to Cross et al. (2006).

Karyotyping was performed using conventional staining techniques with Giemsa (8% in phosphate buffer). Labeling of probes was performed by PCR-based method using the primer sets 1, 3 and 4, for 5S rDNA, 18S rDNA and U2 snRNA respectively. Cycles and PCR conditions, as well as conditions of hybridization and post-hybridization washes, were according to Cross et al. (2003) and Merlo et al. (2007).


RESULTS AND DISCUSSION

5S rDNA

PCR of 5S rDNA yielded a 550 bp amplicon in D. labrax, D. punctatus and the hybrid. This PCR product was purified and cloned, and from 3 to 5 clones of each specimen were sequenced.

The analysis of these sequences showed the amplicon sizes ranged between 546–555 bp in D. labrax, 546–561 bp in D. punctatus and 549–564 bp in the hybrid. This size variability in PCR products was mainly due to the presence, in the NTSs, of a tri-nucleotide microsatellite (GTT) and to hexanucleotide duplication (ATCATT), which was immediately before the microsatellite region (Fig. 1). The duplication was observed only in D. labrax and hybrid clones, not in any D. punctatus clones (11 clones were analyzed). Furthermore, all codifying regions presented conserved Internal Control Regions (A box, Intermediate Element and C box). The Poly-T terminator region was present in all sequences following the 3’ end of 5S rDNA; a ‘TATA-like’ regulator element was also localized in position –30 of the transcription start point. The presence and conservation of these regulatory elements leads to the hypothesis that they are functional genes. We observed that the 5S coding region shows low values of both intra (π) and interspecies nucleotide variation (K) (Table 2), as would be expected. However, the inter-species variation of the NTS region was higher than the intra-species variation, although no fixed differences were found between the two species. The high degree of homogeneity found in the complete 5S rDNA sequences (including coding and spacer) clearly indicates the close relationship between these two species (Table 2). Consequently, this phylogenetic proximity makes the development of hybrid lineages possible. Unlike the 5S rRNA gene, the NTS is selectively neutral and is characterized by high sequence dynamism, by the presence of insertions/deletions (indels), microsatellites and pseudo-genes (Wasko et al., 2001; Ferreira et al., 2007; Gornung et al., 2007). The occurrence of microsatellites in the NTSs has been reported in several marine organisms (Ota et al., 2003; Alves-Costa et al., 2006), and it seems to be a common feature in the Chondrichthyes group (Rocco et al., 2005; Pasolini et al., 2006; Pinhal et al., 2009). Some authors have suggested that the presence of microsatellite sequences favours the maintenance of tandem arrays of multigene families (Liao and Weiner, 1995; Cross and Rebordinos, 2005; Úbeda-Manzanaro et al., 2010b). Microsatellites can stabilize DNA structures, acting as “hot spots” for recombination, and therefore might function as an initiation site for the main mechanisms of concerted evolution, such as gene conversion or unequal cross-over (Liao and Weiner, 1995; Chistiakov et al., 2006). The microsatellite found in D. labrax and D. punctatus could have this role, and this would explain the low intragenomic variability found. Two variants of 5S rDNA have commonly been found in fishes, characterized by length and sequence differences in the NTS (Martins and Wasko, 2004; Campo et al., 2009; Fujiwara et al., 2009; Pinhal et al., 2009).


View Details		Fig. 1
Variable region inside NTSs of each specimens. Shading box highlights the repetition of the trinucleotide GTT. ICR: Internal Control Regions.





View Details		Table 2
Polymorphism (π, bold values), divergence (K, lower diagonal), and fixed differences (upper diagonal) in the different sequence regions studied in D. labrax and D. punctatus


U2 snRNA

PCR products of the U2 snRNA had a length of 1100 bp in the two species studied and in the hybrid. The analysis of sequences finally showed a U2 snRNA sequence of 1057–1089 bp in D. labrax and 1083–1084 bp in D. punctatus. Different species-specific features were observed along this sequence. We found two fixed differences in the sequence of nucleotides (Table 2) and two indel regions. The smaller indel was situated in a poly-G region within the first spacer, in which D. punctatus presents two more Gs than D. labrax. The larger indel was situated between nucleotides 268 and 274; D. labrax presented the sequence with the deletion, and D. punctatus the sequence with the insertion (Fig. 2). To test if this indel was really a species-specific marker, three primers were designed as follows: a forward primer was designed considering the fragment deleted, and another forward primer considering the fragment inserted, while the reverse primer was common to the two species. PCR products were obtained in the two species in the two cases; therefore, the two types of sequence (insertion or deletion) are present in both species and so they could not be considered a specific molecular marker.


View Details		Fig. 2
Indel of 7 nucleotides found in the U2–U5 snRNA spacer. 3’: 3’ box; D: Distal Sequence Element (DSE); P: Proximal Sequence Element (PSE).


The U2 sequences obtained were subjected to BLASTN search in the NCBI database, and this showed a high degree of homology of the first 189 bp with U2 snRNA from other species. In addition to this gene, a high degree of homology with the U5 snRNA was found within the spacer, and, therefore, these two genes are linked in the same array. This is not the first time that this type of linkage has been described. In the flatfish, Solea senegalensis, the 5S rDNA and the U1, U2 and U5 snRNA were found in the same array (Manchado et al., 2006b). Recently, in our laboratory, the U2-U5 snRNA linkage was also seen in three fishes of the Sparidae family (Pagrus pagrus, Pagrus auriga and Diplodus sargus) (Merlo et al., in revision). To date, this linkage has been found in species from several different fish families, which suggests that it is a common feature in Osteichthyes. Manchado et al. (2006a) used the U1-U2 linkage found in S. senegalensis to search for species-specific markers in other flatfish species. Therefore, the detection of this type of linkage will be of great importance for species identification.

Coding regions of the U2 and U5 snRNA genes presented lower intra-specific nucleotide variation in D. punctatus than in D. labrax. The polymorphism observed did not involve differentiation between species, and no fixed differences were found. However, both spacer 1 (from U2 snRNA to U5 snRNA) and spacer 2 (from U5 snRNA to U2 snRNA), showed that inter-specific variation was considerably high and one fixed difference was found in each spacer (Table 2). This could be of interest in the search for species-specific markers. However, it cannot be discounted that some of the observed differences relating to base substitution may be due to mis-incorporation by Taq DNA polymerase, in spite of the error ratio being considered very low (0.25% in frequency after 30 cycles of PCR) (Wasko et al., 2001). The genes which codify the main small nuclear RNA are transcribed by RNA polymerase II, except the U6 snRNA, which is transcribed by RNA polymerase III. However, all these Us snRNA possess promoters with similar characteristics (McNamara-Schroeder et al., 2001). There are basically three regulatory sequences: (i) a Proximal Sequence Element (PSE), which is essential for transcription initiation and is normally localized between 40 and 60 nucleotides upstream of the transcription start point; (ii) a Distal Sequence Element (DSE), which operates as transcription enhancer, and is independent of the position and orientation; and (iii) a 3’ box, which could have a terminator role for the transcription and a role in 3’ processing of the nascent RNA; this box is localized between 9 and 19 nucleotides downstream of the 3’ end of the gene (Kazmaier et al., 1987; Cuello et al., 1999; McNamara-Schroeder et al., 2001; Barzotti et al., 2003). Each of these regulatory sequences has been found in all clones, and a schematic representation is shown in Fig. 2. Comparing the regulatory elements with those obtained from other organisms (Parry et al., 1989; Thomas et al., 1990; Cuello et al., 1999), they show a good homology (Fig. 3). The degree of homology increases when the fish species of the present work are compared with others such as P. pagrus, P. auriga and D. sargus (Merlo et al., in revision) (Fig. 3). The U2 and U5 promoters also showed good homologies between them. The existence and conservation of the protein-binding site within the conserved sequence, and of the regulatory regions in the spacer, indicate the putative functionality of these genes.


View Details		Fig. 3
Putative U2 and U5 snRNA regulatory elements (PSE, DSE and 3’ box) aligned with other from distinct organisms. Dla: Dicentrarchus labrax; Dpu: Dicentrarchus punctatus; Ppa: Pagrus pagrus; Pau: Pagrus auriga; Dsa: Diplodus sargus; Hsa: Homo sapiens; Xla: Xenopus laevis; Cel: Caenorhabditis elegans.


ITS-1

PCR with ITS-1 primers yielded 900 bp products in the two species and the hybrid. Sequencing revealed a real size of 885 bp in D. labrax, while in D. punctatus the size varied between 882 and 934 bp. It should be noted that D. labrax showed two types of ITS-1 sequences, which presented a high nucleotide variability (d = 0.50 ± 0.04; % Identity = 54.68). Within the Osteichthyes group, the size varies within a range from 318 to 1518 bp (average 635.1 ± 159.3) and the percentage of GC content varies from 56.8 to 78.0% (average 68.0 ± 4.2) (Chow et al., 2009). The values obtained in this work are within these ranges and furthermore are close to the average values (Table 3). In this sense, the ITS-1 type II was longer than the ITS-1 type I, and it presented a higher value of GC content. This fact, together with the high nucleotide divergence, supports the conclusion that the sequences can be separated into two types of ITS-1. Dicentrarchus labrax only presented the ITS-1 type I, and its values of intra-specific variation was lower than that found in D. punctatus (Table 2).


View Details		Table 3
Size and GC content of ITS-1 amplicons in Dicentrarchus species


The BLASTN search revealed significant alignments with many ITS-1 sequences of different fish species, especially those belonging to the Percoidei suborder, in which D. labrax and D. punctatus are included. The ITS-1 has regions with secondary structures which are necessary for the maturation process of the rRNA, and a considerable variation in ITS-1 size and sequence is tolerated without inhibiting this function; because of this characteristic, the ITS-1 region has an intermediate rate of evolution (Pleyte et al., 1992). These features make the ITS-1 region a suitable sequence for phylogenetic studies between closely related groups, and for defining sub-species also (Johansen et al., 2006).

We confirmed that the intraspecific value of nucleotide variability in the hybrids, calculated for the three multigene families, was an intermediate value between those of the parental species.

Cytogenetic analysis

Dicentrarchus labrax and D. punctatus present a similar karyotype, which is composed of 48 subtelocentric/acrocentric chromosomes (Sola et al., 1993). In all chromosomal preparations used in this study we observed this same karyotype.

The three probes used in this work (5S rDNA, 18S rDNA and U2 snRNA) were localized in different chromosome pairs (Fig. 4, Table 4); therefore three different chromosomal markers would be obtained. Non-syntenic configuration of 5S rDNA and 18S rRNA gene is the most common situation described in fishes. However, cases have been reported in which the two ribosomal genes are in the same locus (Cross et al., 2006; Fujiwara et al., 2007; Diniz et al., 2008). The U2 snRNA gene has been applied very infrequently as a probe for FISH experiments. Within the fish group, only one work has reported the location of this probe, in four Batrachoididae species, and in that case the authors found three hybridization patterns: concentrated in one chromosome pair; dispersed through the genome; or both concentrated and dispersed (Úbeda-Manzanaro et al., 2010a). Recently, double-FISH studies with the same probes have been carried out in three species of Sparidae family (Merlo et al., in revision). Regarding the differences in the hybridization pattern between the super-order Paracanthopterygii (Batrachoididae family) and Acanthopterygii (Moronidae and Sparidae families) species, it would be interesting to use this probe in more fish species representing a wide range of orders. This should produce more data on the evolution and arrangement of the U2 snRNA cluster. The 5S rDNA has been localized, in both D. labrax and D. punctatus, next to the centromere. This internal position is an ancestral (plesiomorphic) feature in fishes, since this pattern has been described in species of all representing orders (Boron et al., 2006). The results with the 18S rDNA probe have also shown the plesiomorphic condition for this multigene family in fish, i.e. in a terminal position and near the centromere (Merlo et al., 2007). No cytogenetic markers that differentiate between D. labrax and D. punctatus have been detected, because of the conservative pattern of hybridization between these species.


View Details		Fig. 4
Double-FISH realized in D. labrax, D. punctatus y D. labrax (♀) × D. punctatus (♂). Arrows indicate 5S rDNA bearing chromosomes; full arrowheads indicate 45S rDNA bearing chromosomes; and empty arrowheads indicate U2 snRNA bearing chromosomes.





View Details		Table 4
Signal number, chromosome type and localization of probes


Dicentrarchus labrax and D. punctatus are species of considerable commercial interest for both aquaculture and fisheries. We have carried out a preliminary study at the cytogenetic and molecular genetic levels, using three different multigene families; this study has contributed new genetic insights in these two species, and confirms the close relationship between them. Finally, we have also contributed new data on the structure and arrangement of U2 snRNA, which could be a reliable marker for species identification and phylogeny, given the diverse features observed in this gene family, such as the putative linkage with other classes of U snRNA and the variability of the hybridization pattern observed in different species.

This work was supported by grants of the Junta de Andalucía (Spain) to the PAI BIO-219 group.

References
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Alves-Costa, F. A., Wasko, A. P., Oliveira, C., Foresti, F., and Martins, C. (2006) Genomic organization and evolution of the 5S ribosomal DNA in Tilapiini fishes. Genetica 127, 243–252.
Barzotti, R., Pelliccia, F., and Rocchi, A. (2003) Identification and characterization of U1 small nuclear RNA genes from two crustacean isopod species. Chromosome Res. 11, 365–373.
Bonhomme, F., Naciri, M., Bahri-Sfar, L., and Lemaire, C. (2002) Analyse comparée de la structure génétique de deux espèces de poissons marins apprentées et sympatriques Dicentrarchus labrax et Dicentrarchus punctatus. C. R. Biol. 325, 213–220.
Boron, A., Ozouf-Costaz, C., Coutanceau, J.-P., and Woroniecka, K. (2006) Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid-tetraploid population. Genetica 128, 71–79.
Campo, D., Machado-Schiaffino, G., Horreo, J. L., and García-Vázquez, E. (2009) Molecular organization and evolution of 5S rDNA in the genus Merluccius and their phylogenetic implications. J. Mol. Evol. 68, 208–216.
Chistiakov, D. A., Hellemans, B., and Volckaert, F. A. M. (2006) Microsatellites and their genomic distribution, evolution, function and applications: A review with special reference to fish genetics. Aquaculture 255, 1–29.
Chow, S., Ueno, Y., Toyokawa, M., Oohara, I., and Takeyama, H. (2009) Preliminary analysis of length and GC content variation in the ribosomal first Internal Transcribed Spacer (ITS1) of marine animals. Mar. Biotechnol. 11, 301–306.
Cross, I. (2005) Caracterización poblacional y citogenética de Crassostrea angulata. Análisis molecular del ADNr 5S en ostreidos. Tesis Doctoral, p. 133. Universidad de Cádiz, Cádiz, Spain.
Cross, I., and Rebordinos, L. (2005) 5S rDNA and U2 snRNA are linked in the genome of Crassostrea angulata and Crassostrea gigas oysters: does the (CT)n·(GA)n microsatellite stabilize this novel linkage of large tandem arrays? Genome 48, 1116–1119.
Cross, I., Vega, L., and Rebordinos, L. (2003) Nucleolar organizing regions in Crassostrea angulata: chromosomal location and polymorphism. Genetica 119, 65–74.
Cross, I., Díaz, E., Sanchez, I., and Rebordinos, L. (2005) Molecular and cytogenetic characterization of Crassostrea angulata chromosomes. Aquaculture 247, 135–144.
Cross, I., Merlo, A., Manchado, M., Infante, C., Cañavate, J. P., and Rebordinos, L. (2006) Cytogenetic characterization of the sole Solea senegalensis (Teleostei: Pleuronectiformes: Soleidae): Ag-NOR, (GATA)n, (TTAGGG)n and ribosomal genes by one-color and two-color FISH. Genetica 128, 253–259.
Cuello, P., Boyd, D. B., Dye, M. J., Proudfoot, N. J., and Murphy, S. (1999) Transcription of the human U2 snRNA genes continues beyond the 3’ box in vivo. EMBO J. 18, 2867–2877.
Diniz, D., Moreira Filho, O., and Bertollo, L. A. C. (2008) Molecular cytogenetics and characterization of a ZZ/ZW sex chromosome system in Triportheus nematurus (Characiformes, Characidae). Genetica 133, 85–91.
Ferreira, I. A., Oliveira, C., Venere, P. C., Galetti Jr, P. M., and Martins, C. (2007) 5S rDNA variation and its phylogenetic inference in the genus Leporinus (Characiformes: Anostomidae). Genetica 129, 253–257.
Fujiwara, A., Fujiwara, M., Nishida-Umehara, C., Abe, S., and Masaoka, T. (2007) Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers. Genetica 131, 267–274.
Fujiwara, M., Inafuku, J., Takeda, A., Watanabe, A., Fujiwara, A., Kohno, S., and Kubota, S. (2009) Molecular organization of 5S rDNA in bitterlings (Cyprinidae). Genetica 135, 355–365.
Gornung, E., Colangelo, P., and Annesi, F. (2007) 5S ribosomal RNA genes in six species of Mediterranean grey mullets: genomic organization and phylogenetic inference. Genome 50, 787–795.
Hall, T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98.
Jansen, G., Devaere, S., Weekers, P. H. H., and Adriaens, D. (2006) Phylogenetic relationships and divergence time estimate of African anguilliform catfish (Siluriformes: Clariidae) inferred from ribosomal gene and spacer sequences. Mol. Phylogenet. Evol. 38, 65–78.
Johansen, T., Repolho, T., Hellebo, A., and Raae, A. J. (2006) Strict conservation of the ITS regions of the ribosomal RNA genes in Atlantic cod (Gadus morhua L.). DNA sequence 17, 107–114.
Kazmaier, M., Tebb, G., and Mattaj, I. W. (1987) Functional characterization of X. laevis U5 snRNA genes. EMBO J. 6, 3071–3078.
Kuriiwa, K., Hanzawa, N., Yoshino, T., Kimura, S., and Nishida, M. (2007) Phylogenetic relationships and natural hybridization in rabbitfishes (Teleostei: Siganidae) inferred from mitochondrial and nuclear DNA analyses. Mol. Phylogenet. Evol. 45, 69–80.
Liao, D., and Weiner, A. M. (1995) Concerted evolution of the tandemly repeated genes encoding primate U2 small nuclear RNA (the RNU2 locus) does not prevent rapid diversification of the (CT)n·(GA)n microsatellite embedded within the U2 repeat unit. Genomics 30, 583–593.
Librado, P., and Rozas, J. (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452.
Manchado, M., Rebordinos, L., and Infante, C. (2006a) U1 and U2 small nuclear RNA genetic linkage: A novel molecular tool for identification of six sole species (Soleidae, Pleuronectiformes). J. Agr. Food. Chem. 54, 3765–3767.
Manchado, M., Zuasti, E., Cross, I., Merlo, A., Infante, C., and Rebordinos, L. (2006b) Molecular characterization and chromosomal mapping of the 5S rRNA gene in Solea senegalensis: a new linkage to the U1, U2, and U5 small nuclear RNA genes. Genome 49, 79–86.
Martins, C., and Wasko, A. P. (2004) Organization and evolution of 5S ribosomal DNA in the fish genome. In: Focus on Genome Research (ed.: C. R. Williams), pp. 335–363. Nova Science Publishers, New York.
McGinnis, S. M. (2006) Field Guide to Freshwater Fishes of California, p. 539. University of California Press, Los Angeles, California.
McNamara-Schroeder, K. J., Hennessey, R. F., Harding, G. A., Jensen, R. C., and Stumph, W. E. (2001) The Drosophila U1 and U6 gene proximal sequence elements act as important determinants of the RNA polymerase specificity of small nuclear RNA gene promoters in vitro and in vivo. J. Biol. Chem. 276, 31786–31792.
Merlo, A., Cross, I., Palazón, J. L., Sarasquete, C., and Rebordinos, L. (2007) Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae). Genetica 131, 195–200.
Ota, K., Tateno, Y., and Gojobori, T. (2003) Highly differentiated and conserved sex chromosome in fish species (Aulopus japonicus: Teleostei, Aulopidae). Gene 317, 187–193.
Parry, H. D., Tebb, G., and Mattaj, I. W. (1989) The Xenopus U2 gene PSE is a single, compact, element required for transcription initiation and 3’ end formation. Nucleic Acids Res. 17, 3633–3644.
Pasolini, P., Costagliola, D., Rocco, L., and Tinti, F. (2006) Molecular organization of 5S rDNAs in Rajidae (Chondrichthyes): structural features and evolution of piscine 5S rRNA gene and nontranscribed intergenic spacers. J. Mol. Evol. 62, 564–574.
Pendás, A. M., Morán, P., Freije, J. P., and García-Vásquez, E. (1994) Chromosomal location and nucleotide sequence of two tandem repeats of the Atlantic salmon 5S rDNA. Cytogenet. Cell Genet. 67, 31–36.
Pinhal, D., Araki, C. S., Gadig, O. B. F., and Martins, C. (2009) Molecular organization of 5S rDNA in sharks of the genus Rhizoprionodon: insights into the evolutionary dynamics of 5S rDNA in vertebrate genomes. Genet. Res. 91, 61–72.
Pleyte, K. A., Duncan, S. D., and Phillips, R. B. (1992) Evolutionary relationships of the salmonid fish genus Salvelinus inferred from DNA sequences of the First Internal Transcribed Spacer (ITS 1) of ribosomal DNA. Mol. Phylogenet. Evol. 1, 223–230.
Rocco, L., Costagliola, D., Fiorillo, M., Tinti, F., and Stingo, V. (2005) Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, Taeniura lymma and Raja montagui (Chondrichthyes, Batoidea). Genetica 123, 245–253.
Sola, L., Bressanello, S., Rossi, A. R., Iaselli, V., Crosetti, D., and Cataudella, S. (1993) A karyotype analysis of the genus Dicentrarchus by different staining techniques. J. Fish. Biol. 43, 329–337.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599.
Thomas, J., Lea, K., Zucker-Aprison, E., and Blumenthal, T. (1990) The spliceosomal snRNAs of Caenorhabditis elegans. Nucleic Acids Res. 18, 2633–2642.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.
Úbeda-Manzanaro, M., Merlo, M. A., Palazón, J. L., Cross, I., Sarasquete, C., and Rebordinos, L. (2010a) Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and U2 snRNA gene by double-colour FISH in species of the Batrachoididae family. Genetica 138, 787–794.
Úbeda-Manzanaro, M., Merlo, M. A., Palazón, J. L., Sarasquete, C., and Rebordinos, L. (2010b) Sequence characterization and phylogenetic analysis of the 5S ribosomal DNA in species of the Batrachoididae family. Genome 53, 723–730.
Vierna, J., Martínez-Lage, A., and González-Tizón, A. M. (2010) Analysis of ITS1 and ITS2 sequences in Ensis razor shells: suitability as molecular markers at the population and species levels, and evolution of these ribosomal DNA spacers. Genome 53, 23–34.
Wasko, A. P., Martins, C., Wright, J. M., and Galetti Jr, P. M. (2001) Molecular organization of 5S rDNA in fishes of the genus Brycon. Genome 44, 893–902.