Cytogenetic analysis and chromosomal characteristics of the polymorphic 18 S rDNA in the fish Prochilodus lineatus ( Characiformes , Prochilodontidae )

We used differential staining techniques (BSG, GTG, AgNO3, DAPI and CMA3 banding) and fluorescent in situ hybridization (FISH) with 5S and 18S probes to investigated the karyotypic and cytogenetic chracteristics of Prochilodus lineatus specimens from a population in Vila Velha state park (Parque Estadual de Vila Velha, Ponta Grossa, Paraná state, southern Brazil). The specimens studied showed the same karyotype as that found in other P. lineatus populations, i.e. 2n = 54 biarmed chromosomes (40m + 14 sm) and c-positive heterochromatin preferentially located pericentromerically in all chromosomes. The presence of partial or totally heterochromatic supernumerary chromosomes with numeric intra-individual variation was confirmed in the analyzed population. The nucleolar organizing regions (NORs) were interstitially situated on the long arm of chromosome pair 4 directly beneath the centromere. The differential banding techniques and FISH revealed NOR size polymorphism due to structural events such as breaks and duplication of the larger rDNA site cluster. We also observed syntenic localization of the 5S ribosomal genes in the distal segment of the 45S cluster.

As in the majority of Neotropical fish karyotypes, the P. lineatus karyotype contains only one chromosome pair with nucleolar organizing regions (NORs) (Pauls and Bertollo, 1990).The in situ location of ribosomal genes indicates synteny for the 18S and 5S rDNA sites of P. lineatus and Prochilodus argenteus as well as polymorphism in the number of 18S genes (Jesus and Moreira-Filho, 2003;Hatanaka and Galetti Jr., 2004).To investigate the level of conservation of these characters between different P. lineatus populations we analyzed the 18S and 5S rDNA sites of a specific P. lineatus population, giving special attention to polymorphism analysis of the 18S sites as revealed by different chromosome banding methods.
Fluorescent in situ hybridization (FISH) was used to map the major and minor rDNA sites in the chromosomes using 18S rDNA (approximately 1,800 bp) obtained by PCR from the nuclear DNA of Prochilodus argenteus (Hatanaka and Galetti Jr., 2004), using the NS1 5'-GTAGT CATATGCTTGTCTC-3' and NS8 5'-TCCGCAGGTTC ACCTACGGA-3' primers (White et al., 1990) and a 5S rDNA probe from Leporinus elongatus (Martins and Galetti Jr., 1999).The probes were labeled with 14-dATP biotin by nick translation according to the manufacturer's instructions (Bionick Labelling System, Invitrogen).The metaphase chromosomes were treated according to the procedure described by Pinkel et al. (1986) and analyzed using an Olympus BX50 epifluorescence microscope.The chromosome figures were captured using CoolSNAP-pro software (Media Cybernetics).
For karyotyping the chromosomes were arranged into two groups as metacentric (m) or submetacentric (sm) based on their arm ratios (Levan et al., 1964) and arranged in order of decreasing size.

Results and Discussion
The diploid chromosome number of P. lineatus is 2n = 54, corresponding to 40 metacentric and 14 submetacentric chromosomes (Figure 1).This is a basal and conserved condition in Prochilodus, also evidenced in a few other phylogenetically close families such as Parodontidae, Anostomidae and Curimatidae (Galetti Jr. et al., 1994).However, this standard diploid number may be extended to up to 2n = 57 chromosomes due to the presence of B microchromosomes that may vary in number intra-or inter-individually.Various studies (Pauls and Bertollo, 1990;Oliveira et al., 1997;Cavalaro et al., 2000;Jesus et al., 2003) have shown that P. lineatus is a useful Neotropical fish model for studies concerning the origin, behavior and evolution of B chromosomes, which although highly diversified in regards to morphology and distribution of satellite DNAs are always heterochromatic and small in relation to the chromosomes of the standard complement (Artoni et al., 2006).
Besides being present on B chromosomes, constitutive heterochromatin also occurs at the pericentromeric region of all the other chromosomes of the complement and at the NORs of the 4 th metacentric pair (Figure 1b), which appear GC-rich when stained with Chromomycin A 3 (Figures 2a, b and 3a, c, d, e, f).The correlation of NOR sites with GC-rich sites is relatively common among fish, although staining with GC-specific fluorochromes is not considered as a method for the direct determination of ribosomal genes but of GC-rich heterochromatins associated with a gene cluster (Artoni et al., 1999).Pendás et al. (1993) used in situ hybridization with 18S rDNA probes to show that the ribosomal genes of Atlantic salmon (Salmo salar) were interspersed throughout the heterochromatic chromosomal regions and that this resulted in an apparent coincidence between ribosomal genes and heterochromatic regions when the chromosomes subjected to silver nitrate staining and C-banding, these observations being supported by our results with P. lineatus (Figure 3a, e).
The P. lineatus NOR site, located directly beneath the centromere in the interstitial region of the long arm of chromosome pair 4, frequently showed a size polymorphism in one of the homologues (Figures 2a, b, d, and 3).Size polymorphisms of the NORs are relatively common in Neotropical fishes (Foresti et al., 1981;Brum et al., 1998;Vicari et al., 2005) and are sometimes also associated with sex chromosomes (Galetti Jr., 1998;Molina et al., 1998;Born andBertollo, 2000, Artoni andBertollo, 2002;Vicari et al., 2003).Interestingly, a lethal effect in the rainbow trout (Oncorhynchus mykis) has been found to be related to a NORs size polymorphism in homozygotes, with evidence  also showing that heterozygotes present a higher adaptive value (Porto-Foresti et al., 2004).In piscines, the involvement of heterochromatin in the accumulation of rDNA loci through unequal crossing-over or sister chromatid exchanges involving repeated sequences and adjacent loci has been proposed as the main mechanism for these rDNA polymorphisms (Pendás et al. 1993).However, structural chromosomal alterations such as duplications, deletions and dissimilar crossing-over are all mechanisms which may produce structural polymorphisms in NORs (Gold, 1990).Our P. lineatus data permitted a more detailed analysis of NOR polymorphisms, our FISH analysis clearly showing that NOR polymorphisms in this species was due to up to three tandemly repeated 18S rDNA clusters (Figure 3f).Accordingly, the GTG banding pattern showed that this region was formed from three G-negative band segments separated by small G-positive band segments (Figure 3b), indicating that breaks in these segments and localized duplications were responsible for the observed polymorphism.
Pauls and Bertollo (1990) used silver nitrate (Ag) staining to analyze the NORs of members of the genus Prochilodus and found AgNORs in a secondary constriction located interstitially on the long arm of a large meta-centric pair and also described a third metacentric chromosome sporadically bearing active NOR on the telomeric region in Prochilodus marggravii (now P. argenteus) and Prochilodus affinis (now P. costatus).In some P. lineatus specimens from the Mogi Guaçu river (São Paulo State, Brazil) Jesus and Moreira-Filho (2003) detected not only a large metacentric pair but also a variable number of ribosomal sites (one or two additional lower inactive sites) suggesting inter-individual numerical polymorphism of the 18S rDNA regions of the this fish.Hatanaka and Galetti Jr.  (2004) observed a number of additional rDNA clusters in P. argenteus but, on the other hand, Maistro et al. (2000) reported that in P. lineatus the major rDNA region seems to be a large metacentric pair, a finding supported in our present study.
Our data also shows that the 5S ribosomal site is syntenic with the 18S rDNA site, being located subterminally on the long arm of chromosome pair 4m, adjacent and distal to the 18S site (Figures 2c and 3g).The mappings of the 5S rDNA sites in fish have demonstrated a higher frequency for a single chromosome locus, which may correspond to the ancestral condition in this group (Martins and Galetti Jr., 1999).Nevertheless, the occurrence of multiple 5S rDNA sites has also been observed in a few species, such as Astyanax scabripinnis (Ferro et al., 2001) and Hoplerythinus unitaeniatus (Diniz and Bertollo, 2003).On the other hand, the P. argenteus population of the São Francisco River (Minas Gerais State, Brazil), occasionally presented a third hybridization signal corresponding to an additional 5S rDNA site (Hatanaka and Galetti Jr, 2004).Despite being adjacent and distal to the 18S rDNA, the 5S rDNA was not involved in polymorphism of chromosome pair 4 (Figure 3g).Syntenic organization of 18S and 5S rDNAs has also been described in P. lineatus from the Mogi-Guaçu River and in P. argenteus from the São Francisco River (Jesus and Moreira-Filho, 2003;Hatanaka and Galetti Jr, 2004, respectively).In conclusion, the observations regarding the localization and variability of the number of ribosomal genes and the number of sites in P. lineatus show that these may be variable among different populations.The presence of a single 18S rDNA-bearing chromosome pair must be a plesiomorphic condition in relation to the other P. lineatus populations and other species of this genus that present multiple NORs.Similarly, synteny between the two ribosomal families may indicate plesiomorphy in the genus Prochilodus.