Amplification of a GC-rich heterochromatin in the freshwater fish Leporinus desmotes ( Characiformes , Anostomidae )

This is the first description of the karyotype of Leporinus desmotes. The diploid female number was 2n = 54 metaand submetacentric chromosomes. The nucleolar organizing regions (NORs) were studied by silver nitrate staining and rDNA fluorescence in situ hybridization (FISH) and were found to be located in the telomeric region of the long arm of the 9th pair. C-banding revealed centromeric and telomeric heterochromatin segments in most chromosomes. Intercalar blocks of heterochromatin were observed in the long arm of six chromosome pairs. Besides a NOR-adjacent heterochromatin, all of the intercalar heterochromatic segments were brightly fluorescent by mithramycin staining. These data suggest that a unique amplification of a primordial GC-rich heterochromatin, probably NOR-associated, may have taken place in the karyotype diversification of this Leporinus species. Departamento de Ciências Biológicas, Universidade Estadual do Oeste do Paraná, Rua Universitária, 2069, 85814-110 Cascavel, PR, Brasil. Send correspondence to V.P.M. E-mail: vpmargarido@unioeste.br Departamento de Genética e Evolução, Universidade Federal de São Carlos, Via Washington Luís, km 235, 13565-905 São Carlos, SP, Brasil. E-mail: galettip@power.ufscar.br 570 Margarido and Galetti Junior PCR and rDNA probe labeling Genomic DNA was isolated from liver tissue as described by Sambrook et al. (1989). DNA samples were used for PCR amplification with a set of 18S rDNA primers as described by Hizume (1994). PCR products were analyzed in agarose gels, and a fragment of approximately 1.8 kb (corresponding to the 18S rDNA region) was purified using Pharmacia Biotech Bandprep eluation kit. An 18S rDNA probe was labeled by nick translation with biotin-7-dUTP (Roche) according to the manufacturer’s instructions. In situ hybridization and signal detection Fluorescent in situ hybridization (FISH) was performed according to the method of Pinkel et al. (1986). The rDNA probe was detected by avidin-N-fluorescein isothiocyanate (FITC) conjugate, and the signal was enhanced by using biotinilated anti-avidin goat antibodies following a second round of the avidin-FITC detection. Chromosomes were counterstained with propidium iodide. RESULTS Karyotyping and chromosome banding analysis Leporinus desmotes showed a karyotype of 2n = 54 composed of metaand submetacentric chromosomes (Figure 1A). A large heterozygotic secondary constriction was often detected in the end of the long arm of the chromosome pair 9. C-banding revealed heterochromatic blocks in the centromeres and telomeres of most chromosomes. In addition, intercalar heterochromatin blocks were detected in the long arm of the chromosome pairs 2, 5, 6, 9, 10 and 11 (Figure 1B). In the pairs 2 and 6 the intercalar heterochromatin blocks appear duplicated. AgNORs were detected in the end of the long arm of a median-size metacentric, corresponding to pair 9 (Figure 1B). AgNOR homologous sites exhibited size polymorphism in all 30 metaphases analyzed, corroborating the secondary constriction heteromorphism. A large heterochromatin segment was detected adjacent to this secondary constriction. Mithramycin staining revealed a bright MM fluorescent Figure 1 Giemsa-stained (A) and C-banded (B) karyotype of Leporinus desmotes female. The NOR-bearing chromosomes (9th pair) are framed: C-banded (left) and silver nitrate stained (right). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27


INTRODUCTION
The heterochromatin has been reported to serve as an important source of karyotype diversification within and between groups of fish (Ojima and Ueda, 1979;Mayr et al., 1985).The heterochromatin has been described primarily in the centromeric and telomeric areas in most neotropical fish species, but it can be much more abundant in several genera or species (Galetti et al., 1991;Souza et al., 1996;Margarido and Galetti, 1996).
Since the C-banding was described as a method for heterochromatin identification along the chromosomes (Sumner, 1972), additional techniques have been used for investigating this particular chromosome segment (for review, see Sumner, 1990).Some fluorochromes showing base specific DNA-binding properties have revealed qualitative differences in the heterochromatin among the chromosome complement of Astyanax scabripinnis (Souza et al., 1996) and some Leporinus species (Molina et al., 1998).Chromomycin A 3 and mithramycin A are GCrich DNA sequence specific fluorochromes that often promote enhancement of the nucleolar organizing regions (NORs) in fish (Mayr et al., 1985;Amemiya and Gold, 1986) and other lower vertebrates (Schmid and Guttenbach, 1988).However, some heterochromatin segments, which are non-related to NOR sites, can also exhibit fluorescence and brighter signals after GC-specific fluorochrome staining (Artoni et al., 1999).
The heterochromatin has played an important role in the chromosomal diversification of the family Anostomidae (Galetti et al., 1991), including that of sex chro-mosome differentiation in the genus Leporinus (Galetti and Foresti, 1986).Most part of this heterochromatin, however, has shown no differential staining with base-specific fluorochromes.These results suggest an absence of large AT-or GC-rich clusters in these fishes, except for those that are NOR-associated (Galetti et al., 1995) and for the heterochromatic portion of the sex chromosomes observed only in some Leporinus species (Koehler et al., 1997;Molina et al., 1998).
In the present study, we report that C-banding and mithramycin staining revealed large amounts of an intercalar GC-rich heterochromatin in the karyotype of Leporinus desmotes, which could have arisen by amplification of a primordial NOR-associated heterochromatin.Additionally, silver nitrate staining and fluorescent in situ hybridization with 18S rDNA were used to investigate the NOR-heterochromatin relationship.

Chromosome preparation and banding methods
Metaphases were obtained from cephalic kidney cells of two Leporinus desmotes females, collected in the Tocantins River (Porto Nacional, TO), according to the methods of Bertollo et al. (1978).Silver nitrate staining was used to detect AgNOR sites (Howell and Black, 1980).C-banding was visualized by using barium hydroxide (Sumner, 1972) and mithramycin A staining, using distamycin A counterstaining to investigate MM-banding pattern (Schmid, 1980).

PCR and rDNA probe labeling
Genomic DNA was isolated from liver tissue as described by Sambrook et al. (1989).DNA samples were used for PCR amplification with a set of 18S rDNA primers as described by Hizume (1994).PCR products were analyzed in agarose gels, and a fragment of approximately 1.8 kb (corresponding to the 18S rDNA region) was purified using Pharmacia Biotech Bandprep eluation kit.An 18S rDNA probe was labeled by nick translation with biotin-7-dUTP (Roche) according to the manufacturer's instructions.

In situ hybridization and signal detection
Fluorescent in situ hybridization (FISH) was performed according to the method of Pinkel et al. (1986).The rDNA probe was detected by avidin-N-fluorescein isothiocyanate (FITC) conjugate, and the signal was enhanced by using biotinilated anti-avidin goat antibodies following a second round of the avidin-FITC detection.Chromosomes were counterstained with propidium iodide.

Karyotyping and chromosome banding analysis
Leporinus desmotes showed a karyotype of 2n = 54 composed of meta-and submetacentric chromosomes (Figure 1A).A large heterozygotic secondary constriction was often detected in the end of the long arm of the chromosome pair 9. C-banding revealed heterochromatic blocks in the centromeres and telomeres of most chromosomes.In addition, intercalar heterochromatin blocks were detected in the long arm of the chromosome pairs 2, 5, 6, 9, 10 and 11 (Figure 1B).In the pairs 2 and 6 the intercalar heterochromatin blocks appear duplicated.AgNORs were detected in the end of the long arm of a median-size metacentric, corresponding to pair 9 (Figure 1B).AgNOR homologous sites exhibited size polymorphism in all 30 metaphases analyzed, corroborating the secondary constriction heteromorphism.A large heterochromatin segment was detected adjacent to this secondary constriction.Mithramycin staining revealed a bright MM fluorescent  band in this heterochromatin NOR-associated segment and in all of the intercalar heterochromatin segments (Figure 2).Conspicuous duplicated MM + segments were observed in the chromosomes 2 and 6, and were slightly apparent in the chromosome 5.A heteromorphism was detected in the pair 11, in which duplicated intercalar MM + bands were observed only in one element of the pair.
Fluorescent in situ hybridization (rDNA/FISH) 18S rDNA in situ hybridization showed fluorescent signals in only one chromosome pair (9th), confirming the presence of NORs in the end of the long arm of this chromosome pair (Figure 3).NOR size polymorphism was confirmed and no other minor NOR sites could be detected by FISH analysis.

DISCUSSION
The karyotype of Leporinus desmotes is very similar to the karyotype pattern previously described in other Leporinus species (Galetti et al., 1981(Galetti et al., , 1984(Galetti et al., , 1991)).However, a large amount of GC-rich heterochromatin was detected by MM staining among the chromosome complement of L. desmotes.This is an unusual occurrence in the chromosomes in the genus and more generally in fish.Often only NOR sites have been shown with MM + bands in the fish karyotypes (Schmid and Guttenbach, 1980;Mayr et al., 1985;Phillips and Hartley, 1988;Sola et al., 1992).In a few cases in other neotropical fish, some other minor fraction of the heterochromatin not associated with NOR showed MM + bands (Almeida-Toledo et al., 1988;Artoni et al., 1999).
The MM staining also revealed the NORs in the chromosome complement of Leporinus desmotes, confirmed by silver nitrate staining and 18S rDNA in situ hybridization.When a secondary constriction was present in the NOR area, stain intensity differences of both C-and MM banding were observed between the adjacent heterochromatin and the secondary constriction itself.The adjacent heterochromatin revealed C-and MM banding brighter than that of the secondary constriction.It was notable that the propidium iodide counterstaining used in FISH was able to show a differential color (approximately orange) of the NOR adjacent heterochromatin, similar to what has been previously reported to occur in other fish (Ràb et al., 1996).The AgNOR-MM + relationship often observed in fish karyotypes has been explained by the presence of intertwined GC-rich heterochromatin along the rDNA arrays (Pendás et al., 1993).However, the possibility of existence of NOR-associated heterochromatin may not be ruled out among fish (Artoni et al., 1999).At least two categories of heterochromatin could be observed in the chromosomes of L. desmotes.First, there was the centromeric heterochromatin, which showed no fluorescence by MM staining.This is a common finding among fish (Schmid and Guttenbach, 1980;Mayr et al., 1985;Phillips and Hartley, 1988;Galetti et al., 1991;Sola et al., 1992).The second category of heterochromatin was that seen joining the NOR adjacent-heterochromatin and the intercalar ones.This type was revealed by bright fluorescent MM-bands, indicating a typical GC-rich heterochromatin.This characterized the largest amount of heterochromatin observed in the genus Leporinus, outside of that related to sex chromosome differentiation.All Leporinus previously studied showed little heterochromatin, which was limited to the areas of the centromere and/or telomere (Galetti et al., 1991).An exception is the case when the ZW sex chromosome system was detected (Galetti and Foresti, 1986;Galetti et al., 1995;Molina et al., 1998).Considering that no large differences in the euchromatic segments between related species are expected, the unique presence of this intercalar heterochromatin in L. desmotes suggests that these additional chromosome segments may have arisen by an extensive heterochromatin amplification, instead of resulting from chromatin substitution.In this way, it is possible that a primordial GC-rich heterochromatin, most probably the NOR-associated heterochromatin, could spread along some chromosomes by amplification processes.Potential mechanisms for this spread could be unequal chromosome exchanges, transpositions and regional duplications.
Further molecular studies might characterize satellite DNA occurring in this intercalar heterochromatin and eventually reveal a common origin of these chromosome segments in L. desmotes.

ACKNOWLEDGMENTS
The authors are grateful to C.S. Agostinho and E.E.Marques for assistance with specimen collection.This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Publication supported by FAPESP.