Analysis of NOR distribution in cultivated and naturalized stocks of rainbow trout ( Oncorhynchus mykiss )

In salmonid species, nucleolar organizing regions (NORs) have frequently been found on a single chromosome pair. However, some other, smaller yet active NORs have also been described. In the present study, NORs were found to be located in different positions on the chromosomes of 12 rainbow trout samples (11 cultivated and one naturalized stocks). Three phenotypes were found: 1) NORs located on two submetacentric chromosomes, in a subterminal position on the short arm (AA); 2) NORs located on two submetacentric chromosomes, in a subterminal position on the long arm (BB); and 3) NORs located on two submetacentric chromosomes, one in a subterminal position on the short arm, and the other in a subterminal position on the long arm (AB). Phenotype AA was identified in individuals from ten cultivated stocks, phenotype AB was identified in individuals from three cultivated stocks, and phenotype BB was found in one cultivated and one wild stock. Some aspects of the origin and the distribution of the different phenotypes are discussed.


Introduction
Salmonid fish were tentatively introduced in Brazil in the beginning of the twentieth century.In 1962, a large official program was set up, and rainbow trout eggs were imported from California (EUA), in order to start the culture of this species in fish farms, and also to introduce it into some rivers of the Serra da Bocaina (Rio de Janeiro and São Paulo), Serra da Mantiqueira (São Paulo), and other mountain fluvial systems, located in the northern region of the state of Santa Catarina (MacCrimmon, 1971).
Cytogenetic studies have shown that salmonid species frequently have a single chromosome pair with active nucleolus organizer regions (NORs) (Phillips and Ihssen, 1985;Phillips et al., 1986).However, additional NORs have been described in a few specimens (Phillips et al., 1989;Pendás et al., 1993).Fluorescence hybridization studies with 18S probes in the brown trout, Salmo trutta, revealed that, in addition to larger aggregations of rDNA in one chromosome pair, 16 other, smaller, transcriptionally inactive regions were also present (Pendás et al., 1993).
In the rainbow trout Oncorhynchus mykiss, several studies have shown that NORs are located in a subterminal position on the short arm of a submetacentric chromosome pair (Schmid et al., 1982;Phillips and Ihssen, 1985;Mayr et al., 1986;Ueda and Kobayashi, 1988;Lloyd and Thorgaard, 1988).Oliveira et al. (1996) observed, however, that rainbow trouts from Núcleo Experimental de Salmonicultura de Campos do Jordão, imported from California (USA) in the 1960's, exhibited NORs in a subterminal position on the long arm of a submetacentric chromosome pair (Table I).
In the present study, the Ag-NOR technique was used to determine and compare the chromosome location of the NORs in 11 rainbow trout samples obtained from cultivated and naturalized stocks in the states of São Paulo, Rio de Janeiro and Minas Gerais.

Material and Methods
Cytogenetic analysis was performed on rainbow trout (Oncorhynchus mykiss) samples from twelve different cultivated stocks and one sample captured in the wild (naturalized) (Table I).Some specimens of each stock were marked with magnetic tags and kept in tanks at Núcleo Experimental de Salmonicultura de Campos do Jordão, São Paulo, Brazil.
Direct suspensions of kidney cells (Foresti et al., 1993) were used to analyze the chromosomal characteristics of the individuals, after prior injection of the fish with a yeast cell suspension, to increase the number of metaphase cells (Lozano et al., 1988).The procedure used to identify NORs was originally described by Howell and Black (1980).

Results and Discussion
Studies including the identification of NORs have appointed them as important cytogenetic markers for aquaculture research (Fergunson et al., 1995).Moreover, several investigations have demonstrated that polymorphic NOR phenotypes are inherited (Mikelsaar et al., 1977;Markovic et al., 1978;Henderson and Bruere, 1980;Arruda and Monteagudo, 1989).
In the present study, three NOR phenotypes were found: 1) NORs located in a subterminal position on the short arms of two submetacentric chromosomes (AA); 2) NORs located in a subterminal position on the long arms of two submetacentric chromosomes (BB); and 3) NORs located on two submetacentric chromosomes, one in a subterminal position on the short arm, and the other in a subterminal position on the long arm (AB).
The AA phenotype was the most common, and was found in specimens from ten cultivated stocks (Table I and Figures 1a-h, j, l).This observation is in accordance with most of data published on NOR distribution in rainbow trout (Schmid et al., 1982;Phillips and Ihssen, 1985;Mayr et al., 1986;Ueda and Kobayashi, 1988;Lloyd and Thorgaard, 1988;Phillips et al., 1989).Phenotype AB was observed in 20% of the specimens from stock 6, 20% from stock 10, and 30% from stock 11 (Table I and Figures 1i, k,  m).This pattern has not been described in any previous study.
Taking into account that phenotype BB is a characteristic of the rainbow trouts from stock 1 (Oliveira et al., 1996 -Table I, Figure 1n), the occurrence of phenotype AB in some individuals of stocks 6, 10, and 11 may be due to hy-  bridization processes between fish from these stocks and fish from stock 1.Considering the managing process of the stocks involved, this was expected, because eggs and fries of the rainbow trouts from stock 1 have been widely distributed throughout trout farms in the states of São Paulo, Rio de Janeiro and Minas Gerais, where this species is cultivated.Cytogenetic analysis revealed that all fish of stock 12 exhibited phenotype BB (Figure 1o).It was also observed that one of the submetacentric chromosomes had a double mark at the terminal portion of its long arm, similar to that found by Oliveira et al. (1996) in stock 1.
The presence of phenotype BB and of a characteristic chromosome with two NOR segments in the individuals from stock 1 (from Núcleo Experimental de Salmonicultura de Campos do Jordão) and stock 12 (Gavião river) suggests that both stocks have a common ancestor.This observation agrees with MacCrimmon (1971), who reported that the rainbow trout samples introduced in the mountain areas of São Paulo state came from California in the early 1960's.The study of other naturalized populations and the use of alternative molecular markers may lead to a better understanding of the relationships among rainbow trout stocks in Brazil.

Table I -
List of the rainbow trout (Oncorhynchus mykiss) stocks employed for cytogenetic analyses and identification of NOR phenotypes.