P. Moran

Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5S rDNA

Atlantic salmon 5S ribosomal DNA (5S rDNA) was amplified by the polymerase chain reaction, using as primers conserved sequences from the coding region of rainbow trout 5S rRNA. Two amplified products of different molecular weights were obtained, cloned, and sequenced, revealing them to be tandemly arranged. The nucleotide sequences differed between the two clones in the length of the nontranscribed spacer (NTS) and in three nucleotides of the coding sequence. By means of fluorescence in situ hybridization the 5S rDNA was chromosomally located in the heterochromatic arm of the pair bearing the satellite, adjacent to the major ribosomal DNA locus (rDNA).

Sex chromosome linkage of 5S rDNA in rainbow trout (Oncorhynchus mykiss)

The karyotype of the rainbow trout is characterized by a primitive XX/XY sex-determining chromosomal system. (Thorgaard et al., 1977). In the present study using FISH we have physically linked the 5S rRNA genes to the partially undifferentiated X chromosome pair. PCR amplified 5S rDNA was used for FISH and hybridization signals indicated that the genes were duplicated, present in one acrocentric and one metacentric pair of chromosomes. After analyzing several individuals, the female metaphases showed four fluorescent signals whereas males presented only three signals. Two of the three signals obtained in males corresponded to the metacentric pair whereas the single signal was mapped to the heterochromatin that cytologically differentiates the X chromosome from the Y chromosome. Double FISH experiments demonstrated that the 5S rDNA which is not sex linked is located at the NOR bearing arm close to the major ribosomal RNA genes (5.8S, 18S and 28S), similar to the situation observed in Atlantic salmon (Pendas et al., 1994a).

Ribosomal RNA genes are interspersed throughout a heterochromatic chromosome arm in Atlantic salmon

The ribosomal RNA genes (rDNA) have been mapped by fluorescent in situ hybridization (FISH) using four rDNA probes (rDNA/FISH) to Atlantic salmon chromosomes. Signals appeared over the whole heterochromatic chromosome arm displaying the secondary constriction and satellites. The size polymorphism of this sort arm, revealed by C-banding, was confirmed by rDNA/FISH, supporting large interindividual differences in the number of rDNA copies. Conventional techniques for the detection of nucleolar organizer regions are discussed, and their results are compared with those of rDNA/FISH.

Applications of 5S rDNA in Atlantic salmon, brown trout, and in Atlantic salmon brown trout hybrid identification

The members of the salmonid family form perhaps the most economically important group of the world’s fish species. In Europe the most important species employed in fish farming are the introduced rainbow trout (Oncorhynchus mykiss) and the Atlantic salmon (Salmo salnr), whereas interest in the brown trout (Salmo trufta) is based on its ecological diversity and sport fishing. These two Salmo species are very difficult to distinguish using morphological characters, not only during their first months of life (eggs and alevins) but also at the returning adult stage. Isoenzyme genetic analysis, which has usually been used for their identification, has often demonstrated that anglers have confused adults of brown trout with Atlantic salmon, or with salmon x trout hybrids (Leaniz & Verspoor 1989). Caution must therefore be taken in enhancement programmes since the artificial spawning of adults caught in the river could lead to restocking with salmon x trout hybrids. Another area of interest is the identification of manufactured products of these species (i.e. smoked). Genetic analysis potentially provides the only reliable method to unambiguously determine their species identity. In addition, the genetic marker employed should be a nuclear marker if interspe cific hybrids among these species have to be recognized. From a practical point of view, the procedure chosen must be fast, extremely robust, and be able to utilize tiny quantities of any tissue. This last requirement excludes most isoenzyme techniques. At present, PCR-based methodologies constitute the most reliable techniques available for this purpose. Most of these approaches are, however, based on the use of conserved mitochondria1 DNA primers (for the cytochrome b gene) and sequencing of the amplified fragment (Paabo et al. 1989; Bartlett h Davidson 1992). Instead, we have focused on the 5s

Multi-chromosomal location of ribosomal RNA genes and heterochromatin association in brown trout.

The ribosomal rRNA genes have been mapped by fluorescentin situ hybridization (FISH) to brown trout chromosomes. One major NOR chromosome pair and 8 novel minor NOR chromosome pairs have been found. Both major and minor NORs were closely related to polymorphic heterochromatin, as revealed by FISH and C-banding. These results are discussed with respect to NOR expression, the relationship between rDNA and heterochromatin, and evolutionary aspects.

Multiple paternity increases effective size of southern Atlantic salmon populations

Genetic analyses were performed on the progeny of Atlantic salmon (Salmo salar L.) sampled in natural redds of three rivers flowing into the Bay of Biscay, the Nivelle, the Mandeo and the Sella. These rivers are at the southern limit of the European distribution of the species and their populations are small and endangered by human activities. Nine variable number of tandem repeat (VNTR) loci (five minisatellites and four microsatellites) were used for parentage analysis. Multiple male participation was recognized in the fertilization of eggs. A large proportion was fertilized by precociously mature parr. We demonstrate that multiple paternity derived from mature parr is crucial for the conservation of genetic variability in small populations of Atlantic salmon.

Chromosomal localization of the major and 5S rRNA genes in the European eel (Anguilla anguilla)

The major (18S, 5.8S, and 28S) and 5S rRNA genes have been mapped by double fluorescent in situ hybridization to European eel metaphase chromosomes. The major rRNA genes were localized to a submetacentric pair of chromosomes that showed a consistent size polymorphism among the individuals studied. The 5S rRNA genes were clustered in a single locus that mapped to the centromeric region of an acrocentric pair. In contrast to the major rRNA genes, no detectable polymorphism, in either size or intensity of the fluorescent signal, was observed. The chromosomal organization of both families of rRNA genes are discussed in terms of genomic organization and chromosomal evolution.

Genomic analysis of the vitellogenin locus in rainbow trout (Oncorhynchus mykiss) reveals a complex history of gene amplification and retroposon activity.

Abstract Vitellogenins (Vtg) are the major yolk proteins in most oviparous organisms. They are encoded by a small number of genes – between one and four depending on the species. Characterization of the Vtg region in the genome of the rainbow trout reveals unusual features, however, in that this locus contains twenty complete genes and ten pseudogenes per haploid genome. The Vtg genes differ from each other by insertion, deletion and rearrangement events, although, at the sequence level, they show a high degree of similarity. Fluorescent in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE) and Southern analysis indicate that all gene copies are contained in a single 1500-kb region, and that most of the genes form tandem arrays separated by a conserved 4.5-kb intergenic region. The presence of large reiterated fragments indicates that this region has been subjected to several amplification events. The presence of a retroposon element (called I9) in Vtg intron 9 appears to be responsible for the silencing of at least nine of the ten pseudogenes. Two other incomplete retrotransposons (one LTR- and one LINE-type) and sequences derived from a HIV-like retrovirus are inserted into the conserved intergenic region, very close to the transcription start site. Their presence in all Vtg 5′-flanking regions suggests a possible role in gene amplification at this locus.

Genetic assessment of the reproductive success of Atlantic salmon precocious parr by means of VNTR loci

Atlantic salmon precocious parr are dwarf mature males able to mate with adult females making profit of their small size. In this work the reproductive success of the dwarf males in the presence of an adult male and female spawning couple has been estimated in three different situations under simulated natural conditions at the Lapitxuri experimental channel (France). Different hypervariable minisatellite loci were used in the paternity assessment of the progeny. In all the situations tested, precocious parr were able to fertilize between 24.7 and 89.3 per cent of the total eggs. The results are discussed in relation to the important role of Atlantic salmon precocious parr in population structure and the evolution of salmon populations.

Organization and chromosomal location of the major histone cluster in brown trout, Atlantic salmon and rainbow trout

The major histone cluster (hisDNA) was mapped by fluorescent in situ hybridization (FISH) to mitotic chromosomes of Atlantic salmon, brown trout, and rainbow trout. The data reveal that in the three species hisDNA is tandemly repeated in a single locus. Southern blots of genomic DNA indicate that these clusters are representative of the vast majority of the histone genes in these species. Similar reiteration values were found among the three species. Genetic variability in the hisDNA was found only in brown trout for an EcoRI site.