Genomic arrangement of salinity tolerance QTLs in salmonids: A comparative analysis of Atlantic salmon (Salmo salar) with Arctic charr (Salvelinus alpinus) and rainbow trout (

ABSTRACT: BACKGROUND: Quantitative trait locus (QTL) studies show that variation in salinity tolerance in Arctic charr and rainbow trout has a genetic basis, even though both these species have low to moderate salinity tolerance capacities. QTL were observed to localize to homologous linkage group segments within putative chromosomal regions possessing multiple candidate genes. We compared salinity tolerance QTL in rainbow trout and Arctic charr to those detected in a higher salinity tolerant species, Atlantic salmon. The highly derived karyotype of Atlantic salmon allows for the assessment of whether disparity in salinity tolerance in salmonids is associated with differences in genetic architecture. To facilitate these comparisons, we examined the genomic synteny patterns of key candidate genes in the other model teleost fishes that have experienced three whole-genome duplication (3R) events which preceded a fourth (4R) whole genome duplication event common to all salmonid species. RESULTS: Nine linkage groups contained chromosome-wide significant QTL (AS-2, -4p, -4q, -5, -9, -12p, -12q, -14q -17q, -22, and [MINUS SIGN]23), while a single genome-wide significant QTL was located on AS-4q. Salmonid genomes shared the greatest marker homology with the genome of three-spined stickleback. All linkage group arms in Atlantic salmon were syntenic with at least one stickleback chromosome, while 18 arms had multiple affinities. Arm fusions in Atlantic salmon were often between multiple regions bearing salinity tolerance QTL. Nine linkage groups in Arctic charr and six linkage group arms in rainbow trout currently have no synteny alignments with stickleback chromosomes, while eight rainbow trout linkage group arms were syntenic with multiple stickleback chromosomes. Rearrangements in the stickleback lineage involving fusions of ancestral arm segments could account for the 21 chromosome pairs observed in the stickleback karyotype. CONCLUSIONS: Salinity tolerance in salmonids from three genera is to some extent controlled by the same loci. Synteny between QTL in salmonids and candidate genes in stickleback suggests genetic variation at candidate gene loci could affect salinity tolerance in all three salmonids investigated. Candidate genes often occur in pairs on chromosomes, and synteny patterns indicate these pairs are generally conserved in 2R, 3R, and 4R genomes. Synteny maps also suggest that the Atlantic salmon genome contains three larger syntenic combinations of candidate genes that are not evident in any of the other 2R, 3R, or 4R genomes examined. These larger synteny tracts appear to have resulted from ancestral arm fusions that occurred in the Atlantic salmon ancestor. We hypothesize that the superior hypo-osmoregulatory efficiency that is characteristic of Atlantic salmon may be related to these clusters.     

QTL for body weight and condition factor in Atlantic salmon (Salmo salar): comparative analysis with rainbow trout (Oncorhynchus mykiss) and Arctic charr (Salvelinus alpinus)

Genotypes at 91 microsatellite loci in three full-sib families were used to search for QTL affecting body weight (BW) and condition factor in North American Atlantic salmon (Salmo salar). More than one informative marker was identified on 16-18 linkage groups in each family, allowing at least one chromosomal interval to be analyzed per linkage group. Two significant QTL for BW on linkage groups AS-8 and AS-11, and four significant QTL for condition factor on linkage groups AS-2, AS-5, AS-11, and AS-14 were identified. QTL for both BW and condition factor were located on linkage groups AS-1, 6, 8, 11, and 14 when considering both significant and suggestive QTL effects. The largest QTL effects for BW (AS-8) and for condition factor (AS-14) accounted for 20.1 and 24.9% of the trait variation, respectively. Three of the QTL for BW occur on linkage groups where similar effects have been detected on the homologous regions in either rainbow trout (Oncorhynchus mykiss) or Arctic charr (Salvelinus alpinus).     

Quantitative trait loci for body weight, condition factor and age at sexual maturation in Arctic charr (Salvelinus alpinus): comparative analysis with rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In salmonid fishes, life-history changes may often be coupled to early individual growth trajectories. We identified quantitative trait loci (QTL) for body weight (BW), condition factor (K) and age at sexual maturation (MT) in two full-sib families of Arctic charr (Salvelinus alpinus) to ascertain if QTL for MT were confounded with BW QTL intervals. Three significant QTL for BW, three QTL for MT and one significant QTL for K were identified. A BW QTL with major effect was localized to linkage group 8 (AC-8) and explained more than 34% of the phenotypic variation. Markers on AC-8 have previously been identified as being associated with variation in fork length and BW in this species. Similarly, a major QTL (PEV = 23%) with an influence on the female MT was localized to AC-23. Some of these regions are homologous to those in the genomes of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), where similar QTL effects have been detected. Our results also suggest the conservation of MT QTL on the homeologous linkage group pair AC-3/24 in Arctic charr. We further identified chromosomal regions that harbor QTL for multiple traits. In particular, markers on AC-4, -20 and -36 had detectable QTL for all traits studied. Significant MT QTL detected on AC-23, -24, and -27 were autonomous of any BW QTL regions, suggesting that the regulation of MT may be more independent of BW control within this species than in other species of salmonids.     

Evolution of the immunoglobulin M constant region genes of salmonid fish,rainbow trout (Oncorhynchus mykiss) and Arctic charr (Salvelinus alpinus): implications concerning divergence time of species

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X83372 (Oncorhynchus mykiss) and X83373 (Salvelinus alpinus)     

A linkage map for brown trout (Salmo trutta): chromosome homeologies and comparative genome organization with other salmonid fish

We report on the construction of a linkage map for brown trout (Salmo trutta) and its comparison with those of other tetraploid-derivative fish in the family Salmonidae, including Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and Arctic char (Salvelinus alpinus). Overall, we identified 37 linkage groups (2n = 80) from the analysis of 288 microsatellite polymorphisms, 13 allozyme markers, and phenotypic sex in four backcross families. Additionally, we used gene-centromere analysis to approximate the position of the centromere for 20 linkage groups and thus relate linkage arrangements to the physical morphology of chromosomes. Sex-specific maps derived from multiple parents were estimated to cover 346.4 and 912.5 cM of the male and female genomes, respectively. As previously observed in other salmonids, recombination rates showed large sex differences (average female-to-male ratio was 6.4), with male crossovers generally localized toward the distal end of linkage groups. Putative homeologous regions inherited from the salmonid tetraploid ancestor were identified for 10 pairs of linkage groups, including five chromosomes showing evidence of residual tetrasomy (pseudolinkage). Map alignments with orthologous regions in Atlantic salmon, rainbow trout, and Arctic char also revealed extensive conservation of syntenic blocks across species, which was generally consistent with chromosome divergence through Robertsonian translocations.     

Spatial arrangement of fish gill secondary lamellar cells in intact and dissociated tissues from rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Gill structure of rainbow trout and Atlantic salmon was investigated using cell disaggregation and dry fracture techniques for scanning electron microscopy (SEM), allowing new interpreta-tions of the structure of the secondary lamella. The basement membrane underlying the lamellar epithelium (secondary epithelium) was shown to be a tough sheet with numerous depressions corresponding to underlying pillar cells. This membrane is probably the most important structural element of the secondary lamella, capable of withstanding considerable mechanical stress. For the first time the structure of the apical surface of the secondary lamella was shown by SEM to consist of an outer microridged coat overlying a fibrous coat which appears continuous with the extracellular matrix surrounding the rest of the cell. When cells were detached they rounded up and the external microridged coat became more vesicle like, indicating the labile nature of this coat. In cell suspension preparations, epithelial, mucus and chloride cells are present as well as many blood derived cells such as erythrocytes, presumptive leucocytes and thrombocytes.     

A new type of cysteine proteinase inhibitor--the salarin gene from Atlantic salmon (Salmo salar L.) and Arctic charr (Salvelinus alpinus)

Salarin is a 43 kDa glycoprotein which is found so far only in salmonid fish species. It is a strong inhibitor of cysteine proteinases. Here we characterised the salarin gene from Atlantic salmon and cDNA from Arctic charr. The salarin gene has 13 exons and 1026 bp long coding sequence. The translated amino acid sequence has four similar domains. The sequence resembles the proregion of cathepsins, known to inhibit cysteine proteinases. Salarin can be a new type of cysteine proteinase inhibitor.     

A comparative mitogenomic analysis of the potential adaptive value of Arctic charr mtDNA introgression in brook charr populations (Salvelinus fontinalis Mitchill)

Wild brook charr populations (Salvelinus fontinalis) completely introgressed with the mitochondrial genome (mtDNA) of arctic charr (Salvelinus alpinus) are found in several lakes of northeastern Québec, Canada. Mitochondrial respiratory enzymes of these populations are thus encoded by their own nuclear DNA and by arctic charr mtDNA. In the present study we performed a comparative sequence analysis of the whole mitochondrial genome of both brook and arctic charr to identify the distribution of mutational differences across these two genomes. This analysis revealed 47 amino acid replacements, 45 of which were confined to subunits of the NADH dehydrogenase complex (Complex I), one in the cox3 gene (Complex IV), and one in the atp8 gene (Complex V). A cladistic approach performed with brook charr, arctic charr, and two other salmonid fishes (rainbow trout [Oncorhynchus mykiss] and Atlantic salmon [Salmo salar]) revealed that only five amino acid replacements were specific to the charr comparison and not shared with the other two salmonids. In addition, five amino acid substitutions localized in the nad2 and nad5 genes denoted negative scores according to the functional properties of amino acids and, therefore, could possibly have an impact on the structure and functional properties of these mitochondrial peptides. The comparison of both brook and arctic charr mtDNA with that of rainbow trout also revealed a relatively constant mutation rate for each specific gene among species, whereas the rate was quite different among genes. This pattern held for both synonymous and nonsynonymous nucleotide positions. These results, therefore, support the hypothesis of selective constraints acting on synonymous codon usage.     

A comparative study of the relationship between light intensity and feeding ability in brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus)

1. Field observations indicate that the ability to feed at different light intensities may differ between brown trout and Arctic charr, and this is the first study to test this experimentally. To establish a background level of feeding in daylight at midday, trout and charr in two size groups were kept in tanks (one fish per tank) at three constant temperatures (5.0, 10.8 and 13.0 °C) and each fish was offered, one at a time, 50 freshly killed shrimps (Gammarus pulex), the number eaten being recorded. Shrimps could only be taken in the water column because a metal mesh prevented access to dead shrimps on the tank bottom. In a first series of experiments, individual fish were kept at one of 10 natural light intensities (range 0.001–50 lx). In a second series, conditions were similar except that the fish tank was covered in black polyethylene and had a light‐tight lid with a shutter so that light levels could be kept constant, using artificial illumination. In a third series, the fish were fed in total darkness, but the false bottom was removed, allowing access to dead shrimps on the tank bottom as well as in the water column. 2. The results of the first and second series differed interspecifically but were very similar intraspecifically, with no significant differences between the food intake for the two size groups or in the experiments at 10.8 and 13.0 °C. Food intake remained fairly constant at light intensities between 50 lx (dusk or dawn) and 0.03 lx and was similar to that of fish feeding at midday. At 10.8 and 13.0 °C, food intake between 0.03 and 50 lx was higher for trout than for charr, mean values for shrimps eaten per fish being 39.9 for trout (range 36–44, n = 100 fish) and 32.0 for charr (range 28–38, n = 100), but at 5.0 °C, the situation was reversed with mean values of 15.1 for trout (range 11–18, n = 50 fish) and 19.8 for charr (range 17–22, n = 50). 3. As light intensity decreased from 0.04 to 0.001 lx, feeding rate decreased exponentially but was always higher for charr than for trout, with a mean number of shrimps eaten at 0.001 lx of 9.3 for trout (range 5–13, n = 20 fish) and 13.6 for charr (range 9–20, n = 20) at 10.8 and 13.0 °C, and 2.0 for trout (range 1–4, n = 10 fish) and 5.5 for charr (range 2–8, n = 10) at 5.0 °C. In total darkness (false bottom fitted), none of the 50 shrimps was taken by either species. When the false bottom was removed in the third series, the mean number of shrimps consumed over 24 h was eight for trout (range 3–11, n = 20 fish) and 14.9 for charr (range 9–20, n = 20) at 10.8 and 13.0 °C, and two for trout (range 0–4, n = 10 fish) and five for charr (range 3–8, n = 10) at 5.0 °C. 4. Therefore, the feeding ability of trout was superior to that of charr when using photopic vision in daylight and mesotopic vision at dusk and dawn, but inferior to that of charr when using scotopic vision at low light intensity. Charr were also superior at low temperatures and when foraging for food in total darkness. Therefore, as light intensity decreases after dusk in their natural habitat, the advantage in feeding will shift from trout to charr, with the reverse occurring as light intensity increases after dawn.     

Polysialoglycoproteins of Salmonidae fish eggs. Complete structure of 200-kDa polysialoglycoprotein from the unfertilized eggs of rainbow trout (Salmo gairdneri)

Polysialoglycoproteins (PSGP) we first isolated from the unfertilized eggs of rainbow trout (Salmo gairderi) and now found to be a ubiquitous component of Salmonidae fish eggs are a novel type of glycoprotein. PSGP from rainbow trout has a molecular weight of 200 X 10(3), a low protein content (about 15% w/w), and a high sialic acid (N-glycolylneuraminic acid (NeuGc] content (about 60%, w/w). In any evaluation of the biological functions of PSGP, information about the complete structure of this unique macromolecular component is relevant. We have now completed the determination of the overall structural organization of the 200-kDa PSGP, and this is the first report of the complete structural analysis of this novel class of glycoprotein: (Asp)0-2-Ala-Thr*-Ser*-Glu-(Ala-Ala-Thr*-Gly-Pro-Ser-Gly-Asp-Asp-Ala-Thr *-Ser*- Glu)n-Ala-Ala-Thr*-Gly-Pro-Ser-Gly where * indicates the amino acid residues to which oligo- and/or polysialylglycan units are attached and n = 25. Thus the most outstanding structural features of PSGP isolated from the unfertilized eggs of rainbow trout are now the occurrence of (a) tandem repeats of a tridecapeptide and (b) an alpha-2----8-linked oligo(poly)sialyl group on each of the core oligosaccharide chains, i.e. GalNAc- beta 1----4(NeuGc alpha 2----3)GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n ----6)GalNAc alpha 1----Ser (or Thr), GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), and Gal beta 1----3[----8NeuGc alpha 2)n----6) GalNAc alpha 1----Ser (or Thr).     

Identification of the sex chromosomes of brown trout (Salmo trutta) and their comparison with the corresponding chromosomes in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Males are the heterogametic sex in salmonid fishes. In brown trout (Salmo trutta) the sex-determining locus, SEX, has been mapped to the end of linkage group BT-28, which corresponds to linkage group AS-8 and chromosome SSA15 in Atlantic salmon (Salmo salar). We set out to identify the sex chromosomes in brown trout. We isolated Atlantic salmon BAC clones containing microsatellite markers that are on BT-28 and also on AS-8, and used these BACs as probes for fluorescent in situ hybridization (FISH) analysis. SEX is located on the short arm of a small subtelocentric/acrocentric chromosome in brown trout, which is consistent with linkage analysis. The acrocentric chromosome SSA15 in Atlantic salmon appears to have arisen by a centric fusion of 2 small acrocentric chromosomes in the common ancestor of Salmo sp. We speculate that the fusion process that produced Atlantic salmon chromosome SSA15 disrupted the ancestral sex-determining locus in the Atlantic salmon lineage, providing the impetus either for the relocation of SEX or selection pressure for a novel sex-determining gene to arise in this species. Thus, the sex-determining genes may differ in Atlantic salmon and brown trout.     

The second population of Arctic charr Salvelinus alpinus complex (Salmoniformes, Salmonidae) in the Lake Baikal basin, the highest mountain charr population in Russia

We report the finding of the second population of Arctic charr in Lake Baikal basin, in a nameless lake in the Barguzin mountain range in the outlet of the Svetlaya River. The lake is situated at an altitude of 1766 m above sea level; this is the highest lake inhabited by this species in the territory of Russia. Two abundant charr forms, dwarf and small, were recorded in the lake; data on their ecology, morphology, age composition, growth, and microsatellite variation are presented. Both forms feed mainly on zooplankton, though neither has pronounced morphological traits characteristic of specialized plankton-feeders. Dwarf and small charrs are rather close in meristic characters and in the length of gill rakers, but differ substantially in allele frequencies and allele diversity at the microsatellite loci, which is indicative of a high degree of reproductive isolation between them. The discovered population is the only one among Transbaikalian charr populations, that, due to its remoteness, has not been affected by man. For that reason, it can be considered as an etalon of an undisturbed natural charr population. The lake and its surroundings should receive official protected status.     

A new species of trout from the northern drainages of Euphrates River,Turkey (Salmoniformes: Salmonidae)

Salmo fahrettini, a new species, is distributed in the northern tributaries of the Euphrates River. It differs from other Salmo species in adjacent waters by a combination of the following characters: a greyish body; one black spot behind the eye and on the cheek; three to six black spots on the opercle; numerous black spots on the back (missing on the predorsal area), flank and middle part of body, surrounded by a roundish white ring; red spots in the median part of the body, surrounded by a roundish white ring; short and narrow maxilla; increase in the number of black and red spots with an increase in size; adipose fin medium size, no or rarely one red spot at its posterior edge; 109–116 lateral line scales; 27–30 scale rows between dorsal-fin origin and lateral line; 20–23 scale rows between the lateral line and anal-fin origin; maxilla length 8.8–10.0% standard length in males, 8.8–9.6 in females.     

A family of repetitive nucleotide sequences (Sau 3A family) in the genome of 2 forms of the sockeye salmon Oncorhynchus nerka

A family of DNA sequences (Sau3A repeat sequences) has been revealed in genomes of two forms of O. nerka. A DNA fragment belonging to this "family" has been cloned in bacterial plasmid. Its copy number (in genomes of normal (570 +/- 33) and dwarf (66 +/- 12) forms) has been counted. The structural organization of sequences of Sau3A family in genomes of males and females of normal form is not the same. The organization of Sau3A sequences is the same in genomes of dwarf males and dwarf females of normal form, but the quantity of Sau3A sequences copies is smaller. It is supposed that the DNA sequences of Sau3A family bear the sex determination of dwarf form of O. nerka. The fragment of rRNA gene of nerka has been cloned and the number of rRNA gene copies in genomes of normal (200 +/- 62) and dwarf (1690 +/- 24) forms has been defined.     

A putative beta2-adrenoceptor from the rainbow trout (Oncorhynuchus mykiss). Molecular characterization and pharmacology.

Extensive molecular characterization of mammalian beta-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow trout beta2-adrenoceptor gene that codes for a 409-amino-acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout beta2-adrenoceptor shares a high degree of amino-acid sequence conservation with other vertebrate beta2-adrenoceptors. The conclusion that this sequence is a rainbow trout beta2-adrenoceptor is further supported by phylogenetic analysis of vertebrate beta-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout beta2-adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-protein-binding domain of the rainbow trout beta2-adrenoceptor sequence suggests that the in vivo control of trout beta2-adrenoceptor signaling differs substantially from that of mammals.     

Isolation, characterization, and chromosomal location of the tRNA(Met) genes in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta).

This work describes the isolation, characterization, and physical location of the methionine tRNA in the genome of Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.). An Atlantic salmon genomic library was screened using a tRNA(Met) probe from Xenopus laevis. Two cosmid clones containing the Atlantic salmon tRNA(Met) gene were isolated, subcloned and sequenced. The tRNA(Met) was mapped to metaphase chromosomes by fluorescence in situ hybridization (FISH). Chromosomal data indicated that the tDNA of methionine is tandemly repeated in a single locus in both species. Analysis of genomic DNA by Southern hybridization confirmed the tandem organization of this gene.     

The fine structure of the epidermis of two species of salmonid fish,the Atlantic salmon (Salmo salar L.) and the brown trout (Salmo trutta L.)

The fine structure of epidermal mucous cells of two species of salmonid fish has been described. Mucous cells are, next to filament-containing cells, the most commonly encountered cells in fish epidermis. The development of the cells as they progress to the periphery has been characterised. They are initially difficult to distinguish from filament-containing cells: later, they can be recognised by the presence of much smooth-surfaced E.R. The mucigenesis and the subsequent secretion of mucus has been observed and it is essentially comparable to that which occurs in the mucous cells of the mammalian intestine. The mucous layer of the epidermal surface seems to mainly comprise of the products of these mucous cells and the "cuticle" seen in other species has not yet been observed in the salmonid species investigated here.     

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells.

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.