Quantitative trait loci for magnitude of the plasma cortisol response to confinement in rainbow trout.

Better understanding of the mechanisms underlying interindividual variation in stress responses and their links with production traits is a key issue for sustainable animal breeding. In this study, we searched for quantitative trait loci (QTL) controlling the magnitude of the plasma cortisol stress response and compared them to body size traits in five F2 full-sib families issued from two rainbow trout lines divergently selected for high or low post-confinement plasma cortisol level. Approximately 1000 F2 individuals were individually tagged and exposed to two successive acute confinement challenges (1 month interval). Post-stress plasma cortisol concentrations were determined for each fish. A medium density genome scan was carried out (268 markers, overall marker spacing less than 10 cM). QTL detection was performed using qtlmap software, based on an interval mapping method (http://www.inra.fr/qtlmap). Overall, QTL of medium individual effects on cortisol responsiveness (<10% of phenotypic variance) were detected on 18 chromosomes, strongly supporting the hypothesis that control of the trait is polygenic. Although a core array of QTL controlled cortisol concentrations at both challenges, several QTL seemed challenge specific, suggesting that responses to the first and to a subsequent exposure to the confinement stressor are distinct traits sharing only part of their genetic control. Chromosomal location of the steroidogenic acute regulatory protein (STAR) makes it a good potential candidate gene for one of the QTL. Finally, comparison of body size traits QTL (weight, length and body conformation) with cortisol-associated QTL did not support evidence for negative genetic relationships between the two types of traits.

Sex differentiation in an all-female (XX) rainbow trout population with a genetically governed masculinization phenotype.

Sex determination is known to be male heterogametic in the rainbow trout, Oncorhynchus mykiss; however, scattered observations that deviate from this rather strict genetic control have been reported. Here, we provide a detailed morphological and histological characterization of the gonadal differentiation and development (from 43 days postfertilization to 11 months of age) in an all-female (XX) population with a genetically governed masculinization phenotype. In comparison with control males and females, the gonadal differentiation in these animals was characterized by many perturbations, including significantly fewer germ cells. This decrease in germ cells was confirmed by the significantly decreased expression of 2 germ cell maker genes (vasa and sycp3) in the masculinized XX populations as compared with the control females and control males. Although only a proportion of the total adult population was partially or fully masculinized, this early differentiating phenotype affected nearly all the sampled animals. This suggests that the adult masculinization phenotype is the consequence of an early functional imbalance in ovarian differentiation in the entire population. We hypothesize that the lower number of germ cells that we observed in this population could be one cause of their masculinization.

Evidence of two contrasting brown trout Salmo trutta populations spatially separated in the River Borne (France) and shift in management towards conservation of the native lineage.

A multidisciplinary study was made of brown trout Salmo trutta in the Borne River, a typical fast-flowing mountain stream in the Northern French Alps, in the geographical range of the Mediterranean lineages (ML). Information on (1) the proportion of stocked fluoro-marked fish in the angling harvest, (2) the introgression of introduced DNA microsatellite alleles into the native gene pool and (3) the demography of the population in situ in autumn revealed two contrasting populations separated by a physical barrier to upstream migration. A native S. trutta population (c. 10 000 adults) lives downstream of the barrier and is characterized by a large frequency of ML alleles (82-97%) and high densities (43-55 fish 100 m(-2)). This population is maintained predominantly by natural recruitment of juveniles (51-82%). In contrast, the upstream population is characterized by a large frequency of Atlantic lineage (AL) alleles (78-100%) and low densities (1-2 fish 100 m(-2)) and appears to be maintained by restocking (90-100%). The origins of these sharply contrasting populations appear to reflect isolation by an impassable barrier, catastrophic flooding, a downstream gradient in water quality, stocking and fishing pressure. The native downstream population has been resilient to large sudden floods and to intensive stockings of domesticated AL fish. The results of this study justify a shift in management towards conservation and rehabilitation of the native population.

Large extent of mitochondrial DNA transfer from Oreochromis aureus to O. niloticus in West Africa.

Introgressive hybridization has an important evolutionary significance in terms of gene diversity and speciation. Among the major groups of vertebrates, fish show a strong propensity to hybridize. In order to highlight the possible occurrence of gene flow between two tilapia species, Oreochromis niloticus and O. aureus, a comparison of allozyme and mitochondrial DNA (mtDNA) polymorphism was performed on sympatric and allopatric populations of these two species. Nuclear data were congruent with the morphological identification of O. niloticus and O. aureus populations. In opposition, the mtDNA analysis resulted in two strictly differentiated groups which did not follow the morphological and nuclear DNA classification. The first group consisted of East African O. niloticus populations and the second included all the O. aureus populations and the West African O. niloticus populations. Moreover, in some cases, the same sequences were detected in both species. These data strongly support a differential introgression of mtDNA from O. aureus to O. niloticus involving all the West African area. This work points out the risk of misinterpretation of mtDNA or nuclear DNA data when only one single class of marker is used.

Genetic structure and phylogeography of European catfish (Silurus glanis) populations.

The genetic structure of Silurus glanis (Europe's largest freshwater fish species) across most of its natural distribution was investigated using 10 microsatellite loci. The revealed levels of genetic diversity were much higher than previous allozyme and restriction fragment length polymorphism mitochondrial DNA analyses had shown; relative levels of variability among populations were however, in good agreement with the previous studies. Populations from large basins (Volga and Danube rivers) were the most polymorphic, while samples from the smaller Greek rivers, which are more prone to genetic bottleneck, exhibited the lowest levels of genetic diversity. Microsatellite multilocus genotyping permitted the assignment of individual fish to their population of origin with a score as high as 98.3%. Despite the great genetic differentiation of S. glanis populations, no consistent pattern of geographical structuring was revealed, in contrast to previous studies of European freshwater fish species. A model of isolation by distance seems more probable and a hypothesis of recent dispersion from only one glacial refugium is proposed. The discovery of the highest levels of microsatellite and mitochondrial diversity in the Volga sample and the presence of river connections, during the Pleistocene, between this area and all major areas of the present catfish distribution, place this refugium around the Ponto-Caspian region. Combining these data with those from previous studies, a number of markers are now available to monitor wild and hatchery populations even at the individual level.

Juxtaposed microsatellite systems as diagnostic markers for admixture: an empirical evaluation with brown trout (Salmo trutta) as model organism.

A juxtaposed microsatellite system (JMS) is composed of two microsatellite repeat arrays separated by a sequence of less than 200 bp and more than 20 bp. This paper presents the first empirical evaluation of JMSs for the study of genetic admixture induced by man, with brown trout (Salmo trutta) as model organism. Two distinct admixture situations were studied: native populations from streams of the Atlantic basin and of the Mediterranean basin, respectively, all stocked with domestic strains originating from the Atlantic basin. For these two situations, we first evaluated by simulation the ability of JMSs to differentiate between alien alleles and naturally shared homoplasious or ancestral alleles, and thus to behave as diagnostic markers for admixture. Simulations indicated that JMSs are expected to be reliable diagnostic markers in most divergent (i.e. Mediterranean) populations and nonreliable diagnostic markers in most closely related (i.e. Atlantic) populations. Three JMSs were genotyped in domestic strains as well as in nonstocked and stocked populations of brown trout sampled in different rivers of the Mediterranean and Atlantic basins. The observed distributions of JMS haplotypes were consistent with simulation predictions confirming that JMSs were reliable diagnostic markers only over a given proportion of the species range, i.e. in substantially divergent populations. JMSs also reinforced the diagnostic character of three microsatellite sites for the studied Mediterranean populations. This last result is consistent with our simulation results which showed that, although much less frequently than at JMSs, diagnostic markers are likely to be found at single site microsatellites provided that the native Mediterranean population has a sufficiently small effective population size. For each population of the Mediterranean basin admixture coefficients did not differ significantly across JMSs and mean admixture coefficients sometimes differ among populations. The interpretation of the origin of JMS haplotypes based on the allele length variants was supported by nucleotide sequence analysis.

A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates.

We constructed a genetic linkage map for a tetraploid derivative species, the rainbow trout (Oncorhynchus mykiss), using 191 microsatellite, 3 RAPD, 7 ESMP, and 7 allozyme markers in three backcross families. The linkage map consists of 29 linkage groups with potential arm displacements in the female map due to male-specific pseudolinkage arrangements. Synteny of duplicated microsatellite markers was used to identify and confirm some previously reported pseudolinkage arrangements based upon allozyme markers. Fifteen centromeric regions (20 chromosome arms) were identified with a half-tetrad analysis using gynogenetic diploids. Female map length is approximately 10 M, but this is a large underestimate as many genotyped segments remain unassigned at a LOD threshold of 3.0. Extreme differences in female:male map distances were observed (ratio F:M, 3.25:1). Females had much lower recombination rates (0.14:1) in telomeric regions than males, while recombination rates were much higher in females within regions proximal to the centromere (F:M, 10:1). Quadrivalent formations that appear almost exclusively in males are postulated to account for the observed differences.

Comparative analysis of microsatellite and allozyme markers: a case study investigating microgeographic differentiation in brown trout (Salmo trutta).

A comparative study between microsatellite and allozyme markers was conducted on natural populations of resident brown trout (Salmo trutta) sampled over a reduced geographical scale and on hatchery strains. The higher level of polymorphism observed at microsatellite loci resulted in higher power of statistical tests for differentiation among population samples and for genotypic linkage disequilibrium. Genetic distances of Cavalli-Sforza and Edwards were on average two times larger for microsatellites than for allozymes but multilocus FST estimates computed over the entire set of populations were not significantly different for both categories of markers. Assignment tests of individual fish to the set of sampled populations demonstrated a much higher efficiency of microsatellites compared to allozymes. Pairwise multilocus FST estimates were significantly correlated to waterway distances and there was a significant tendency for the incorrectly classified individuals to be assigned to one of the nearest populations, indicating that isolation-by-distance acted significantly on brown trout populations. This increase of differentiation with distance was higher for allozymes than for microsatellites. Traditional measures of genetic differentiation (Cavalli-Sforza and Edwards' chord distance and FST) were compared for microsatellites to recently proposed statistics taking into account allele size differences (Goldstein's distance and PST). Using Goldstein's distance for neighbour-joining analysis did not improve the tree structure resolution. Multilocus estimates of PST and FST were not significantly different when computed over the entire set of populations but no significant correlation was detected between matrices of pairwise multilocus PST estimates and waterway distances.

Mitochondrial DNA differentiation among East and West African Nile tilapia populations

Variation of the ND5/6 mtDNA fragment was studied in six Nile tilapia populations using PCR and RFLP analysis. The observed variation allows a strict discrimination between eastern and western African populations.

Mitochondrial control region and protein coding genes sequence variation among phenotypic forms of brown trout Salmo trutta from northern Italy.

The Pô River basin of northern Italy is the home of distinctive and endemic morphological forms of brown trout Salmo trutta. We used PCR-direct sequencing and RFLP techniques to study variation in the mitochondrial control region of 225 trout in order to assess genetic relatedness among 18 populations from that region. The distribution analysis of these genotypes among north Italian populations confirmed the phylogenetic differentiation of marbled trout Salmo trutta marmoratus populations and the postglacial origin of S. t. carpio. Extensive genetic heterogeneity was observed among morphologically identical S. t. fario populations. Introgression with domestic strains of Atlantic basin origin was detected in all forms. In order to assess the phylogenetic congruence detected in coding and noncoding regions of the mitochondrial genome, we also analysed sequence variation in segments of the cytochrome b and ATPase subunit VI genes among representatives of all variants detected in the analysis of the control region. Variation in protein coding genes was only slightly less than that observed in the control region of the same individuals, both in terms of number of variants detected and of pairwise sequence divergence estimates among variants. Phylogenetic analysis based on protein coding genes sequences identified the same phylogenetic groupings defined by the control region analysis and also allowed a partial resolution of their phyletic relationships that was previously unresolved. However, coding and noncoding segments differed substantially in the transition-transversion ratio (17:0 in coding segments vs. 17:6 in control region segments).

(CT)n and (GT)n microsatellites: a new class of genetic markers for Salmo trutta L. (brown trout).

Thirteen (GT)n and four (CT)n microsatellite loci (n = 10 or more and n = 20 or more, respectively) have been isolated from a partial genomic library of brown trout and sequenced. On average, a (GT)n repeat sequence occurs approximately every 23 kb and a (CT)n repeat sequence every 76 kb in brown trout genome. Primers for DNA amplifications using the polymerase chain reaction (PCR) were synthesized for three single locus microsatellites. Mendelian inheritance of the observed polymorphisms was confirmed in full-sib families. Four brown trout populations (10 unrelated individuals per population) were screened for polymorphism with these three microsatellite loci. The total number of alleles detected in the four populations is five at one locus, six at the other two microsatellite loci and is three, on average, per population. Heterozygosities range from 0.18 to 0.74. The largest differences in allelic frequencies occurred between the Mediterranean and the Atlantic populations: this result is congruent with previous allozymic data. The gene-centromere distances of the three microsatellite markers were determined on gynogenetic lines: post-reduction rates range from 0.17 to 0.60. For all the three microsatellite loci, the primers designed from brown trout sequences can be used in another closely related species of salmonid, the rainbow trout (Oncorhynchus mykiss). This last aspect supports the view that microsatellite markers may have wide application in genetic studies in salmonid species and fishes in general.

DNA sequence variation of the mitochondrial control region among geographically and morphologically remote European brown trout Salmo trutta populations.

Throughout its natural range, the brown trout Salmo trutta L. exhibits a complex pattern of morphological and life-history variation. This has led to considerable taxonomic confusion, hampering the understanding of the evolutionary history of the species. To document the phylogenetic relationships among morphologically and geographically remote brown trout populations across western Europe, we determined the DNA sequence variation in segments of the mitochondrial control region for 151 individuals representing 24 populations. DNA was prepared for double-stranded sequencing by the polymerase chain reaction (PCR). Twenty-one variable nucleotide positions within a 640-bp fragment surveyed defined 12 genotypes differing by a mean of 7 nucleotide substitutions (range 1-12). Five major phylogenetic assemblages differing by mean sequence divergence estimates of 0.96 to 1.44% were identified. These groupings exhibited a strong spatial partitioning but lacked congruence with either ecological or morphological differentiation. Complete mitochondrial DNA (mtDNA) monomorphism across all Atlantic basin populations contrasted with the high interdrainage genetic diversity observed in more southerly populations. This study exemplified the usefulness of mitochondrial DNA sequence analysis for estimating phylogenetic relationships within S. trutta populations.

Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs.

Persistence, integration into host genome, germ line transmission and expression of foreign genes microinjected into cytoplasm of fertilized rainbow trout eggs has been examined. Foreign DNA persisted as large random concatenates in approximately 50% of 6 to 12 month-old trout and exhibited a mosaic pattern between tissues. In some cases, free concatenates were observed indicating that extrachromosomal replication occurred in trout. Approximately 50% of the males had the foreign sequences in sperm DNA and all the examined animals transmitted these sequences to their progeny. The percentage of transgenic offsprings ranged from 10 to 30% and putative junction fragments were identified in Southern blot analysis in some of them. These results strongly support the hypothesis that the injected genes became integrated into the genome host, most likely after the first round of chromosomal replication. We also examined the expression of the microinjected plasmids which contained viral or mammalian promoters linked to human or rat growth hormone gene. In no case could exogenous growth hormone be detected.

Gene segregation in induced tetraploid rainbow trout: genetic evidence of preferential pairing of homologous chromosomes.

Gene segregation at six protein loci was analysed in progeny from tetraploid males and females obtained by suppression of first mitosis. The triploid full-sib families from five tetraploid males and the diploid gynogenetic lines from four tetraploid females were examined. The proportions of heterozygous gametes (0.83 on the average) were significantly higher than expected from tetrasomic inheritance (0.667) at all the loci studied. This was explained by preferential pairing of homologous chromosomes. The proportions of heterozygous gametes were significantly different between loci, but the variations were not correlated with the gene--centromere distances. Our results showed that, at least for one locus, the homozygous gametes mainly resulted from pairing of homologous chromosomes rather than from pairing of homologous chromosomes, quadrivalent formation, and chromatin exchanges between homologous chromosomes.

High level of residual heterozygosity in gynogenetic rainbow trout, Salmo gairdneri, Richardson.

Nine gynogenetic lines of rainbow trout and their full-sib controls were used to study genetic variation at 14 protein loci and at one body color locus. The maternal genotypes could be clearly determined from the analysis of the control full-sibs. Gynogenetic off springs only possessed the alleles of the mother. In these gynogenetic individuals, obtained by retention of the second polar body, the residual heterozygosity (r) was calculated at 8 loci. The following results were obtained: (1) The values of r varied greatly from one locus to another (from 0.11 to 1.00); (2) On the other hand, for a given locus, variations of r between females were not significant; (3) Four loci reached residual heterozygosity close to 1. The following hypotheses are put forward to explain these results: total interference, atypical meiosis and partial tetraploidization. The high residual heterozygosity mean observed in rainbow trout (0.71±0.11) is discussed with respect to inbred line production and management and a breeding scheme alternating gynogenesis and full-sib mating is suggested.

Electrophoretic variation in six populations of brown trout (Salmo trutta L.).

Starch gel electrophoretic studies of 16 enzymes encoded by 34 Loci were performed on six brown trout populations. One new polymorphism is described at the Pmi-2 locus. Breeding data were analysed for both single and joint segregation of six loci: Aat-1, Cpk-1, G3p-2, Mdh-2, Mdh-3, and Pmi-2. All the loci are shown to segregate in simple mendelian ratios and one nonrandom joint segregation was observed. The polymorphism level, heterozygosities, and genetic distances were estimated and compared with those reported in other studies on brown trout and closely related salmonid species. The polymorphism level (25%) and average heterozygosity (9%) were high. Significant genetic distances were observed, but the average degree of differentiation between populations appeared to be small (9% of the total heterozygosity).

[Electrophoretic evidence of a strong genetic differentiation between brown trout populations of Corsica]. / Mise en évidence électrophorétique d'une forte différenciation génétique entre populations de truite fario de Corse.

Electrophoretic variations of 18 enzyme systems encoded by 37 loci have revealed a high degree of genetic differentiation between three brown trout populations from Corsica. One of these populations could be Salmo trutta macrostigma Dum.