Domestication shapes morphology in rainbow trout Oncorhynchus mykiss.

In this study, clonal lines from North American resident and migratory populations of rainbow trout Oncorhynchus mykiss adapted to different geographical conditions and with different domestication histories were characterized morphologically. Lines reared in a common-garden experiment were characterized for external shape and meristic values, searching for a general pattern of morphological variation due to exposure to captive conditions. A sharp distinction was identified between wild and captive lines. The body profile was deeper in captive lines, with longer dorsal and anal fins and shorter and deeper caudal peduncles. Highly significant differences were also identified in meristic values among the lines but no consistent relation between meristic values and domestication status was detected. This morphological characterization will facilitate the selection of lines with divergent phenotypes for subsequent quantitative trait loci analysis, aimed at identifying genome regions linked with morphological adaptive response to captive conditions.

Heteromorphic sex chromosomes in male rainbow trout.

A pair of subtelocentric chromosomes differs in the size of the short arm in male, but not female, rainbow trout (Salmo gairdneri). The morphological similarity of the X and Y chromosomes, and the observation of Y chromosomes intermediate between the X and normal Y, suggest that the sex chromosomes are at an early stage of differentiation in this species.

Recombination is suppressed over a large region of the rainbow trout Y chromosome.

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus (SEX), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy-163 marker, which maps close to SEX, was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.

The cutthroat trout Y chromosome is conserved with that of rainbow trout.

Five genetic markers previously shown to be located on the sex chromosomes of rainbow trout (Oncorhynchus mykiss) were tested for linkage with the sex locus of Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) in a genetic cross created from a rainbow x cutthroat male hybrid. We show that the sex locus of both rainbow and cutthroat trout is on the same homologous linkage group. Fluorescence in situ hybridization (FISH) using a probe for the microsatellite marker Omm1665, which maps close to the sex locus of Yellowstone cutthroat trout, was used to identify the Y chromosome of cutthroat trout in the hybrid. The Y chromosome of cutthroat trout is sub-telocentric and lacks a DAPI band found on the short arm of the Y chromosome of some rainbow trout males.

Use of androgenesis for estimating maternal and mitochondrial genome effects on development and oxygen consumption in rainbow trout, Oncorhynchus mykiss.

Chromosome set manipulation was used to produce rainbow trout, Oncorhynchus mykiss, with identical nuclear backgrounds, but different maternal backgrounds to determine mitochondrial effects on development rate and oxygen consumption. Significant differences in development rate and oxygen consumption were observed between groups from different females. Development rates ranged from a mean of 317.97 degree days ( degrees d) to 335.25 degrees d in progeny from different females. Mean oxygen consumption rates ranged from 3.31 micromol O2 g(-1) wet mass h(-1) to 9.66 micromol O2 g(-1) wet mass h(-1). Oxygen consumption and development rate analysis revealed the two slowest developing groups had the highest oxygen consumption rates. Development rate differences between second generation clonal females indicate that mitochondrial genomes play a significant role on early development and are comparable to development rate differences between clonal lines of rainbow trout. These results indicate that selection for mitochondrial genomes could increase growth rates and possibly food conversion ratios in aquaculture species.

Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout (Oncorhynchus mykiss).

Utilizing a splenic cDNA library and rapid amplification of cDNA 5' ends (5'-RACE), a C-type lectin gene was cloned from a homozygous cloned rainbow trout. The 1176 bp cDNA contains a 714 bp open reading frame from which a 238-amino-acid (aa) (27 kDa) protein was deduced. It was confirmed that this protein belongs to the C-type animal lectins, and is a type II membrane receptor. The predicted protein from this sequence contains a 48 aa cytoplasmic domain, a 20 aa transmembrane domain (TM), a 46 aa stalk region and a 124 aa carbohydrate-recognition domain (CRD). The stalk region contains a leucine-zipper, and an N-glycosylation site was also found in the CRD. Sequence alignment and phylogenetic analysis of the CRD indicate that the protein has similarity with human dendritic cell immunoreceptor (DCIR), gp120 binding C-type lectin (gp120BCL) and mammalian hepatic lectins. The N-terminus (aa 4-183) has similarity with NKG2, a group of C-type lectin receptors important in human natural killer cell function. The genomic DNA (gDNA) containing this gene was amplified and sequenced. The 4569 bp gDNA contains five exons and four introns. The first three exons encode the cytoplasmic domain, the TM and stalk region, respectively. Unlike the other type II C-type lectin receptors in which the CRD was encoded by three exons, the CRD of this lectin was encoded by two exons. A transposon Tc1-like fragment was found in intron III. Intron IV is composed of a simple repeat. Tissue-specific expression of the gene was studied by RT-PCR, and it was mainly expressed in spleen and peripheral blood leukocyte (PBL). Using AluI to digest the fragment containing exon I, intron I and exon II, an RFLP was produced between the sequences of this gene in two cloned fish, OSU 142 and Arlee (AR). Seventy-one doubled haploids (DH) of OSU X AR were screened, and the gene was mapped to linkage group XIV on the published map (Young et al., Genetics 148 (1998) 839).

Adult triploids in a rainbow trout family.

Six triploid individuals were found in a full-sib family of 11 adult rainbow trout (Salmo gairdneri) from a domesticated hatchery stock. The triploid individuals were normal in size and external appearance, had underdeveloped gonads, and showed no evidence of 3n/2n chimerism or mosaicism. XXY triploids were males, suggesting that the Y chromosome is male determining in trout. Because they may avoid production losses associated with sexual maturation in normal fish, triploid trout and salmon could potentially be useful in fish culture.

Gene-Centromere Mapping in Rainbow Trout: High Interference over Long Map Distances.

Ten enzyme loci were mapped in relation to their centromeres in gynogenetic diploid rainbow trout. Gene-centromere map distances, calculated under the assumption of complete interference, range from 1.1 cM for Ldh4 to 50 cM for Sod1. The Idh2 and Est1 loci are linked on the same chromosome arm.-The observation of close to 100% heterozygous gynogenetic diploids for the Sod1 and Mdh3,4 loci suggests that near-complete interference occurs on the chromosome arms carrying these loci. The high interference observed in this study and in several other species of fish may be related to the small size of fish chromosome arms.-Comparisons of map locations for the Ldh3 and Ldh4 and the Mdh3 and Mdh4 loci, which were duplicated by a tetraploid event in the evolution of salmonid fish, reveal that they are located at similar distances from their centromeres. Comparative mapping of loci duplicated longer ago shows more variation in map location.-The high proportion of heterozygotes for some loci after gynogenesis involving second polar body retention demonstrates that this is not a practical method for producing homozygous inbred lines in rainbow trout; treatments suppressing the first cell division are more promising for this purpose.

A detailed linkage map of rainbow trout produced using doubled haploids.

We report the first detailed genetic linkage map of rainbow trout (Oncorhynchus mykiss). The segregation analysis was performed using 76 doubled haploid rainbow trout produced by androgenesis from a hybrid between the "OSU" and "Arlee" androgenetically derived homozygous lines. Four hundred and seventy-six markers segregated into 31 major linkage groups and 11 small groups (< 5 markers/group). The minimum genome size is estimated to be 2627.5 cM in length. The sex-determining locus segregated to a distal position on one of the linkage groups. We analyzed the chromosomal distribution of three classes of markers: (1) amplified fragment length polymorphisms, (2) variable number of tandem repeats, and (3) markers obtained using probes homologous to the 5' or 3' end of salmonid-specific small interspersed nuclear elements. Many of the first class of markers were clustered in regions that appear to correspond to centromeres. The second class of markers were more telomeric in distribution, and the third class were intermediate. Tetrasomic inheritance, apparently related to the tetraploid ancestry of salmonid fishes, was detected at one simple sequence repeat locus and suggested by the presence of one extremely large linkage group that appeared to consist of two smaller groups linked at their tips. The double haploid rainbow trout lines and linkage map present a foundation for further genomic studies.

Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA.

A full-length cDNA for retinoblastoma (RB1) has been cloned from a cDNA library prepared from 3-week-old rainbow trout (Oncorhynchus mykiss) eyed embryos. The trout RB1 cDNA encodes a predicted protein of 910 amino acids and is the most divergent cloned retinoblastoma gene sequence to date. RT-PCR studies reveal high levels of RB1 expression by the second week of embryogenesis, which remains uniformly expressed until hatching. Expression studies of adult fish tissues show the RB1 gene to be expressed in all tissues examined, including the oesophagus, eye, liver, intestine, posterior and anterior kidney, skin, stomach, muscle, spleen, gill, swim bladder, gonads and brain. The RB1 gene appears to be a single copy gene based on Southern analysis, and maps to linkage group XVI in the trout genome map. Polymorphisms in the RB1 gene and in closely linked markers should facilitate LOH analysis of RB1.

Does differential selection on the 5S rDNA explain why the rainbow trout sex chromosome heteromorphism is not linked to the SEX locus?

Many but not all rainbow trout strains have morphologically distinguishable sex chromosomes. In these strains, the short arm of the X has multiple copies of 5S rDNA and a bright DAPI band near the centromere, both of which are missing from the Y chromosome, which has a very small short arm. We examined the presence of these markers using fluorescence in situ hybridization (FISH) in four different YY clonal lines derived from different strains and compared the results with sexed fish of the Donaldson strain with the normal X/Y heteromorphism. The Y chromosome in two of the YY clonal lines (Arlee and Swanson) is indistinguishable from the X chromosome and it is positive for 5S rDNA and the DAPI bright band. On the other hand, both 5S rDNA sequences and the DAPI band were not found on the Y chromosome in Hot Creek and Clearwater which have the normal Y. Thus the presence of these two cytogenetic markers may account for the size difference between the short arm of the X and Y chromosome found in most rainbow trout strains. In fishes the expression of one type of 5S rRNA is restricted to oocytes and previous work suggests that although XX males are fairly common, XY females are rare, implying a selective disadvantage for XY females. A hypothesis is presented to explain why this sex chromosome heteromorphism is not closely linked to the SEX locus, which is found on the long arm of the Y chromosome in rainbow trout.

Cloning, characterization and genomic structure of the natural killer cell enhancement factor (NKEF)-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss).

Natural killer cell enhancement factor (NKEF) belongs to the antioxidant protein family. In the human, NKEF has the ability to enhance natural killer cell cytotoxic activity in vitro. In the present work, the cDNAs of NKEF from three strains of homozygous clones of rainbow trout were cloned from the splenic cDNA library of one of the strains, OSU142, and then by RT-PCR for the Hot Creek (HC) and Arlee (AR) strains. The HC sequence has 99% sequence identity with both OSU142 and AR. OSU142 and AR have only one nucleotide difference in the cDNA sequence. All three sequences have the same deduced NKEF peptide, which contains 199 amino acids. The 6. 5 kb genomic DNA of OSU142 containing NKEF was sequenced and contains six exons and five introns. Tissue specific expression of NKEF was studied by RT-PCR in eight different tissues of OSU142 and revealed that all tissues expressed NKEF. A southern blot revealed that the gene for NKEF is present in a single copy. The cDNA and amino acid sequences of trout NKEF have high similarity with human, rat, mouse and carp sequences, therefore, indicating that NKEF is a very conserved gene.

Arlee line of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity.

Nonspecific cytotoxic cell (NCC) activity was assessed in the peripheral blood of four isogenic lines of rainbow trout (Oncorhynchus mykiss) which were derived by the chromosome set manipulation technique of androgenesis. In these fish, whose isogenicity was previously confirmed by multilocus DNA fingerprint analysis, NCC activity was studied by the release of 51Cr from YAC-1 targets. Two groups of trout (the homozygous Arlee 12 line and the heterozygous hybrid of the Arlee 63 and Arlee 12 lines) had significantly lower levels of NCC activity in peripheral blood than either outbred rainbow trout or other lines with Hot Creek or hybrid Arlee x Hot Creek ancestry. The low NCC activity in the Arlee line appears to be inherited as a recessive trait. Peripheral blood cells of the trout mediated lectin dependent cellular cytotoxicity (LDCC) with the addition of phytohemagglutinin to co-cultures of effector cells and YAC-1 cells. The low NCC activity in the peripheral blood of these fish is not due to a condition analogous to the NCC-deficient Chediak-Higashi syndrome of man or the beige mutation of mice.

Coding sequences of the MHC II beta chain of homozygous rainbow trout (Oncorhynchus mykiss).

Six lines of homozygous rainbow trout (Oncorhynchus mikiss) from different genetic and geographical backgrounds have been produced as aquatic models for biomedical research by the chromosome set manipulation techniques of androgenesis and gynogenesis. Messenger RNA from spleens was extracted. and the MHC II B cDNA sequences, amplified by RT PCR, were cloned into plasmids. Sequences of the MHC II beta2 domains were highly conserved between the different plasmids from the same and different lines of trout. Most of the variability among sequences was found in the amino terminal half of the beta1 domain, which corresponds with the peptide binding region of the MHC II molecule. This diversity suggests that the different lines of trout may exhibit differences in immune response. Rainbow trout MHC II B sequences were similar to the MHC II B sequences of the Pacific salmon (O. gorbuscha, O. tshawytscha, O. nerka, O. miasou, O. kisutch). Southern blot analysis performed on the restricted DNA of the OSU and Hot Creek trout, and the doubled haploid progeny produced by androgenesis from OSU x Hot Creek hybrids indicates that two distinct genes encode the MHC II B sequences and that these genes are unlinked.

High incidence of a male-specific genetic marker in phenotypic female chinook salmon from the Columbia River.

Numerous populations of anadromous salmonids in the northwestern United States have been declining for many years, resulting in Endangered Species Act listings and in some cases extinction. The degradation of river ecosystems has been proposed as one of the major reasons for the inability of salmon to maintain their populations. However, the specific factors interfering with the reproduction and survival of salmon during the freshwater phase of their life cycle have not been fully described. This study was initiated to determine the incidence of phenotypic sex reversal in wild, fall chinook salmon (Oncorhynchus tshawytcha) that returned to spawn in the Columbia River. Fish were sampled at different locations within this watershed to determine whether they were faithfully expressing their genotype. We report a high incidence (84%) of a genetic marker for the Y chromosome in phenotypic females sampled from the wild, which was not observed in female fish raised in hatcheries. It appears likely that female salmon with a male genotype have been sex reversed, creating the potential for an abnormal YY genotype in the wild that would produce all-male offspring and alter sex ratios significantly.

Responses of cloned rainbow trout Oncorhynchus mykiss to an attenuated strain of infectious hematopoietic necrosis virus.

The objective of this work was to examine the response of homozygous clones of rainbow trout to vaccination by an attenuated strain (Nan Scott Lake; NSL) of infectious hematopoietic necrosis virus (IHNV). Adult rainbow trout of the Hot Creek Strain (YY males maintained in a recirculating system at 12 degrees C) were injected 3 times with 10(5) to 10(7) plaque forming units (pfu) of NSL. Intraperitoneal injections were given at Day 0 and at 2 and 4 mo post-infection. All fish were nonlethally bled at monthly intervals for 18 mo. Serum from each fish was analyzed by the complement-dependent neutralization assay and by western blot against purified NSL virus. The highest virus neutralization titers were detected 4 mo after the first injection, and peaked at 1280. When sera were analyzed by western blot, the predominating responses of the serum from immunized fish on the reduced western blot were against M1, a matrix protein of the virus and to a 90 kDa stress protein. The 90 kDa protein was identified by a monoclonal antibody as a stress protein derived from the CHSE-214 cells in which the purified IHN virus was grown and which associates with the virus during purification.

Acceptance of skin grafts by isogenic rainbow trout (Oncorhynchus mykiss).

OBJECTIVE: To test the immunocompetence of isogenic families of rainbow trout by measuring their ability to accept or reject skin grafts. ANIMALS: 3 families of isogenic rainbow trout (Oncorhynchus mykiss), produced by mating homozygous females and homozygous males, plus 4 chinook salmon (O tshawytscha) were used in these experiments. PROCEDURE: Grafts (allografts, members of the same family; autografts, donor and recipient were the same fish; and xenografts, O tshawytscha as donor) were exchanged. Grafts were applied on day 0 and removed on day 21, placed in neutral-buffered formalin, and embedded in paraffin. Lymphocytes and nuclei were counted in representative stained sections in the epidermis, dermis, and hypodermis. Results were analyzed by univariate analysis, using the Shapiro-Wilk statistic. RESULTS: Autografts were retained and minimal histologic changes were apparent. Allografts were histologically similar to autografts. Xenografts were rejected. CONCLUSIONS: Results indicate that the immune system of isogenic rainbow trout is unable to distinguish between family members within isogenic families, but that a vigorous response is mounted against chinook salmon xenografts. The isogenic rainbow trout are immunocompetent with respect to the phenomenon of graft rejection.

Robertsonian polymorphism and constitutive heterochromatin distribution in chromosomes of the rainbow trout (Salmo gairdneri).

White-blood-cell culture was used to examine the chromosomes of 53 rainbow trout (Salmo gairdneri) from three locations in the Pacific Northwest of the United States. A Robertsonian chromosome polymorphism is present, resulting in diploid numbers of 60, 59, or 58 in different individuals with 104 chromosome arms. The low level of intraindividual Robertsonian variation, differences in the number of subtelocentric chromosomes between individuals with different chromosome numbers, and frequencies of fish with different chromosome numbers in one population suggest that the interindividual differences are inherited and not somatic. C-banding shows that constitutive heterochromatin is localized near the centromeres and near the secondary constriction one chromosome pair.

Sex chromosomes in the sockeye salmon: a Y-autosome fusion.

Chromosomes of 21 sockeye salmon [Oncorhynchus nerka (Walbaum)] from three locations in Washington state were examined. All males had 57 chromosomes, while all females had 58 chromosomes. Both sexes had 104 chromosome arms. It appears that in males of this species the Y chromosome and an autosome have fused to form a metacentric chromosome.