Characterization of 38 polymorphic microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Thirty‐eight new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage of heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Five markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.     

Polymorphisms in major histocompatibility complex class IIα genes are associated with resistance to infectious hematopoietic necrosis in rainbow trout,Oncorhynchus mykiss (Walbaum, 1792)

Infectious hematopoietic necrosis is a serious viral disease of salmonids, including rainbow trout Oncorhynchus mykiss, and causes tremendous economic losses to the rainbow trout farming industry. Major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity in vertebrate organisms and have been linked with the resistance to numerous pathogenic diseases. In this study, polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by cloning and sequencing were used to examine polymorphisms in the DAA genes (specifically DAA exon 2 of MHC class IIα) of rainbow trout and investigate their association with the infectious hematopoietic necrosis virus (IHNV) resistance in rainbow trout. Seventeen alleles were resolved, including 13 novel alleles. Individuals possessed between two and five alleles, indicating that the genome harbours at least three closely‐related DAA exon 2 loci. The ratio of non‐synonymous to synonymous nucleotide substitutions suggested that DAA exon 2 is under positive selection. A greater variability of amino acids and non‐synonymous nucleotide substitution rate was evident in the peptide‐binding region (PBR) than in the non‐PBR (27.75%). Importantly, the analyses revealed that certain MHC class IIα alleles appear to confer resistance to IHNV in rainbow trout, while others confer susceptibility. The most common alleles in the resistant populations of rainbow trout, Onmy‐DAA*1301 and Onmy‐DAA*0304, confer resistance to IHNV and were not present in the susceptible population. Hence, these alleles may be ideal molecular markers that can assist the breeding of IHNV resistance in rainbow trout.     

The development and characterization of a 57K single nucleotide polymorphism array for rainbow trout

In this study, we describe the development and characterization of the first high‐density single nucleotide polymorphism (SNP) genotyping array for rainbow trout. The SNP array is publically available from a commercial vendor (Affymetrix). The SNP genotyping quality was high, and validation rate was close to 90%. This is comparable to other farm animals and is much higher than previous smaller scale SNP validation studies in rainbow trout. High quality and integrity of the genotypes are evident from sample reproducibility and from nearly 100% agreement in genotyping results from other methods. The array is very useful for rainbow trout aquaculture populations with more than 40 900 polymorphic markers per population. For wild populations that were confounded by a smaller sample size, the number of polymorphic markers was between 10 577 and 24 330. Comparison between genotypes from individual populations suggests good potential for identifying candidate markers for populations' traceability. Linkage analysis and mapping of the SNPs to the reference genome assembly provide strong evidence for a wide distribution throughout the genome with good representation in all 29 chromosomes. A total of 68% of the genome scaffolds and contigs were anchored through linkage analysis using the SNP array genotypes, including ~20% of the genome assembly that has not been previously anchored to chromosomes.     

Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O. clarki (cutthroat trout) subspecies

A suite of 26 PCR‐based markers was developed that differentiates rainbow (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki). The markers also differentiated rainbow from other cutthroat trout subspecies (O. clarki), and several of the markers differentiated between cutthroat trout subspecies. This system has numerous positive attributes, including: nonlethal sampling, high species‐specificity and products that are easily identified and scored using agarose gel electrophoresis. The methodology described for developing the markers can be applied to virtually any system in which numerous markers are desired for identifying or differentiating species or subspecies.     

Characterization of sex chromosomes in rainbow trout and coho salmon using fluorescence in situ hybridization (FISH)

With the aim of characterizing the sex chromosomes of rainbow trout (Oncorhynchus mykiss) and to identify the sex chromosomes of coho salmon (O. kisutch), we used molecular markers OmyP9, 5S rDNA, and a growth hormone gene fragment (GH2), as FISH probes. Metaphase chromosomes were obtained from lymphocyte cultures from farm specimens of rainbow trout and coho salmon. Rainbow trout sex marker OmyP9 hybridizes on the sex chromosomes of rainbow trout, while in coho salmon, fluorescent signals were localized in the medial region of the long arm of one subtelocentric chromosome pair. This hybridization pattern together with the hybridization of a GH2 intron probe on a chromosome pair having the same morphology, suggests that a subtelocentric pair could be the sex chromosomes in this species. We confirm that in rainbow trout, one of the two loci for 5S rDNA genes is on the X chromosome. In males of this species that lack a heteromorphic sex pair (XX males), the 5S rDNA probe hybridized to both subtelocentrics This finding is discussed in relation to the hypothesis of intraspecific polymorphism of sex chromosomes in rainbow trout.     

Development and characterization of novel tetra‐, tri‐, and dinucleotide microsatellite markers in rainbow trout (Oncorhynchus mykiss)

We discuss the development and characterization of 40 polymorphic rainbow trout (Oncorhynchus mykiss) microsatellite loci. We used enriched libraries to isolate 14 dinucleotide, seven trinucleodide, eight compound di/tetranucleotide, and 11 tetranucleotide loci. These markers will be useful for selective breeding via marker‐assisted selection, population genetics studies, parentage analysis, and have already been used for genome mapping.     

Transcriptome Profiling of Embryonic Development Rate in Rainbow Trout Advanced Backcross Introgression Lines

In rainbow trout (Oncorhynchus mykiss) and other fishes, embryonic development rate is an ecologically and evolutionarily important trait that is closely associated with survival and physiological performance later in life. To identify genes differentially regulated in fast and slow-developing embryos of rainbow trout, we examined gene expression across developmental time points in rainbow trout embryos possessing alleles linked to a major quantitative trait loci (QTL) for fast versus slow embryonic development rate. Whole genome expression microarray analyses were conducted using embryos from a fourth generation backcross family, whereby each backcross generation involved the introgression of the fast-developing alleles for a major development rate QTL into a slow-developing clonal line of rainbow trout. Embryos were collected at 15, 19, and 28 days post-fertilization; sex and QTL genotype were determined using molecular markers, and cDNA from 48 embryos were used for microarray analysis. A total of 183 features were identified with significant differences between embryonic development rate genotypes. Genes associated with cell cycle growth, muscle contraction and protein synthesis were expressed significantly higher in embryos with the fast-developing allele (Clearwater) than those with the slow-developing allele (Oregon State University), which may associate with fast growth and early body mass construction in embryo development. Across time points, individuals with the fast-developing QTL allele appeared to have earlier onset of these developmental processes when compared to individuals with the slow development alleles, even as early as 15 days post-fertilization. Differentially expressed candidate genes chosen for linkage mapping were localized primarily to regions outside of the major embryonic development rate QTL, with the exception of a single gene (very low-density lipoprotein receptor precursor).     

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.     

Development and evaluation of 200 novel SNP assays for population genetic studies of westslope cutthroat trout and genetic identification of related taxa

DNA sequence data were collected and screened for single nucleotide polymorphisms (SNPs) in westslope cutthroat trout (Oncorhynchus clarki lewisi) and also for substitutions that could be used to genetically discriminate rainbow trout (O. mykiss) and cutthroat trout, as well as several cutthroat trout subspecies. In total, 260 expressed sequence tag‐derived loci were sequenced and allelic discrimination genotyping assays developed from 217 of the variable sites. Another 50 putative SNPs in westslope cutthroat trout were identified by restriction‐site‐associated DNA sequencing, and seven of these were developed into assays. Twelve O. mykiss SNP assays that were variable within westslope cutthroat trout and 12 previously published SNP assays were also included in downstream testing. A total of 241 assays were tested on six westslope cutthroat trout populations (N = 32 per population), as well as collections of four other cutthroat trout subspecies and a population of rainbow trout. All assays were evaluated for reliability and deviation from Hardy–Weinberg and linkage equilibria. Poorly performing and duplicate assays were removed from the data set, and the remaining 200 assays were used in tests of population differentiation. The remaining markers easily distinguished the various subspecies tested, as evidenced by mean G_ST of 0.74. A smaller subset of the markers (N = 86; average G_ST = 0.40) was useful for distinguishing the six populations of westslope cutthroat trout. This study increases by an order of magnitude the number of genetic markers available for the study of westslope cutthroat trout and closely related taxa and includes many markers in genes (developed from ESTs).     

Habitat use by Nonnative Rainbow Trout, Oncorhynchus mykiss, and Native Little Colorado spinedace, Lepidomeda vittata

We evaluated overlap in microhabitat use between nonnative rainbow trout, Oncorhynchus mykiss, and native Little Colorado spinedace, Lepidomeda vittata, a federally threatened cyprinid, in natural and experimental settings. In natural settings, we also examined occurrence and microhabitat use of two other native fishes, speckled dace, Rhinichthys osculus, and bluehead sucker, Catostomus discobolus. Native species co-occurred, as did rainbow trout and bluehead sucker. However, occurrences of Little Colorado spinedace and speckled dace were not significantly correlated with occurrence of rainbow trout. Total lengths of all three native species were significantly smaller at allopatric sites than at sites sympatric with rainbow trout. Microhabitat characteristics at sites with rainbow trout did not differ from those where the other three species were found, but did differ among the native species. In laboratory experiments with Little Colorado spinedace and rainbow trout, rainbow trout used the lower depth zone most, and spinedace increased use of the lower depth zone upon addition of rainbow trout. In addition, species tended to co-occur in zones, but used cover independently of one-another, suggesting a low level of agonistic interactions. However, after addition of a high density of rainbow trout, spinedace tended to use cover less than before. We suggest that the species can coexist at low rainbow trout densities. Potential negative effects of rainbow trout on Little Colorado spinedace likely increase with increasing densities of rainbow trout, and rainbow trout likely affect smaller size classes of Little Colorado spinedace more than larger ones.     

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.     

A consolidated linkage map for rainbow trout (Oncorhynchus mykiss)

Androgenetic doubled haploid progeny produced from a cross between the Oregon State University and Arlee clonal rainbow trout (Oncorhynchus mykiss) lines, used for a previous published rainbow trout map, were used to update the map with the addition of more amplified fragment length polymorphic (AFLP) markers, microsatellites, type I and allozyme markers. We have added more than 900 markers, bringing the total number to 1359 genetic markers and the sex phenotype including 799 EcoRI AFLPs, 174 PstI AFLPs, 226 microsatellites, 72 VNTR, 38 SINE markers, 29 known genes, 12 minisatellites, five RAPDs, and four allozymes. Thirty major linkage groups were identified. Synteny of linkage groups in our map with the outcrossed microsatellite map has been established for all except one linkage group in this doubled haploid cross. Putative homeologous relationships among linkage groups, resulting from the autotetraploid nature of the salmonid genome, have been revealed based on the placement of duplicated microsatellites and type I loci.     

Parallel introgression and selection on introduced alleles in a native species

As humans cause the redistribution of species ranges, hybridization between previously allopatric species is on the rise. Such hybridization can have complex effects on overall fitness of native species as new allelic combinations are tested. Widespread species introductions provide a unique opportunity to study selection on introgressed alleles in independent, replicated populations. We examined selection on alleles that repeatedly introgressed from introduced rainbow trout (Oncorhynchus mykiss) into native westslope cutthroat trout (Oncorhynchus clarkii lewisi) populations in western Canada. We found that the degree of introgression of individual single nucleotide polymorphisms from the invasive species into the native is correlated between independent watersheds. A number of rainbow trout alleles have repeatedly swept to high frequency in native populations, suggesting parallel adaptive advantages. Using simulations, we estimated large selection coefficients up to 0.05 favoring several rainbow trout alleles in the native background. Although previous studies have found reduced hybrid fitness and genome‐wide resistance to introgression in westslope cutthroat trout, our results suggest that some introduced genomic regions are strongly favored by selection. Our study demonstrates the utility of replicated introductions as case studies for understanding parallel adaptation and the interactions between selection and introgression across the genome. We suggest that understanding this variation, including consideration of beneficial alleles, can inform management strategies for hybridizing species.     

Screening of population genetic SCAR markers for mykiss <Emphasis Type="Italic">Parasalmo</Emphasis> (<Emphasis Type="Italic">Oncorhynchus</Emphasis>) <Emphasis Type="Italic">mykiss</Emphasis> from Kamchatka

Using AP-PCR, the genome of Kamchatka mykiss (Parasalmo (O.) mykiss) was examined. Polymorphic fragments, implying geographic differences among the samples, were selected, cloned, and sequenced. Based on these sequences, longer, specific SCAR primers were selected and constructed. Using the BLAST software program, the sequences were analyzed for analogy to those from the GenBank database. It seemed likely that all sequences obtained belonged to earlier unexamined repeated sequences, variable in the populations of the species of interest. A total of seven SCAR markers, characterized by population-significant variability of the DNA products in Kamchatka geographic group of rainbow trout were constructed. These markers can be used for further investigation of the species Parasalmo (O.) mykiss. The SCAR marker sequences were deposited in GenBank under the accession numbers EU805500 to EU805506.     

A resource of single‐nucleotide polymorphisms for rainbow trout generated by restriction‐site associated DNA sequencing of doubled haploids

Salmonid genomes are considered to be in a pseudo‐tetraploid state as a result of a genome duplication event that occurred between 25 and 100 Ma. This situation complicates single‐nucleotide polymorphism (SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants (PSVs) and not simple allelic variants. To differentiate PSVs from simple allelic variants, we used 19 homozygous doubled haploid (DH) lines that represent a wide geographical range of rainbow trout populations. In the first phase of the study, we analysed SbfI restriction‐site associated DNA (RAD) sequence data from all the 19 lines and selected 11 lines for an extended SNP discovery. In the second phase, we conducted the extended SNP discovery using PstI RAD sequence data from the selected 11 lines. The complete data set is composed of 145 168 high‐quality putative SNPs that were genotyped in at least nine of the 11 lines, of which 71 446 (49%) had minor allele frequencies (MAF) of at least 18% (i.e. at least two of the 11 lines). Approximately 14% of the RAD SNPs in this data set are from expressed or coding rainbow trout sequences. Our comparison of the current data set with previous SNP discovery data sets revealed that 99% of our SNPs are novel. In the support files for this resource, we provide annotation to the positions of the SNPs in the working draft of the rainbow trout reference genome, provide the genotypes of each sample in the discovery panel and identify SNPs that are likely to be in coding sequences.     

A Second Generation Integrated Map of the Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Genome: Analysis of Conserved Synteny with Model Fish Genomes

DNA fingerprints and end sequences from bacterial artificial chromosomes (BACs) from two new libraries were generated to improve the first generation integrated physical and genetic map of the rainbow trout (Oncorhynchus mykiss) genome. The current version of the physical map is composed of 167,989 clones of which 158,670 are assembled into contigs and 9,319 are singletons. The number of contigs was reduced from 4,173 to 3,220. End sequencing of clones from the new libraries generated a total of 11,958 high quality sequence reads. The end sequences were used to develop 238 new microsatellites of which 42 were added to the genetic map. Conserved synteny between the rainbow trout genome and model fish genomes was analyzed using 188,443 BAC end sequence (BES) reads. The fractions of BES reads with significant BLASTN hits against the zebrafish, medaka, and stickleback genomes were 8.8%, 9.7%, and 10.5%, respectively, while the fractions of significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 6.2%, 5.8%, and 5.5%, respectively. The overall number of unique regions of conserved synteny identified through grouping of the rainbow trout BES into fingerprinting contigs was 2,259, 2,229, and 2,203 for stickleback, medaka, and zebrafish, respectively. These numbers are approximately three to five times greater than those we have previously identified using BAC paired ends. Clustering of the conserved synteny analysis results by linkage groups as derived from the integrated physical and genetic map revealed that despite the low sequence homology, large blocks of macrosynteny are conserved between chromosome arms of rainbow trout and the model fish species.     

Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki)

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.