Characterization of 13 single nucleotide polymorphism markers for chum salmon

We report the characterization of 13 single nucleotide polymorphism (SNP) genotyping assays for chum salmon (Oncorhynchus keta). These assays are based on the 5′‐nuclease reaction and thus facilitate high‐throughput genotyping with minimal optimization time. Because data generated using these markers may be transported and combined across laboratories, SNPs offer the potential to reduce the amount of redundant work being done in mixture and migratory studies of chum salmon.     

Discovery and characterization of single nucleotide polymorphisms in coho salmon,Oncorhynchus kisutch

Molecular population genetic analyses have become an integral part of ecological investigation and population monitoring for conservation and management. Microsatellites have been the molecular marker of choice for such applications over the last several decades, but single nucleotide polymorphism (SNP) markers are rapidly expanding beyond model organisms. Coho salmon (Oncorhynchus kisutch) is native to the north Pacific Ocean and its tributaries, where it is the focus of intensive fishery and conservation activities. As it is an anadromous species, coho salmon typically migrate across multiple jurisdictional boundaries, complicating management and requiring shared data collection methods. Here, we describe the discovery and validation of a suite of novel SNPs and associated genotyping assays which can be used in the genetic analyses of this species. These assays include 91 that are polymorphic in the species and one that discriminates it from a sister species, Chinook salmon. We demonstrate the utility of these SNPs for population assignment and phylogeographic analyses, and map them against the draft trout genome. The markers constitute a large majority of all SNP markers described for coho salmon and will enable both population‐ and pedigree‐based analyses across the southern part of the species native range.     

Development of 54 novel single-nucleotide polymorphism (SNP) assays for sockeye and coho salmon and assessment of available SNPs to differentiate stocks within the Columbia River

Single-nucleotide polymorphisms (SNPs) have potential for broad application in population and conservation genetics, but availability of these markers is limited in many nonmodel species. In this study, genomic and expressed sequence tagged (EST) sequences from closely related salmonids (Chinook salmon and rainbow trout) were used to design primers for amplification and sequencing of sockeye (Oncorhynchus nerka) and coho (Oncorhynchus kisutch) salmon DNA for SNP discovery. One hundred and six primer sets were designed and tested for amplification in each species. An ascertainment panel of 32 diverse individuals from each species was used as template for PCR amplification and Sanger sequencing. In total, 21,647 bases of consensus sequence were screened in sockeye salmon and 20,784 bases in coho salmon with 93 and 149 SNP sites identified, respectively. Sixty-four SNP sites were chosen for assay development, and 54 of the assays were validated by comparison with genotype and sequence data (O. nerka = 23; O. kisutch = 31). These validated SNP assays along with 142 other available SNP assays [O. nerka = 103 (126 total); O. kisutch = 30 (61 total)] were used to genotype collections of O. nerka (N = 5) and O. kisutch (N = 4) from various sites in the Columbia River to evaluate the utility of these markers in this region. Results from factorial correspondence analysis indicate that these SNP markers are capable of distinguishing O. nerka populations, but O. kisutch collections were less distinct because of their common ancestry.     

BAC-based upgrading and physical integration of a genetic SNP map in Atlantic salmon

A better understanding of the genotype–phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups.     

Next-generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout

The increased numbers of genetic markers produced by genomic techniques have the potential to both identify hybrid individuals and localize chromosomal regions responding to selection and contributing to introgression. We used restriction-site-associated DNA sequencing to identify a dense set of candidate SNP loci with fixed allelic differences between introduced rainbow trout (Oncorhynchus mykiss) and native westslope cutthroat trout (Oncorhynchus clarkii lewisi). We distinguished candidate SNPs from homeologs (paralogs resulting from whole-genome duplication) by detecting excessively high observed heterozygosity and deviations from Hardy-Weinberg proportions. We identified 2923 candidate species-specific SNPs from a single Illumina sequencing lane containing 24 barcode-labelled individuals. Published sequence data and ongoing genome sequencing of rainbow trout will allow physical mapping of SNP loci for genome-wide scans and will also provide flanking sequence for design of qPCR-based TaqMan(?) assays for high-throughput, low-cost hybrid identification using a subset of 50-100 loci. This study demonstrates that it is now feasible to identify thousands of informative SNPs in nonmodel species quickly and at reasonable cost, even if no prior genomic information is available.     

The complete sequence of the mitochondrial genome of the Chinook salmon, Oncorhynchus tshawytscha

The complete sequence of the mitochondrial genome of Chinook salmon, Oncorhynchus tshawytscha, has been determined. The circular genome consisting of 16,644 base pairs encodes thirteen proteins, the 12S and 16S ribosomal RNAs, and 22 transfer RNAs. These genes are ordered in the same way as most other vertebrates. The nucleotide and amino acid sequences of the ribosomal RNAs and the thirteen protein-coding genes were compared with those of other salmonids such as Oncorhynchus mykiss, Salmo salar, Salvelinus fontinalis, Salvelinus alpinus and Coregonus lavaretus. The sequence features of the control region (D-loop), the origin of L-strand replication and a putative peptide codified by the 16S mitochondrial RNA are described and discussed.     

Assembling a dual purpose TaqMan-based panel of single-nucleotide polymorphism markers in rainbow trout and steelhead (Oncorhynchus mykiss) for association mapping and population genetics analysis

We establish a TaqMan-based assay panel for genotyping single-nucleotide polymorphisms in rainbow trout and steelhead (Oncorhynchus mykiss). We develop 22 novel single-nucleotide polymorphism markers based on new steelhead sequence data and on assays from sister taxa. Additionally, we adapt 154 previously developed markers to the TaqMan platform. At the beginning of this study, 59 SNPs with TaqMan assays were available to the scientific community. By adding 176 additional TaqMan assays to this number, we greatly expand the biological applications of TaqMan genotyping within both population genetics and quantitative genetics.     

Whole genome duplication: challenges and considerations associated with sequence orthology assignment in Salmoninae

To illustrate some of the challenges and considerations in assigning correct orthology necessary for any comparative genomic investigation among salmonids, sequence data from the non-coding regions of different chromosomes in three members of the subfamily Salmoninae, rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and Arctic charr Salvelinus alpinus, were compared. By analysing c. 55 distinct loci, corresponding to c. 142 kbp sequence information per species, 18 duplicated patterns representative of the two sequential rounds of teleost-specific whole genome duplications (i.e. 3R and 4R WGD) were identified. Sequence similarities between the 4R paralogues were c. 90%, which was slightly lower than those of the 4R orthologues and c. 60% for the 3R products. Through careful examination of the sequence data, however, only 14 loci could reliably be assigned as true orthologues. Locus-specific trees were constructed through maximum parsimony, maximum likelihood and neighbour-joining methods and were rooted using the information from a close relative, lake whitefish Coregonus clupeaformis. All approaches generated congruent trees supporting the {Coregonus [Salmo (Oncorhynchus, Salvelinus)]} topology. The general phenotypic characteristics of sequences, however, were highly suggestive of the basal position of Oncorhynchus, raising the hypothesis of an accelerated rate of nucleotide evolution in this species.     

Sequence-based genotyping for marker discovery and co-dominant scoring in germplasm and populations

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n?=?222 samples) and lettuce (n?=?87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike.     

The effect of stress and exercise on post-mortem biochemistry of Atlantic salmon and rainbow trout

Freshwater Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss responded similarly to increase in water flow (exercise), reduction in holding tank water level (stress), or 30 min chasing with water level reduction (stress and exercise). Stress generally resulted in elevated plasma cortisol, above the control. Fish responded to stress and exercise combined, with elevated lactate and [H⁺] which was sometimes associated with elevated plasma cortisol. These changes were combined with a depletion of the muscle adenylate pool. Post-mortem, this resulted in an increase in the rate of onset of rigor, and a higher and sometimes sustained muscle proton load. Both species produced predominantly inosine as opposed to hypoxanthine, for up to 72 h of ice storage. This study shows that the physiological disruption in Atlantic salmon and rainbow trout caused by simulated harvest conditions of stress and exercise, results in mostly transient changes in post-mortem muscle biochemistry. These changes lead to an earlier onset and resolution of rigor, and lower post-mortem muscle pH in comparison to the control.     

Effects of species, culture history, size and residency on relative competitive ability of salmonids

The relative competitive ability of juvenile farm and wild salmonids was investigated to provide insight into the potential effects of introduction of cultured salmon on wild Pacific salmonid ( Oncorhynchus ) species. Aquarium experiments involving equal contests ( i.e. size matched, simultaneously introduced individuals) indicated that two wild coho salmon Oncorhynchus kisutch populations were competitively equal to a farm coho salmon population. In equal contests between farm Atlantic salmon Salmo salar (Mowi strain) and these wild coho salmon populations or coastal cutthroat trout Oncorhynchus clarki clarki , Atlantic salmon were subordinate in all cases. When Atlantic salmon were given a residence advantage, however, they were competitively equal to both wild coho salmon populations, but remained subordinate to coastal cutthroat trout. When Atlantic salmon were given a 10–30% length advantage, they were competitively equal to one wild coho salmon population but remained subordinate to the other. In equal contests in semi-natural stream channels, both wild coho and farm Atlantic salmon grew significantly more in the presence of the other species than when alone. It appears that coho salmon obtain additional food ration by out competing Atlantic salmon, whereas Atlantic salmon were stimulated to feed more in the presence of coho salmon competitors. These results suggest that wild coho salmon and cutthroat trout should out compete farm Atlantic salmon of a similar size in nature. As the relative competitive ability of Atlantic salmon improves when they have a size and residence advantage, should feral populations become established, they may exist on a more equal competitive footing owing to the long freshwater residence of Atlantic salmon.     

Use of differential expression data for identification of novel immune relevant expressed sequence tag-linked microsatellite markers in Atlantic salmon (Salmo salar L.)

Despite the large number of genes contributing to the immune response, wildlife immunogenetic studies have tended to focus mostly on the major histocompatibility complex-related genes. Here, we utilized previously published microarray and competitive RNA hybridization information to identify 3750 immune relevant Atlantic salmon (Salmo salar) expressed sequence tags. We then identified those expressed sequence tags containing microsatellites and subsequently designed 48 primer pairs and tested them for polymorphism in Atlantic salmon. Altogether, 16 polymorphic markers were characterized, with allele numbers ranging from two to 18, and these 16 loci were further tested in five other salmonid species.     

The importance of genetic verification for determination of Atlantic salmon in north Pacific waters

Genetic analyses of two unknown but putative Atlantic salmon Salmo salar captured in the Copper River drainage, Alaska, demonstrated the need for validation of morphologically unusual fishes. Mitochondrial DNA sequences (control region and cytochrome b) and data from two nuclear genes [first internal transcribed spacer (ITS-1) sequence and growth hormone (GH1) amplification product] indicated that the fish caught in fresh water on the Martin River was a coho salmon Oncorhynchus kisutch , while the other fish caught in the intertidal zone of the Copper River delta near Grass Island was an Atlantic salmon. Determination of unusual or cryptic fish based on limited physical characteristics and expected seasonal spawning run timing will add to the controversy over farmed Atlantic salmon and their potential effects on native Pacific species. It is clear that determination of all putative collections of Atlantic salmon found in Pacific waters requires validation. Due to uncertainty of fish identification in the field using plastic morphometric characters, it is recommended that genetic analyses be part of the validation process.     

Characterization and PCR multiplexing of novel highly variable tetranucleotide Atlantic salmon (Salmo salar L.) microsatellites

Seven novel and highly variable tetranucleotide microsatellite markers, and conditions for multiplexing and simultaneous genotyping six of these in a single run, are described for Atlantic salmon. These provide a highly informative and cost‐effective set of molecular markers for genetic studies on cultured and wild populations of the species. The primers sets showed cross‐species amplification of appropriately sized amplified products in a number of other salmonid species and suggests the primer sets may have wider application.     

Comparison of GNRH3 genes across salmonid genera

Genomic sequences of gonadotropin-releasing hormone genes were amplified and examined for sequence divergence among members of three different genera of the subfamily Salmoninae: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and Arctic charr (Salvelinus alpinus). Sequences of GNRH3A and GNRH3B (formerly known as sGnRH1 and sGnRH2) were 97-99% similar in coding regions and 94-98% similar in non-coding regions among genera, but comparisons within species between GNRH3A and GNRH3B were only 90-92% similar in coding regions and 83-89% similar in non-coding regions. Polymorphisms in the parents of mapping families for each species allowed for linkage mapping of the GNRH3B gene in all three species and the GNRH3A gene in rainbow trout. GNRH3B maps to linkage group 6 in rainbow trout, linkage group 16 in Atlantic salmon and linkage group 25 in Arctic charr. GNRH3A mapped to linkage group 30 in rainbow trout.     

Detection of salmonid alphavirus RNA in wild marine fish: implications for the origins of salmon pancreas disease in aquaculture

Salmonid alphaviruses (SAVs), which include the aetiological agents of salmon pancreas disease (SPD) in farmed Atlantic salmon Salmo salar L. and sleeping disease (SD) in rainbow trout Oncorhynchus mykiss (Walbaum), are significant viral pathogens of European salmonid aquaculture. SAV is horizontally transmitted and the virus can survive for extended periods in seawater. A lack of convincing evidence for vertical transmission coupled to the fact that the SPD virus (SPDV) occurs in historically infected sites irrespective of fallow period duration suggests that a substantial reservoir of infection exists in the marine environment. We used a highly sensitive real-time PCR (qPCR) assay targeting a region of the SAV nsP1 gene to screen wild marine fish species for the presence of SAV in an attempt to identify such a potential reservoir. Screened fish species were caught in the vicinity of aquaculture activity in an area with a previous history of SAV infection (Shetland Isles, Scotland). SAV RNA was detected in internal organs (kidney and heart) from the flatfish species common dab Limanda limanda, long rough dab Hippoglossoides platessoides, and plaice Pleuronectes platessa. Based on these findings, sampling was extended to an area remote from aquaculture activity (Stonehaven Bay, NE coast of Scotland) from where heart tissues obtained from common dab also tested positive. While no virus could be cultivated from these samples, qPCR detections were shown to be SAV-specific by sequencing of an alternative gene region (E2) to that targeted by the qPCR assay. Analysis of these nucleotide sequences revealed minor differences to those previously obtained from farmed salmon, and subsequent phylogenetic analysis of an E2 dataset demonstrated a subtype V-like sequence.     

IIb‐RAD‐sequencing coupled with random forest classification indicates regional population structuring and sex‐specific differentiation in salmon lice (Lepeophtheirus salmonis)

The aquaculture industry has been dealing with salmon lice problems forming serious threats to salmonid farming. Several treatment approaches have been used to control the parasite. Treatment effectiveness must be optimized, and the systematic genetic differences between subpopulations must be studied to monitor louse species and enhance targeted control measures. We have used IIb‐RAD sequencing in tandem with a random forest classification algorithm to detect the regional genetic structure of the Norwegian salmon lice and identify important markers for sex differentiation of this species. We identified 19,428 single nucleotide polymorphisms (SNPs) from 95 individuals of salmon lice. These SNPs, however, were not able to distinguish the differential structure of lice populations. Using the random forest algorithm, we selected 91 SNPs important for geographical classification and 14 SNPs important for sex classification. The geographically important SNP data substantially improved the genetic understanding of the population structure and classified regional demographic clusters along the Norwegian coast. We also uncovered SNP markers that could help determine the sex of the salmon louse. A large portion of the SNPs identified to be under directional selection was also ranked highly important by random forest. According to our findings, there is a regional population structure of salmon lice associated with the geographical location along the Norwegian coastline.     

Characterization of 19 single nucleotide polymorphism markers for coho salmon

We report 39 single nucleotide polymorphisms (SNPs) observed in 23 nuclear DNA sequences in coho salmon Oncorhynchus kisutch. High‐throughput genotyping assays based on the 5′‐nuclease reaction were developed for 17 of these nuclear SNPs and for two previously published mitochondrial DNA SNPs. Minor allele frequency differences (Δq) among collections were between 5.2% and 51.2%, resulting in per locus F_ST estimates of 0.00–0.24 with an average of 0.09.