Discovery and characterization of single nucleotide polymorphisms in Chinook salmon, Oncorhynchus tshawytscha

Molecular population genetics of non-model organisms has been dominated by the use of microsatellite loci over the last two decades. The availability of extensive genomic resources for many species is contributing to a transition to the use of single nucleotide polymorphisms (SNPs) for the study of many natural populations. Here we describe the discovery of a large number of SNPs in Chinook salmon, one of the world's most important fishery species, through large-scale Sanger sequencing of expressed sequence tag (EST) regions. More than 3 Mb of sequence was collected in a survey of variation in almost 132 kb of unique genic regions, from 225 separate ESTs, in a diverse ascertainment panel of 24 salmon. This survey yielded 117 TaqMan (5' nuclease) assays, almost all from separate ESTs, which were validated in population samples from five major stocks of salmon from the three largest basins on the Pacific coast of the contiguous United States: the Sacramento, Klamath and Columbia Rivers. The proportion of these loci that was variable in each of these stocks ranged from 86.3% to 90.6% and the mean minor allele frequency ranged from 0.194 to 0.236. There was substantial differentiation between populations with these markers, with a mean F(ST) estimate of 0.107, and values for individual loci ranging from 0 to 0.592. This substantial polymorphism and population-specific differentiation indicates that these markers will be broadly useful, including for both pedigree reconstruction and genetic stock identification applications.     

Rearing and migration of juvenile Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) in a large river floodplain

Off-channel habitat has become increasingly recognized as key for migratory fishes such as juvenile Chinook salmon (Oncorhynchus tshawytscha). Hence, floodplain habitat has been identified as critical for the continued persistence of California’s Central Valley salmon, particularly the Yolo Bypass, the primary floodplain of the Sacramento River. To provide insight into factors supporting juvenile salmon use of this 240 km², partially leveed floodplain, we examined inter- and intra-annual relationships between environmental correlates and residency time, apparent growth, emigration, migratory phenotype, and survival over more than a decade for natural-origin (“wild”) fish and experimentally-released hatchery fish. Flood duration was positively associated with hatchery juveniles residing longer and achieving larger size. Wild juveniles grew larger and emigrated later with cumulative temperature experience (accumulated thermal units) and warmer average annual temperatures during flood years. Within years, both wild and hatchery salmon departed the floodplain as flood waters receded. Parr-sized juveniles dominated outmigrant composition, though fry and smolt-sized juveniles were also consistently observed. Survival to the ocean fishery was not significantly different between hatchery fish that reared in the Yolo Bypass versus those that reared in the main stem Sacramento River. Our study indicates improved frequency and duration of connectivity between the Sacramento River and the Yolo Bypass could increase off-channel rearing opportunities that expand the life history diversity portfolio for Central Valley Chinook salmon.     

Signals of large scale climate drivers,hatchery enhancement,and marine factors in Yukon River Chinook salmon survival revealed with a Bayesian life history model

Understanding how species might respond to climate change involves disentangling the influence of co‐occurring environmental factors on population dynamics, and is especially problematic for migratory species like Pacific salmon that move between ecosystems. To date, debate surrounding the causes of recent declines in Yukon River Chinook salmon (Oncorhynchus tshawytscha) abundance has centered on whether factors in freshwater or marine environments control variation in survival, and how these populations at the northern extremity of the species range will respond to climate change. To estimate the effect of factors in marine and freshwater environments on Chinook salmon survival, we constructed a stage‐structured assessment model that incorporates the best available data, estimates incidental marine bycatch mortality in trawl fisheries, and uses Bayesian model selection methods to quantify support for alternative hypotheses. Models fitted to two index populations of Yukon River Chinook salmon indicate that processes in the nearshore and marine environments are the most important determinants of survival. Specifically, survival declines when ice leaves the Yukon River later in the spring, increases with wintertime temperature in the Bering Sea, and declines with the abundance of globally enhanced salmon species consistent with competition at sea. In addition, we found support for density‐dependent survival limitations in freshwater but not marine portions of the life cycle, increasing average survival with ocean age, and age‐specific selectivity of bycatch mortality in the Bering Sea. This study underscores the utility of flexible estimation models capable of fitting multiple data types and evaluating mortality from both natural and anthropogenic sources in multiple habitats. Overall, these analyses suggest that mortality at sea is the primary driver of population dynamics, yet under warming climate Chinook salmon populations at the northern extent of the species’ range may be expected to fare better than southern populations, but are influenced by foreign salmon production.     

Identification of multiple genetically distinct populations of Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) in a small coastal watershed

Management and restoration planning for Pacific salmon is often characterized by efforts at broad multi-basin scales. However, finer-scale genetic and phenotypic variability may be present within individual basins and can be overlooked in such efforts, even though it may be a critical component for long-term viability. Here, we investigate Chinook salmon (Oncorhynchus tshawytscha) within the Siletz River, a small coastal watershed in Oregon, USA. Adult Chinook salmon were genotyped using neutral microsatellite markers, single nucleotide polymorphisms and “adaptive” loci, associated with temporal variation in migratory behavior in many salmon populations, to investigate genetic diversity based upon both spatial and temporal variation in migratory and reproductive behavior. Results from all three marker types identified two genetically distinct populations in the basin, corresponding to early returning fish that spawn above a waterfall, a spring-run population, and later returning fish spawning below the waterfall, a fall-run population. This finding is an important consideration for management of the species, as spring-run populations generally only have been recognized in large watersheds, and highlights the need to evaluate population structure of salmon within smaller watersheds, and thereby increase the probability of successful conservation of salmon species.     

Finding Markers That Make a Difference: DNA Pooling and SNP-Arrays Identify Population Informative Markers for Genetic Stock Identification

Genetic stock identification (GSI) using molecular markers is an important tool for management of migratory species. Here, we tested a cost-effective alternative to individual genotyping, known as allelotyping, for identification of highly informative SNPs for accurate genetic stock identification. We estimated allele frequencies of 2880 SNPs from DNA pools of 23 Atlantic salmon populations using Illumina SNP-chip. We evaluated the performance of four common strategies (global F _ST, pairwise F _ST, Delta and outlier approach) for selection of the most informative set of SNPs and tested their effectiveness for GSI compared to random sets of SNP and microsatellite markers. For the majority of cases, SNPs selected using the outlier approach performed best followed by pairwise F _ST and Delta methods. Overall, the selection procedure reduced the number of SNPs required for accurate GSI by up to 53% compared with randomly chosen SNPs. However, GSI accuracy was more affected by populations in the ascertainment group rather than the ranking method itself. We demonstrated for the first time the compatibility of different large-scale SNP datasets by compiling the largest population genetic dataset for Atlantic salmon to date. Finally, we showed an excellent performance of our top SNPs on an independent set of populations covering the main European distribution range of Atlantic salmon. Taken together, we demonstrate how combination of DNA pooling and SNP arrays can be applied for conservation and management of salmonids as well as other species.     

Genomewide single nucleotide polymorphism discovery in Atlantic salmon (Salmo salar): validation in wild and farmed American and European populations

A considerable number of single nucleotide polymorphisms (SNPs) are required to elucidate genotype–phenotype associations and determine the molecular basis of important traits. In this work, we carried out de novo SNP discovery accounting for both genome duplication and genetic variation from American and European salmon populations. A total of 9 736 473 nonredundant SNPs were identified across a set of 20 fish by whole‐genome sequencing. After applying six bioinformatic filtering steps, 200 K SNPs were selected to develop an Affymetrix Axiom^® myDesign Custom Array. This array was used to genotype 480 fish representing wild and farmed salmon from Europe, North America and Chile. A total of 159 099 (79.6%) SNPs were validated as high quality based on clustering properties. A total of 151 509 validated SNPs showed a unique position in the genome. When comparing these SNPs against 238 572 markers currently available in two other Atlantic salmon arrays, only 4.6% of the SNP overlapped with the panel developed in this study. This novel high‐density SNP panel will be very useful for the dissection of economically and ecologically relevant traits, enhancing breeding programmes through genomic selection as well as supporting genetic studies in both wild and farmed populations of Atlantic salmon using high‐resolution genomewide information.     

Generic genetic differences between farmed and wild Atlantic salmon identified from a 7K SNP-chip

Genetic interactions between farmed and wild conspecifics are of special concern in fisheries where large numbers of domesticated individuals are released into the wild. In the Atlantic salmon (Salmo salar), selective breeding since the 1970's has resulted in rapid genetic changes in commercially important traits, such as a doubling of the growth rate. Each year, farmed salmon escape from net pens, enter rivers, and interbreed with wild salmon. Field experiments demonstrate that genetic introgression may weaken the viability of recipient populations. However, due to the lack of diagnostic genetic markers, little is known about actual rates of gene flow from farmed to wild populations. Here we present a panel of 60 single nucleotide polymorphisms (SNPs) that collectively are diagnostic in identifying individual salmon as being farmed or wild, regardless of their populations of origin. These were sourced from a pool of 7000 SNPs comparing historical wild and farmed salmon populations, and were distributed on all but two of the 29 chromosomes. We suggest that the generic differences between farmed and wild salmon at these SNPs have arisen due to domestication. The identified panel of SNPs will permit quantification of gene flow from farmed to wild salmon populations, elucidating one of the most controversial potential impacts of aquaculture. With increasing global interest in aquaculture and increasing pressure on wild populations, results from our study have implications for a wide range of species.     

Differentiating diploid and triploid individuals using single nucleotide polymorphisms genotyped by amplicon sequencing

Triploidy can occur naturally or be induced in fish and shellfish during artificial propagation in order to produce sterile individuals. Fisheries managers often stock these sterile triploids as a means of improving angling opportunities without risking unwanted reproduction of the stocked fish. Additionally, the rearing of all‐triploid individuals has been suggested as a means to reduce the possibility of escaped aquaculture fish interbreeding with wild populations. Efficient means of determining if an individual is triploid or diploid are therefore needed both to monitor the efficacy of triploidy‐inducing treatments and, when sampling fish from a body of water that has a mixture of diploids and triploids, to determine the ploidy of a fish prior to further analyses. Currently, ploidy is regularly measured through flow cytometry, but this technique typically utilizes a fresh blood sample. This study presents an alternative, cost‐effective method of determining ploidy by analysing amplicon‐sequencing data for biallelic single‐nucleotide polymorphisms (SNPs). For each sample, heterozygous genotypes are identified and the likelihoods of diploidy and triploidy are calculated based on the read counts for each allele. The accuracy of this method is demonstrated using triploid and diploid brook trout (Salvelinus fontinalis) genotyped with a panel of 234 SNPs and Chinook salmon (Oncorhynchus tshawytscha) genotyped with a panel of 298 SNPs following the GT‐seq methodology of amplicon sequencing.     

Understanding the behavior and ecology of Chinook salmon (Oncorhynchus tshawytscha) on an important feeding ground in the Gulf of Alaska

Population declines and demographic changes of Chinook salmon (Oncorhynchus tshawytscha), have been documented throughout this species’ range, though information on natural and anthropogenic mechanisms related to these changes are not fully understood. To provide insights into marine behaviors and survival of Chinook salmon, 40 pop-up satellite archival tags (PSATs), that collected environmental data, were attached to large (69–100 cm FL) Chinook salmon caught in the marine waters of Cook Inlet, Alaska. PSATs provided evidence of predation on tagged Chinook salmon by ectothermic and unconfirmed predators, and provided valuable information about the migratory characteristics and occupied depths and temperatures of this species while occupying Cook Inlet and the Gulf of Alaska. The results from this study suggest that late-marine mortality of Chinook salmon of a variety of stock-origins by apex predators is more common in Cook Inlet than previously thought, and may be used to improve our understanding this species’ population dynamics. Furthermore, results from this study adds to the existing knowledge of marine habitat use by Chinook Salmon and may be useful in assessing the vulnerability and interactions between this species and anthropogenic activities.

     

Influence of environmental conditions at spawning sites and migration routes on adaptive variation and population connectivity in Chinook salmon

Many species that undergo long breeding migrations, such as anadromous fishes, face highly heterogeneous environments along their migration corridors and at their spawning sites. These environmental challenges encountered at different life stages may act as strong selective pressures and drive local adaptation. However, the relative influence of environmental conditions along the migration corridor compared with the conditions at spawning sites on driving selection is still unknown. In this study, we performed genome–environment associations (GEA) to understand the relationship between landscape and environmental conditions driving selection in seven populations of the anadromous Chinook salmon (Oncorhynchus tshawytscha)—a species of important economic, social, cultural, and ecological value—in the Columbia River basin. We extracted environmental variables for the shared migration corridors and at distinct spawning sites for each population, and used a Pool‐seq approach to perform whole genome resequencing. Bayesian and univariate GEA tests with migration‐specific and spawning site‐specific environmental variables indicated many more candidate SNPs associated with environmental conditions at the migration corridor compared with spawning sites. Specifically, temperature, precipitation, terrain roughness, and elevation variables of the migration corridor were the most significant drivers of environmental selection. Additional analyses of neutral loci revealed two distinct clusters representing populations from different geographic regions of the drainage that also exhibit differences in adult migration timing (summer vs. fall). Tests for genomic regions under selection revealed a strong peak on chromosome 28, corresponding to the GREB1L/ROCK1 region that has been identified previously in salmonids as a region associated with adult migration timing. Our results show that environmental variation experienced throughout migration corridors imposed a greater selective pressure on Chinook salmon than environmental conditions at spawning sites.     

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.     

Population assignment reveals low migratory connectivity in a weakly structured songbird

Understanding migratory connectivity is essential for determining the drivers behind population dynamics and for implementing effective conservation strategies for migratory species. Genetic markers provide a means to describe migratory connectivity; however, they can be uninformative for species with weak population genetic structure, which has limited their application. Here, we demonstrated a genomic approach to describing migratory connectivity in the prothonotary warbler, Protonotaria citrea, a Neotropical songbird of conservation concern. Using 26,189 single nucleotide polymorphisms (SNPs), we revealed regional genetic structure between the Mississippi River Valley and the Atlantic Seaboard with overall weak genetic differentiation among populations (F_ST = 0.0055; 95% CI: 0.0051–0.0059). Genetic variation had a stronger association with geographic rather than environmental factors, with each explaining 14.5% and 8.2% of genetic variation, respectively. By varying the numbers of genomic markers used in population assignment models with individuals of known provenance, we identified a maximum assignment accuracy (89.7% to site, 94.3% to region) using a subset of 600 highly differentiated SNPs. We then assigned samples from nonbreeding sites to breeding region and found low migratory connectivity. Our results highlight the importance of filtering markers for informative loci in models of population assignment. Quantifying migratory connectivity for weakly structured species will be useful for expanding studies to a wider range of migratory species across taxonomic groups and may contribute to a deeper understanding of the evolution of migratory strategies.     

Genetic signals of artificial and natural dispersal linked to colonization of South America by non‐native Chinook salmon (Oncorhynchus tshawytscha)

Genetics data have provided unprecedented insights into evolutionary aspects of colonization by non‐native populations. Yet, our understanding of how artificial (human‐mediated) and natural dispersal pathways of non‐native individuals influence genetic metrics, evolution of genetic structure, and admixture remains elusive. We capitalize on the widespread colonization of Chinook salmon Oncorhynchus tshawytscha in South America, mediated by both dispersal pathways, to address these issues using data from a panel of polymorphic SNPs. First, genetic diversity and the number of effective breeders (N_b) were higher among artificial than natural populations. Contemporary gene flow was common between adjacent artificial and natural and adjacent natural populations, but uncommon between geographically distant populations. Second, genetic structure revealed four distinct clusters throughout the Chinook salmon distributional range with varying levels of genetic connectivity. Isolation by distance resulted from weak differentiation between adjacent artificial and natural and between natural populations, with strong differentiation between distant Pacific Ocean and Atlantic Ocean populations, which experienced strong genetic drift. Third, genetic mixture analyses revealed the presence of at least six donor geographic regions from North America, some of which likely hybridized as a result of multiple introductions. Relative propagule pressure or the proportion of Chinook salmon propagules introduced from various geographic regions according to government records significantly influenced genetic mixtures for two of three artificial populations. Our findings support a model of colonization in which high‐diversity artificial populations established first; some of these populations exhibited significant admixture resulting from propagule pressure. Low‐diversity natural populations were likely subsequently founded from a reduced number of individuals.     

A mobile sex‐determining region,male‐specific haplotypes and rearing environment influence age at maturity in Chinook salmon

Variation in age at maturity is an important contributor to life history and demographic variation within and among species. The optimal age at maturity can vary by sex, and the ability of each sex to evolve towards its fitness optimum depends on the genetic architecture of maturation. Using GWAS of RAD sequencing data, we show that age at maturity in Chinook salmon exhibits sex‐specific genetic architecture, with age at maturity in males influenced by large (up to 20 Mb) male‐specific haplotypes. These regions showed no such effect in females. We also provide evidence for translocation of the sex‐determining gene between two different chromosomes. This has important implications for sexually antagonistic selection, particularly that sex linkage of adaptive genes may differ within and among populations based on chromosomal location of the sex‐determining gene. Our findings will facilitate research into the genetic causes of shifting demography in Chinook salmon as well as a better understanding of sex determination in this species and Pacific salmon in general.     

Testing advances in molecular discrimination among Chinook salmon life histories: evidence from a blind test

The application of DNA‐based markers toward the task of discriminating among alternate salmon runs has evolved in accordance with ongoing genomic developments and increasingly has enabled resolution of which genetic markers associate with important life‐history differences. Accurate and efficient identification of the most likely origin for salmon encountered during ocean fisheries, or at salvage from fresh water diversion and monitoring facilities, has far‐reaching consequences for improving measures for management, restoration and conservation. Near‐real‐time provision of high‐resolution identity information enables prompt response to changes in encounter rates. We thus continue to develop new tools to provide the greatest statistical power for run identification. As a proof of concept for genetic identification improvements, we conducted simulation and blind tests for 623 known‐origin Chinook salmon (Oncorhynchus tshawytscha) to compare and contrast the accuracy of different population sampling baselines and microsatellite loci panels. This test included 35 microsatellite loci (1266 alleles), some known to be associated with specific coding regions of functional significance, such as the circadian rhythm cryptochrome genes, and others not known to be associated with any functional importance. The identification of fall run with unprecedented accuracy was demonstrated. Overall, the top performing panel and baseline (HMSC21) were predicted to have a success rate of 98%, but the blind‐test success rate was 84%. Findings for bias or non‐bias are discussed to target primary areas for further research and resolution.     

Zooplankton ecology and trophic resources for rearing native fish on an agricultural floodplain in the Yolo Bypass California,USA

Out-migrating juvenile Chinook Salmon Oncorhynchus tshawytscha in California’s Central Valley lack frequent access to historical off-channel habitats such as floodplains. However, many regions have agricultural floodplains that may provide habitat value to young salmon. To determine the suitability of agricultural floodplain, this study tested whether winter-inundated rice fields in a historic flood basin in California’s Central Valley could provide adequate food resources for rearing juvenile Chinook Salmon. We examined the suitability of flooded rice fields for three post-harvest habitat types: stubble, fallow, and disced. Soil emergent and pelagic zooplankton communities were compared to determine colonization sources. Winter-inundated rice fields had high densities of zooplankton, which increased over the course of the study. Daphnia pulex, a large-bodied cladoceran and an excellent forage species of juvenile Chinook Salmon, was abundant in our study. Cladocerans colonized via source water while ostracods likely colonized from a soil egg bank. Overall, there was no discernable effect of habitat type on zooplankton community structure or density, except for D. pulex. Our results suggest that flooded agricultural rearing habitat can support juvenile Chinook Salmon based on high densities of zooplankton and other suitable habitat conditions have the potential to support a robust aquatic food web.     

Genetic differentiation of Alaska Chinook salmon: the missing link for migratory studies

Most information about Chinook salmon genetic diversity and life history originates from studies from the West Coast USA, western Canada and southeast Alaska; less is known about Chinook salmon from western and southcentral Alaska drainages. Populations in this large area are genetically distinct from populations to the south and represent an evolutionary legacy of unique genetic, phenotypic and life history diversity. More genetic information is necessary to advance mixed stock analysis applications for studies involving these populations. We assembled a comprehensive, open-access baseline of 45 single nucleotide polymorphisms (SNPs) from 172 populations ranging from Russia to California. We compare SNP data from representative populations throughout the range with particular emphasis on western and southcentral Alaska. We grouped populations into major lineages based upon genetic and geographic characteristics, evaluated the resolution for identifying the composition of admixtures and performed mixed stock analysis on Chinook salmon caught incidentally in the walleye pollock fishery in the Bering Sea. SNP data reveal complex genetic structure within Alaska and can be used in applications to address not only regional issues, but also migration pathways, bycatch studies on the high seas, and potential changes in the range of the species in response to climate change.     

New tetranucleotide microsatellites for fine‐scale discrimination among endangered chinook salmon (Oncorhynchus tshawytscha)

The unambiguous identification of Central Valley spring‐run chinook salmon has become imperative since their proposed listing in 1998. The accuracy of methods used to assign individuals to their stock of origin is critical for understanding juvenile migration patterns and determining the success of protection measures. Existing microsatellites discriminate between the endangered winter‐run and other chinook but are insufficient to characterize phylogenetically less distinct runs. Here, we isolated and developed highly variable tetranucleotide microsatellites for the specific goal of increasing discriminatory power among closely related populations, providing a new power towards the reliable differentiation of nonwinter runs