Kerry A. Naish

Single‐nucleotide polymorphism (SNP) discovery and applications of SNP genotyping in nonmodel organisms

A supplemental issue on the topic of single-nucleotidepolymorphism-enabled (SNP) research in nonmodel organisms is especially timely. In this issue, organisms with reference genomes are considered to be ‘model’; ‘nonmodel’ organisms are those whose genomes are yet to be sequenced. Advances in DNA sequencing and SNP genotyping have provided profound insights into the genetics of model organisms, but until recently, studies of nonmodel species lagged behind because of the scarcity of sequence and markers (see Fig. 1). In the past year, Tautz et al. (2010) and associated papers in supplemental issue of Molecular Ecology described a revolutionary transition from studies of ‘molecular ecology’ to studies of ‘ecological genomics’. Concurrently, Allendorf et al. (2010) grappled with placing the new-found wealth of sequence and SNP information into a ‘conservation genomics’ context. This revolution in molecular genetics studies would have been difficult to forecast a few years ago. Molecular genetic studies provide exceptional insight into relationships, migration and evolution of natural populations (Morin et al. 2004). During the origins of molecular ecology, in the 1960s and 1970s, it became clear that techniques such as allozyme electrophoresis would provide a basic framework for understanding species interactions and adaptation and for conserving natural genetic variability (Utter et al. 1966, 1974; Avise et al. 1975). Technical limitations at the time restricted both the ability to explore the dynamics of genetic diversity in species exhibiting low levels of variation as well as the direct analysis of adaptive variation in the wild. During the following years, innovators began to dream of potential applications for conservation and management of economically exploited species that included using molecular markers to determine the population-of-origin of migrating animals (see papers in Ryman & Utter 1987; Waples & Aebersold 1990), an important focus of many papers in this issue. Recent decades were punctuated by improvements in molecular and statistical techniques that produced an array of tools relevant to ecological and evolutionary studies such as assignment tests, estimates of effective population size, fine-scale population structure, kinship analyses (e.g. Helyar et al. this issue; Waples & Waples this issue) and genome-wide surveys based upon an ever increasing resolution of individuals and populations. The advantages of genotyping polymorphic SNPs with high-throughput assays have created much interest (Vignal et al. 2002; Brumfield et al. 2003; Morin et al. 2004; Schlötterer 2004). Until recently, however, the scarcity of available DNA sequence data for nonmodel species limited marker development. Further, because of comparatively low mutation rates, cross-species amplification of primers for SNP analyses did not yield the same results as for microsatellites. For example, Miller et al. (2010) tested the OvineSNP50 BeadChip, developed for domestic sheep, in two related ungulates and found only about 1% of the nearly 50 000 SNP loci to be polymorphic. Therefore, the SNP assays or probes developed for one species were not likely to be useful in others, even though primers may cross-amplify. The current supplemental issue contains 22 papers that underline the advantages of SNPs, advocate the need for SNP research in nonmodel organisms, and chart advances in discovery and applications. Although progress is apparent across a broad array of taxa, most papers presented here focus upon species of fish. This outcome, beyond the bias of the workshop organizers, may be in part because of the well-developed multinational collaborations that coordinate the sharing of DNA Correspondence: J. E. Seeb, Fax: (206) 543 5728; E-mail: jseeb@uw.edu

An empirical verification of population assignment methods by marking and parentage data: hatchery and wild steelhead (Oncorhynchus mykiss) in Forks Creek, Washington, USA

Assignment tests are increasingly applied in ecology and conservation, although empirical comparisons of methods are still rare or are restricted to few of the available approaches. Furthermore, the performance of assignment tests in cases with low population differentiation, violations of Hardy–Weinberg equilibrium and unbalanced sampling designs has not been verified. The release of adult hatchery steelhead to spawn in Forks Creek in 1996 and 1997 provided an opportunity to compare the power of different assignment methods to distinguish their offspring from those of sympatric wild steelhead. We compared standard assignment methods requiring baseline samples (frequency, distance and Bayesian) and clustering approaches with and without baseline information, using six freely available computer programs. Assignments were verified by parentage data obtained for a subset of returning offspring. All methods provided similar assignment success, despite low differentiation between wild and hatchery fish (FST = 0.02). Bayesian approaches with baseline data performed best, whereas the results of clustering methods were variable and depended on the samples included in the analysis and the availability of baseline information. Removal of a locus with null alleles and equalizing sample sizes had little effect on assignments. Our results demonstrate the robustness of most assignment tests to low differentiation and violations of assumptions, as well as their utility for ecological studies that require correct classification of different groups.

A Dense Linkage Map for Chinook salmon (Oncorhynchus tshawytscha) Reveals Variable Chromosomal Divergence After an Ancestral Whole Genome Duplication Event

Comparisons between the genomes of salmon species reveal that they underwent extensive chromosomal rearrangements following whole genome duplication that occurred in their lineage 58−63 million years ago. Extant salmonids are diploid, but occasional pairing between homeologous chromosomes exists in males. The consequences of re-diploidization can be characterized by mapping the position of duplicated loci in such species. Linkage maps are also a valuable tool for genome-wide applications such as genome-wide association studies, quantitative trait loci mapping or genome scans. Here, we investigated chromosomal evolution in Chinook salmon (Oncorhynchus tshawytscha) after genome duplication by mapping 7146 restriction-site associated DNA loci in gynogenetic haploid, gynogenetic diploid, and diploid crosses. In the process, we developed a reference database of restriction-site associated DNA loci for Chinook salmon comprising 48528 non-duplicated loci and 6409 known duplicated loci, which will facilitate locus identification and data sharing. We created a very dense linkage map anchored to all 34 chromosomes for the species, and all arms were identified through centromere mapping. The map positions of 799 duplicated loci revealed that homeologous pairs have diverged at different rates following whole genome duplication, and that degree of differentiation along arms was variable. Many of the homeologous pairs with high numbers of duplicated markers appear conserved with other salmon species, suggesting that retention of conserved homeologous pairing in some arms preceded species divergence. As chromosome arms are highly conserved across species, the major resources developed for Chinook salmon in this study are also relevant for other related species.

Long-term effects of translocation and release numbers on fine-scale population structure among coho salmon (Oncorhynchus kisutch)

Management actions, such as translocations, reintroductions and supportive breeding, can have both negative and positive effects on population recovery. Several studies have examined the incidence of introgression following such actions, but few studies have explored the effect of release numbers on gene flow between closely related recipient populations. We examined population structure of coho salmon in Puget Sound (Washington State, USA) to evaluate the relationship between the number of individuals transferred between rivers, and the number released within rivers, on inter‐ and intrariver population divergence. Eleven microsatellite loci were surveyed in 23 hatchery and wild samples collected from 11 rivers within and one hatchery outside Puget Sound. Pairwise genetic divergences between most populations were significant, but the population structure could not be explained by an isolation‐by‐distance model (Mantel test, P > 0.05). In contrast, we detected significant hatchery influence on population structure. The numbers of fish transferred among rivers between 1952 and 2004 was negatively correlated with differentiation between rivers (partial Mantel test, P = 0.005) but not within rivers (t‐test, P = 0.41). Number of fish released from hatcheries that collect broodstock locally was negatively correlated with population structure within rivers (t‐test P = 0.002), and between nearby rivers (partial Mantel P = 0.04). Our results indicate that the population structure can, to some degree, be altered by the number of individuals transferred and by local release number of individuals in ongoing artificial propagation programs. The findings presented here emphasize the need to control the number of individuals that are either inadvertently introduced, or are deliberately released under conservation scenarios.

Integration of Random Forest with population-based outlier analyses provides insight on the genomic basis and evolution of run timing in Chinook salmon (Oncorhynchus tshawytscha).

Anadromous Chinook salmon populations vary in the period of river entry at the initiation of adult freshwater migration, facilitating optimal arrival at natal spawning. Run timing is a polygenic trait that shows evidence of rapid parallel evolution in some lineages, signifying a key role for this phenotype in the ecological divergence between populations. Studying the genetic basis of local adaptation in quantitative traits is often impractical in wild populations. Therefore, we used a novel approach, Random Forest, to detect markers linked to run timing across 14 populations from contrasting environments in the Columbia River and Puget Sound, USA. The approach permits detection of loci of small effect on the phenotype. Divergence between populations at these loci was then examined using both principle component analysis and FST outlier analyses, to determine whether shared genetic changes resulted in similar phenotypes across different lineages. Sequencing of 9107 RAD markers in 414 individuals identified 33 predictor loci explaining 79.2% of trait variance. Discriminant analysis of principal components of the predictors revealed both shared and unique evolutionary pathways in the trait across different lineages, characterized by minor allele frequency changes. However, genome mapping of predictor loci also identified positional overlap with two genomic outlier regions, consistent with selection on loci of large effect. Therefore, the results suggest selective sweeps on few loci and minor changes in loci that were detected by this study. Use of a polygenic framework has provided initial insight into how divergence in a trait has occurred in the wild.

Comparative mapping between coho salmon (Oncorhynchus kisutch) and three other salmonids suggests a role for chromosomal rearrangements in the retention of duplicated regions following a whole genome duplication event

Whole genome duplication has been implicated in evolutionary innovation and rapid diversification. In salmonid fishes, however, whole genome duplication significantly pre-dates major transitions across the family, and re-diploidization has been a gradual process between genomes that have remained essentially collinear. Nevertheless, pairs of duplicated chromosome arms have diverged at different rates from each other, suggesting that the retention of duplicated regions through occasional pairing between homeologous chromosomes may have played an evolutionary role across species pairs. Extensive chromosomal arm rearrangements have been a key mechanism involved in re-dipliodization of the salmonid genome; therefore, we investigated their influence on degree of differentiation between homeologs across salmon species. We derived a linkage map for coho salmon and performed comparative mapping across syntenic arms within the genus Oncorhynchus, and with the genus Salmo, to determine the phylogenetic relationship between chromosome arrangements and the retention of undifferentiated duplicated regions. A 6596.7 cM female coho salmon map, comprising 30 linkage groups with 7415 and 1266 nonduplicated and duplicated loci, respectively, revealed uneven distribution of duplicated loci along and between chromosome arms. These duplicated regions were conserved across syntenic arms across Oncorhynchus species and were identified in metacentric chromosomes likely formed ancestrally to the divergence of Oncorhynchus from Salmo. These findings support previous studies in which observed pairings involved at least one metacentric chromosome. Re-diploidization in salmon may have been prevented or retarded by the formation of metacentric chromosomes after the whole genome duplication event and may explain lineage-specific innovations in salmon species if functional genes are found in these regions.

An integrated linkage map reveals candidate genes underlying adaptive variation in Chinook salmon (Oncorhynchus tshawytscha)

Salmonids are an important cultural and ecological resource exhibiting near worldwide distribution between their native and introduced range. Previous research has generated linkage maps and genomic resources for several species as well as genome assemblies for two species. We first leveraged improvements in mapping and genotyping methods to create a dense linkage map for Chinook salmon Oncorhynchus tshawytscha by assembling family data from different sources. We successfully mapped 14 620 SNP loci including 2336 paralogs in subtelomeric regions. This improved map was then used as a foundation to integrate genomic resources for gene annotation and population genomic analyses. We anchored a total of 286 scaffolds from the Atlantic salmon genome to the linkage map to provide a framework for the placement 11 728 Chinook salmon ESTs. Previously identified thermotolerance QTL were found to colocalize with several candidate genes including HSP70, a gene known to be involved in thermal response, as well as its inhibitor. Multiple regions of the genome with elevated divergence between populations were also identified, and annotation of ESTs in these regions identified candidate genes for fitness related traits such as stress response, growth and behaviour. Collectively, these results demonstrate the utility of combining genomic resources with linkage maps to enhance evolutionary inferences.

Chromosome rearrangements, recombination suppression, and limited segregation distortion in hybrids between Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss)

BackgroundIntrogressive hybridization is an important evolutionary process that can lead to the creation of novel genome structures and thus potentially new genetic variation for selection to act upon. On the other hand, hybridization with introduced species can threaten native species, such as cutthroat trout (Oncorhynchus clarkii) following the introduction of rainbow trout (O. mykiss). Neither the evolutionary consequences nor conservation implications of rainbow trout introgression in cutthroat trout is well understood. Therefore, we generated a genetic linkage map for rainbow-Yellowstone cutthroat trout (O. clarkii bouvieri) hybrids to evaluate genome processes that may help explain how introgression affects hybrid genome evolution.ResultsThe hybrid map closely aligned with the rainbow trout map (a cutthroat trout map does not exist), sharing all but one linkage group. This linkage group (RYHyb20) represented a fusion between an acrocentric (Omy28) and a metacentric chromosome (Omy20) in rainbow trout. Additional mapping in Yellowstone cutthroat trout indicated the two rainbow trout homologues were fused in the Yellowstone genome. Variation in the number of hybrid linkage groups (28 or 29) likely depended on a Robertsonian rearrangement polymorphism within the rainbow trout stock. Comparison between the female-merged F1 map and a female consensus rainbow trout map revealed that introgression suppressed recombination across large genomic regions in 5 hybrid linkage groups. Two of these linkage groups (RYHyb20 and RYHyb25_29) contained confirmed chromosome rearrangements between rainbow and Yellowstone cutthroat trout indicating that rearrangements may suppress recombination. The frequency of allelic and genotypic segregation distortion varied among parents and families, suggesting few incompatibilities exist between rainbow and Yellowstone cutthroat trout genomes.ConclusionsChromosome rearrangements suppressed recombination in the hybrids. This result supports several previous findings demonstrating that recombination suppression restricts gene flow between chromosomes that differ by arrangement. Conservation of synteny and map order between the hybrid and rainbow trout maps and minimal segregation distortion in the hybrids suggest rainbow and Yellowstone cutthroat trout genomes freely introgress across chromosomes with similar arrangement. Taken together, these results suggest that rearrangements impede introgression. Recombination suppression across rearrangements could enable large portions of non-recombined chromosomes to persist within admixed populations.

Functional Annotation of All Salmonid Genomes (FAASG): An international initiative supporting future salmonid research, conservation and aquaculture

We describe an emerging initiative - the ‘Functional Annotation of All Salmonid Genomes’ (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

Genetic and Evolutionary Considerations in Fishery Management: Research Needs for the Future

Genetic methods have become indispensable for sound fishery management and will become even more so in the twenty-first century. Selectively neutral genetic markers are widely used in stock identification, mixed-stock fishery analysis, monitoring levels of genetic diversity within populations and levels of connectivity among populations, and for a range of other applications. We expect that future research will continue to provide incremental improvements in the number and type of genetic markers available, as well as in the methods for data analysis and the necessary computational resources. Topics that will merit special consideration include: (1) developing a better understanding of the various flavors of demographic independence and how genetic markers can provide relevant insights; (2) more powerful ways to deal with the low signal-to-noise ratio of population differentiation found in many marine species (the signal of genetic differences is small compared to various sources of random noise); (3) better integration of genetic information, biological information, and information about physical features of the habitat to provide a fuller picture of dynamic marine ecosystems; (4) whether the tiny effective size to census size ratios reported for some marine species are accurate, and if so what this means for conservation of large marine populations. In contrast to the situation with neutral genetic markers, evolutionary changes involving traits related to fitness have only recently attracted much attention in fishery management. In general, any changes to marine ecosystems alter the selective regimes that component species experience and hence can be expected to produce an evolutionary response. Three general topics are particularly important in this regard: harvest, artificial propagation, and climate change. One key challenge is to disentangle the effects of genetics versus environment in determining observed patterns of phenotypic change. Quantitative genetic and molecular genetic approaches can accomplish this, and our capabilities for examining functional parts of the genome are rapidly expanding. However, these methods are logistically challenging and resource-intensive, so in the near future will be feasible Chapter 23 Genetic and Evolutionary Considerations in Fishery Management: Research Needs for the Future Robin S. Waples and Kerry A. Naish R.S. Waples ( ) Northwest Fisheries Science Center, 2725 Montlake Blvd. East, Seattle, WA 98112, USA e-mail: robin.waples@noaa.gov K.A. Naish School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195, USA R.J. Beamish and B.J. Rothschild (eds.), The Future of Fisheries Science in North America, 427 Fish & Fisheries Series,