James E. Seeb

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 comparison of SNPs and microsatellites for parentage and kinship assignment in a wild sockeye salmon (Oncorhynchus nerka) population.

Because of their high variability, microsatellites are still considered the marker of choice for studies on parentage and kinship in wild populations. Nevertheless, single nucleotide polymorphisms (SNPs) are becoming increasing popular in many areas of molecular ecology, owing to their high‐throughput, easy transferability between laboratories and low genotyping error. An ongoing discussion concerns the relative power of SNPs compared to microsatellites—that is, how many SNP loci are needed to replace a panel of microsatellites? Here, we evaluate the assignment power of 80 SNPs (HE = 0.30, 80 independent alleles) and 11 microsatellites (HE = 0.85, 192 independent alleles) in a wild population of about 400 sockeye salmon with two commonly used software packages (Cervus3, Colony2) and, for SNPs only, a newly developed software (SNPPIT). Assignment success was higher for SNPs than for microsatellites, especially for parent pairs, irrespective of the method used. Colony2 assigned a larger proportion of offspring to at least one parent than the other methods, although Cervus and SNPPIT detected more parent pairs. Identification of full‐sib groups without parental information from relatedness measures was possible using both marker systems, although explicit reconstruction of such groups in Colony2 was impossible for SNPs because of computation time. Our results confirm the applicability of SNPs for parentage analyses and refute the predictability of assignment success from the number of independent alleles.

Genotyping by sequencing resolves shallow population structure to inform conservation of Chinook salmon (Oncorhynchus tshawytscha)

Recent advances in population genomics have made it possible to detect previously unidentified structure, obtain more accurate estimates of demographic parameters, and explore adaptive divergence, potentially revolutionizing the way genetic data are used to manage wild populations. Here, we identified 10 944 single‐nucleotide polymorphisms using restriction‐site‐associated DNA (RAD) sequencing to explore population structure, demography, and adaptive divergence in five populations of Chinook salmon (Oncorhynchus tshawytscha) from western Alaska. Patterns of population structure were similar to those of past studies, but our ability to assign individuals back to their region of origin was greatly improved (>90% accuracy for all populations). We also calculated effective size with and without removing physically linked loci identified from a linkage map, a novel method for nonmodel organisms. Estimates of effective size were generally above 1000 and were biased downward when physically linked loci were not removed. Outlier tests based on genetic differentiation identified 733 loci and three genomic regions under putative selection. These markers and genomic regions are excellent candidates for future research and can be used to create high‐resolution panels for genetic monitoring and population assignment. This work demonstrates the utility of genomic data to inform conservation in highly exploited species with shallow population structure.

Short reads and nonmodel species: exploring the complexities of next‐generation sequence assembly and SNP discovery in the absence of a reference genome

How practical is gene and SNP discovery in a nonmodel species using short read sequences? Next‐generation sequencing technologies are being applied to an increasing number of species with no reference genome. For nonmodel species, the cost, availability of existing genetic resources, genome complexity and the planned method of assembly must all be considered when selecting a sequencing platform. Our goal was to examine the feasibility and optimal methodology for SNP and gene discovery in the sockeye salmon (Oncorhynchus nerka) using short read sequences. SOLiD short reads (up to 50 bp) were generated from single‐ and pooled‐tissue transcriptome libraries from ten sockeye salmon. The individuals were from five distinct populations from the Wood River Lakes and Mendeltna Creek, Alaska. As no reference genome was available for sockeye salmon, the SOLiD sequence reads were assembled to publicly available EST reference sequences from sockeye salmon and two closely related species, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Additionally, de novo assembly of the SOLiD data was carried out, and the SOLiD reads were remapped to the de novo contigs. The results from each reference assembly were compared across all references. The number and size of contigs assembled varied with the size reference sequences. In silico SNP discovery was carried out on contigs from all four EST references; however, discovery of valid SNPs was most successful using one of the two conspecific references.

SNP Genotyping by the 5′-Nuclease Reaction: Advances in High-Throughput Genotyping with Nonmodel Organisms

Population genetics studies play an increasingly important role in the management and conservation of nonmodel organisms. Unlike studies with model organisms, a typical population genetics study of a nonmodel organism may be conducted by analyzing thousands or hundreds of thousands of individuals for several dozen single nucleotide polymorphisms (SNPs). The use of robust, robotically mediated TaqMan reactions provides substantial advantages in these types of studies. We describe the methods and laboratory setup for analyzing a sustained high throughput of SNP assays in routine university or natural resource agency laboratories with a handful of thermal cyclers. Agencies sustain rates of nearly 150,000 assays per week using uniplex reactions with the Applied Biosystems 7900HT Fast Real-Time PCR System (AB 7900HT). We further describe the medium-density array run on the BioMark from Fluidigm, which increases this rate to over 500,000 assays per week by multiplexing 96 samples for 96 SNPs.

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.

Summer-Fall Distribution of Stocks of Immature Sockeye Salmon in the Bering Sea as Revealed by Single-Nucleotide Polymorphisms

Abstract We report stock composition estimates for immature (ocean-age .1 and .2) sockeye salmon Oncorhynchus nerka distributed across the Bering Sea in late summer and fall. We establish a baseline data set composed of single-nucleotide polymorphism markers that can achieve very high accuracies in identifying sockeye salmon stocks from throughout their range in Asia and North America. We demonstrate the capabilities of this data set to address high-seas salmon issues by analyzing samples collected by researchers from Russia, Japan, and the United States during late summer and fall 2002–2004 as part of the Bering–Aleutian Salmon International Survey. According to our findings, (1) Gulf of Alaska (GOA) stocks formed a significant portion of the immature sockeye salmon migrating in the eastern and central Bering Sea in summer and fall, and western GOA stocks had a broader distribution in the Bering Sea than eastern GOA stocks; (2) Asian stocks migrated as far east as the western Aleutian Islands and the Don...

Transcriptome sequencing and high‐resolution melt analysis advance single nucleotide polymorphism discovery in duplicated salmonids

Until recently, single nucleotide polymorphism (SNP) discovery in nonmodel organisms faced many challenges, often depending upon a targeted‐gene approach and Sanger sequencing of many individuals. The advent of next‐generation sequencing technologies has dramatically improved discovery, but validating and testing SNPs for use in population studies remain labour intensive. Here, we detail a SNP discovery and validation pipeline that incorporates 454 pyrosequencing, high‐resolution melt analysis (HRMA) and 5′ nuclease genotyping. We generated 4.59 × 108 bp of redundant sequence from transcriptomes of two individual chum salmon, a highly valued species across the Pacific Rim. Nearly 26 000 putative SNPs were identified—some as heterozygotes and some as homozygous for different nucleotides in the two individuals. For validation, we selected 202 templates containing single putative SNPs and conducted HRMA on 10 individuals from each of 19 populations from across the species range. Finally, 5′ nuclease genotyping validated 37 SNPs that conformed to Hardy–Weinberg equilibrium expectations. Putative SNPs expressed as heterozygotes in an ascertainment individual had more than twice the validation rate of those homozygous for different alleles in the two fish, suggesting that many of the latter may have been paralogous sequence variants. Overall, this validation rate of 37/202 suggests that we have found more than 4500 templates containing SNPs for use in this population set. We anticipate using this pipeline to significantly expand the number of SNPs available for the studies of population structure and mixture analyses as well as for the studies of adaptive genetic variation in nonmodel organisms.

Meiotic maps of sockeye salmon derived from massively parallel DNA sequencing

BackgroundMeiotic maps are a key tool for comparative genomics and association mapping studies. Next-generation sequencing and genotyping by sequencing are speeding the processes of SNP discovery and the development of new genetic tools, including meiotic maps for numerous species. Currently there are limited genetic resources for sockeye salmon, Oncorhynchus nerka. We develop the first dense meiotic map for sockeye salmon using a combination of novel SNPs found in restriction site associated DNA (RAD tags) and SNPs available from existing expressed sequence tag (EST) based assays.ResultsWe discovered and genotyped putative SNPs in 3,430 RAD tags. We removed paralogous sequence variants leaving 1,672 SNPs; these were combined with 53 EST-based SNP genotypes for linkage mapping. The map contained 29 male and female linkage groups, consistent with the haploid chromosome number expected for sockeye salmon. The female map contains 1,057 loci spanning 4,896 cM, and the male map contains 1,118 loci spanning 4,220 cM. Regions of conservation with rainbow trout and synteny between the RAD based rainbow trout map and the sockeye salmon map were established.ConclusionsUsing RAD sequencing and EST-based SNP assays we successfully generated the first high density linkage map for sockeye salmon.

Spawning Habitat and Geography Influence Population Structure and Juvenile Migration Timing of Sockeye Salmon in the Wood River Lakes, Alaska

Abstract The strict homing of sockeye salmon Oncorhynchus nerka results in reproductively isolated populations that often spawn in close proximity and share rearing habitat. High spawning fidelity enables these populations to adapt to local conditions, resulting in a wide range of life history characteristics and genetic variation within individual watersheds. The Wood River system in southwestern Alaska provides a pristine, well-studied system in which to examine fine-scale population structure and its influences on juvenile life histories. Adult sockeye salmon spawn in lake beaches, rivers, and small tributaries throughout this watershed, and juveniles rear in five nursery lakes. We genotyped 30 spawning populations and 6,066 migrating smolts at 45 single nucleotide polymorphism loci, two of which are candidates for positive selection in the study system. We show that there is significant genetic structure (F ST = 0.032) in the Wood River lakes and that divergence is generally related to spawning rather...