Garrett J. McKinney

RADseq provides unprecedented insights into molecular ecology and evolutionary genetics: comment on Breaking RAD by Lowry et al. (2016)

In their recently corrected manuscript, “Breaking RAD: An evaluation of the utility of restriction site associated DNA sequencing for genome scans of adaptation”, Lowry et al. argue that genome scans using RADseq will miss many loci under selection due to a combination of sparse marker density and low levels of linkage disequilibrium in most species. We agree that marker density and levels of LD are important considerations when designing a RADseq study; however, we dispute that RAD‐based genome scans are as prone to failure as Lowry et al. suggest. Their arguments ignore the flexible nature of RADseq; the availability of different restriction enzymes and capacity for combining restriction enzymes ensures that a well‐designed study should be able to generate enough markers for efficient genome coverage. We further believe that simplifying assumptions about linkage disequilibrium in their simulations are invalid in many species. Finally, it is important to note that the alternative methods proposed by Lowry et al. have limitations equal to or greater than RADseq. The wealth of studies with positive impactful findings that have used RAD genome scans instead supports the argument that properly conducted RAD genome scans are an effective method for gaining insight into ecology and evolution, particularly for non‐model organisms and those with large or complex genomes.

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.

Identification of Multiple QTL Hotspots in Sockeye Salmon ( Oncorhynchus nerka ) Using Genotyping-by-Sequencing and a Dense Linkage Map

Understanding the genetic architecture of phenotypic traits can provide important information about the mechanisms and genomic regions involved in local adaptation and speciation. Here, we used genotyping-by-sequencing and a combination of previously published and newly generated data to construct sex-specific linkage maps for sockeye salmon (Oncorhynchus nerka). We then used the denser female linkage map to conduct quantitative trait locus (QTL) analysis for 4 phenotypic traits in 3 families. The female linkage map consisted of 6322 loci distributed across 29 linkage groups and was 4082 cM long, and the male map contained 2179 loci found on 28 linkage groups and was 2291 cM long. We found 26 QTL: 6 for thermotolerance, 5 for length, 9 for weight, and 6 for condition factor. QTL were distributed nonrandomly across the genome and were often found in hotspots containing multiple QTL for a variety of phenotypic traits. These hotspots may represent adaptively important regions and are excellent candidates for future research. Comparing our results with studies in other salmonids revealed several regions with overlapping QTL for the same phenotypic trait, indicating these regions may be adaptively important across multiple species. Altogether, our study demonstrates the utility of genomic data for investigating the genetic basis of important phenotypic traits. Additionally, the linkage map created here will enable future research on the genetic basis of phenotypic traits in salmon.

Ontogenetic changes in embryonic and brain gene expression in progeny produced from migratory and resident Oncorhynchus mykiss

Little information has been gathered regarding the ontogenetic changes that contribute to differentiation between resident and migrant individuals, particularly before the onset of gross morphological and physiological changes in migratory individuals. The aim of this study was to evaluate gene expression during early development in Oncorhynchus mykiss populations with different life histories, in a tissue known to integrate environmental cues to regulate complex developmental processes and behaviours. We sampled offspring produced from migrant and resident parents, collecting whole embryos prior to the beginning of first feeding, and brain tissue at three additional time points over the first year of development. RNA sequencing for 32 individuals generated a reference transcriptome of 30 177 genes that passed count thresholds. Differential gene expression between migrant and resident offspring was observed for 1982 genes. The greatest number of differentially expressed genes occurred at 8 months of age, in the spring a full year before the obvious physiological transformation from stream‐dwelling parr to sea water‐adaptable smolts begins for migrant individuals. Sex and age exhibited considerable effects on differential gene expression between migrants and resident offspring. Differential gene expression was observed in genes previously associated with migration, but also in genes previously unassociated with early life history divergence. Pathway analysis revealed coordinated differential expression in genes related to phototransduction, which could modulate photoperiod responsiveness and variation in circadian rhythms. The role for early differentiation in light sensitivity and biological rhythms is particularly intriguing in understanding early brain processes involved in differentiation of migratory and resident life history types.

Paralogs are revealed by proportion of heterozygotes and deviations in read ratios in genotyping by sequencing data from natural populations

Whole‐genome duplications have occurred in the recent ancestors of many plants, fish, and amphibians, resulting in a pervasiveness of paralogous loci and the potential for both disomic and tetrasomic inheritance in the same genome. Paralogs can be difficult to reliably genotype and are often excluded from genotyping‐by‐sequencing (GBS) analyses; however, removal requires paralogs to be identified which is difficult without a reference genome. We present a method for identifying paralogs in natural populations by combining two properties of duplicated loci: (i) the expected frequency of heterozygotes exceeds that for singleton loci, and (ii) within heterozygotes, observed read ratios for each allele in GBS data will deviate from the 1:1 expected for singleton (diploid) loci. These deviations are often not apparent within individuals, particularly when sequence coverage is low; but, we postulated that summing allele reads for each locus over all heterozygous individuals in a population would provide sufficient power to detect deviations at those loci. We identified paralogous loci in three species: Chinook salmon (Oncorhynchus tshawytscha) which retains regions with ongoing residual tetrasomy on eight chromosome arms following a recent whole‐genome duplication, mountain barberry (Berberis alpina) which has a large proportion of paralogs that arose through an unknown mechanism, and dusky parrotfish (Scarus niger) which has largely rediploidized following an ancient whole‐genome duplication. Importantly, this approach only requires the genotype and allele‐specific read counts for each individual, information which is readily obtained from most GBS analysis pipelines.

Genomic islands of divergence linked to ecotypic variation in sockeye salmon

Regions of the genome displaying elevated differentiation (genomic islands of divergence) are thought to play an important role in local adaptation, especially in populations experiencing high gene flow. However, the characteristics of these islands as well as the functional significance of genes located within them remain largely unknown. Here, we used data from thousands of SNPs aligned to a linkage map to investigate genomic islands of divergence in three ecotypes of sockeye salmon (Oncorhynchus nerka) from a single drainage in southwestern Alaska. We found ten islands displaying high differentiation among ecotypes. Conversely, neutral structure observed throughout the rest of the genome was low and not partitioned by ecotype. One island on linkage group So13 was particularly large and contained six SNPs with FST > 0.14 (average FST of neutral SNPs = 0.01). Functional annotation revealed that the peak of this island contained a nonsynonymous mutation in a gene involved in growth in other species (TULP4). The islands that we discovered were relatively small (80–402 Kb), loci found in islands did not show reduced levels of diversity, and loci in islands displayed slightly elevated linkage disequilibrium. These attributes suggest that the islands discovered here were likely generated by divergence hitchhiking; however, we cannot rule out the possibility that other mechanisms may have produced them. Our results suggest that islands of divergence serve an important role in local adaptation with gene flow and represent a significant advance towards understanding the genetic basis of ecotypic differentiation.

Recombination patterns reveal information about centromere location on linkage maps

Linkage mapping is often used to identify genes associated with phenotypic traits and for aiding genome assemblies. Still, many emerging maps do not locate centromeres – an essential component of the genomic landscape. Here, we demonstrate that for genomes with strong chiasma interference, approximate centromere placement is possible by phasing the same data used to generate linkage maps. Assuming one obligate crossover per chromosome arm, information about centromere location can be revealed by tracking the accumulated recombination frequency along linkage groups, similar to half‐tetrad analyses. We validate the method on a linkage map for sockeye salmon (Oncorhynchus nerka) with known centromeric regions. Further tests suggest that the method will work well in other salmonids and other eukaryotes. However, the method performed weakly when applied to a male linkage map (rainbow trout; O. mykiss) characterized by low and unevenly distributed recombination – a general feature of male meiosis in many species. Further, a high frequency of double crossovers along chromosome arms in barley reduced resolution for locating centromeric regions on most linkage groups. Despite these limitations, our method should work well for high‐density maps in species with strong recombination interference and will enrich many existing and future mapping resources.

Identification and Characterization of Sex-Associated Loci in Sockeye Salmon Using Genotyping-by-Sequencing and Comparison with a Sex-Determining Assay Based on the sdY Gene

Loci that can be used to screen for sex in salmon can provide important information for study of both wild and cultured populations. Here, we tested for associations between sex and genotypes at thousands of loci available from a genotyping-by-sequencing (GBS) dataset to discover sex-associated loci in sockeye salmon (Oncorhynchus nerka). We discovered 7 sex-associated loci, developed high-throughput assays for 2 loci, and tested the utility of these 2 assays in 8 collections of sockeye salmon sampled throughout North America. We also screened an existing assay based on the master sex-determining gene in salmon (sdY) in these collections. The ability of GBS-derived loci to assign fish to their phenotypic sex varied substantially among collections suggesting that recombination between the loci that we discovered and the sex-determining gene has occurred. Assignment accuracy to phenotypic sex was much higher with the sdY assay but was still less than 100%. Alignment of sequences from GBS-derived loci to draft genomes for 2 salmonids provided strong evidence that many of these loci are found on chromosomes orthologous to the known sex chromosome in sockeye salmon. Our study is the first to describe the approximate location of the sex-determining region in sockeye salmon and indicates that sdY is also the master sex-determining gene in this species. However, discordances between sdY genotypes and phenotypic sex and the variable performance of GBS-derived loci warrant more research.

Using Linkage Maps as a Tool To Determine Patterns of Chromosome Synteny in the Genus Salvelinus

Next generation sequencing techniques have revolutionized the collection of genome and transcriptome data from nonmodel organisms. This manuscript details the application of restriction site-associated DNA sequencing (RADseq) to generate a marker-dense genetic map for Brook Trout (Salvelinus fontinalis). The consensus map was constructed from three full-sib families totaling 176 F1 individuals. The map consisted of 42 linkage groups with a total female map size of 2502.5 cM, and a total male map size of 1863.8 cM. Synteny was confirmed with Atlantic Salmon for 38 linkage groups, with Rainbow Trout for 37 linkage groups, Arctic Char for 36 linkage groups, and with a previously published Brook Trout linkage map for 39 linkage groups. Comparative mapping confirmed the presence of 8 metacentric and 34 acrocentric chromosomes in Brook Trout. Six metacentric chromosomes seem to be conserved with Arctic Char suggesting there have been at least two species-specific fusion and fission events within the genus Salvelinus. In addition, the sex marker (sdY; sexually dimorphic on the Y chromosome) was mapped to Brook Trout BC35, which is homologous with Atlantic Salmon Ssa09qa, Rainbow Trout Omy25, and Arctic Char AC04q. Ultimately, this linkage map will be a useful resource for studies on the genome organization of Salvelinus, and facilitates comparisons of the Salvelinus genome with Salmo and Oncorhynchus.

Resolving allele dosage in duplicated loci using genotyping-by-sequencing data: A path forward for population genetic analysis

Whole‐genome duplications have occurred in the recent ancestors of many plants, fish and amphibians. Signals of these whole‐genome duplications still exist in the form of paralogous loci. Recent advances have allowed reliable identification of paralogs in genotyping‐by‐sequencing (GBS) data such as that generated from restriction‐site‐associated DNA sequencing (RADSeq); however, excluding paralogs from analyses is still routine due to difficulties in genotyping. This exclusion of paralogs may filter a large fraction of loci, including loci that may be adaptively important or informative for population genetic analyses. We present a maximum‐likelihood method for inferring allele dosage in paralogs and assess its accuracy using simulated GBS, empirical RADSeq and amplicon sequencing data from Chinook salmon. We accurately infer allele dosage for some paralogs from a RADSeq data set and show how accuracy is dependent upon both read depth and allele frequency. The amplicon sequencing data set, using RADSeq‐derived markers, achieved sufficient depth to infer allele dosage for all paralogs. This study demonstrates that RADSeq locus discovery combined with amplicon sequencing of targeted loci is an effective method for incorporating paralogs into population genetic analyses.