Complex patterns of genetic population structure in the mouthbrooding marine catfish, Bagre marinus, in the Gulf of Mexico and U.S. Atlantic.

Patterns of genetic variation reflect interactions among microevolutionary forces that vary in strength with changing demography. Here, patterns of variation within and among samples of the mouthbrooding gafftopsail catfish (Bagre marinus, Family Ariidae) captured in the U.S. Atlantic and throughout the Gulf of Mexico were analyzed using genomics to generate neutral and non-neutral SNP data sets. Because genomic resources are lacking for ariids, linkage disequilibrium network analysis was used to examine patterns of putatively adaptive variation. Finally, historical demographic parameters were estimated from site frequency spectra. The results show four differentiated groups, corresponding to the (1) U.S. Atlantic, and the (2) northeastern, (3) northwestern, and (4) southern Gulf of Mexico. The non-neutral data presented two contrasting signals of structure, one due to increases in diversity moving west to east and north to south, and another to increased heterozygosity in the Atlantic. Demographic analysis suggested that recently reduced long-term effective population size in the Atlantic is likely an important driver of patterns of genetic variation and is consistent with a known reduction in population size potentially due to an epizootic. Overall, patterns of genetic variation resemble that of other fishes that use the same estuarine habitats as nurseries, regardless of the presence/absence of a larval phase, supporting the idea that adult/juvenile behavior and habitat are important predictors of contemporary patterns of genetic structure.

Temperature-associated selection linked to putative chromosomal inversions in king scallop (Pecten maximus).

The genomic landscape of divergence-the distribution of differences among populations or species across the genome-is increasingly characterized to understand the role that microevolutionary forces such as natural selection and recombination play in causing and maintaining genetic divergence. This line of inquiry has also revealed chromosome structure variation to be an important factor shaping the landscape of adaptive genetic variation. Owing to a high prevalence of chromosome structure variation and the strong pressure for local adaptation necessitated by their sessile nature, bivalve molluscs are an ideal taxon for exploring the relationship between chromosome structure variation and local adaptation. Here, we report a population genomic survey of king scallop (Pecten maximus) across its natural range in the northeastern Atlantic Ocean, using a recent chromosome-level genome assembly. We report the presence of at least three large (12-22 Mb), putative chromosomal inversions associated with sea surface temperature and whose frequencies are in contrast to neutral population structure. These results highlight a potentially large role for recombination-suppressing chromosomal inversions in local adaptation and suggest a hypothesis to explain the maintenance of differences in reproductive timing found at relatively small spatial scales across king scallop populations.

Disentangling complex genomic signals to understand population structure of an exploited, estuarine-dependent flatfish.

Interpreting contemporary patterns of population structure requires an understanding of the interactions among microevolutionary forces and past demographic events. Here, 4,122 SNP-containing loci were used to assess structure in southern flounder (Paralichthys lethostigma) sampled across its range in the US Atlantic Ocean (Atlantic) and Gulf of Mexico (Gulf) and relationships among components of genomic variation and spatial and environmental variables were assessed across estuarine population samples in the Gulf. While hierarchical amova revealed significant heterogeneity within and between the Atlantic and Gulf, pairwise comparisons between samples within ocean basins demonstrated that all significant heterogeneity occurred within the Gulf. The distribution of Tajima's D estimated at a genome-wide scale differed significantly from equilibrium in all estuaries, with more negative values occurring in the Gulf. Components of genomic variation were significantly associated with environmental variables describing individual estuaries, and environment explained a larger component of variation than spatial proximity. Overall, results suggest that there is genetic spatial autocorrelation caused by shared larval sources for proximal nurseries (migration/drift), but that it is modified by environmentally driven differentiation (selection). This leads to conflicting signals in different parts of the genome and creates patterns of divergence that do not correspond to paradigms of strong local directional selection.

Demonstration of the Use of Environmental DNA for the Non-Invasive Genotyping of a Bivalve Mollusk, the European Flat Oyster (Ostrea edulis).

Accurate SNP (single nucleotide polymorphism) genotype information is critical for a wide range of selective breeding applications in aquaculture, including parentage assignment, marker-assisted, and genomic selection. However, the sampling of tissue for genetic analysis can be invasive for juvenile animals or taxa where sampling tissue is difficult or may cause mortality (e.g. bivalve mollusks). Here, we demonstrate a novel, non-invasive technique for sampling DNA based on the collection of environmental DNA using European Flat Oysters (Ostrea edulis) as an example. The live animals are placed in individual containers until sufficient genetic material is released into the seawater which is then recovered by filtration. We compared the results of tissue and eDNA derived SNP genotype calls using a PCR based genotyping platform. We found that 100% accurate genotype calls from eDNA are possible, but depend on appropriate filtration and the dilution of the sample throughout the workflow. We also developed an additional low-cost DNA extraction technique which provided >99% correct SNP genotype calls in comparison to tissue. It was concluded that eDNA sampling can be used in hatchery and selective breeding programs applicable to any aquatic organism for which direct tissue sampling may result in animal welfare concerns or mortality.

Evolution of population structure in an estuarine-dependent marine fish.

Restriction site-associated DNA (RAD) sequencing was used to characterize neutral and adaptive genetic variation among geographic samples of red drum, Sciaenops ocellatus, an estuarine-dependent fish found in coastal waters along the southeastern coast of the United States (Atlantic) and the northern Gulf of Mexico (Gulf). Analyses of neutral and outlier loci revealed three genetically distinct regional clusters: one in the Atlantic and two in the northern Gulf. Divergence in neutral loci indicated gradual genetic change and followed a linear pattern of isolation by distance. Divergence in outlier loci was at least an order of magnitude greater than divergence in neutral loci, and divergence between the regions in the Gulf was twice that of divergence between other regions. Discordance in patterns of genetic divergence between outlier and neutral loci is consistent with the hypothesis that the former reflects adaptive responses to environmental factors that vary on regional scales, while the latter largely reflects drift processes. Differences in basic habitat, initiated by glacial retreat and perpetuated by contemporary oceanic and atmospheric forces interacting with the geomorphology of the northern Gulf, followed by selection, appear to have led to reduced gene flow among red drum across the northern Gulf, reinforcing differences accrued during isolation and resulting in continued divergence across the genome. This same dynamic also may pertain to other coastal or nearshore fishes (18 species in 14 families) where genetically or morphologically defined sister taxa occur in the three regions.

Genomic Tools and Selective Breeding in Molluscs.

The production of most farmed molluscs, including mussels, oysters, scallops, abalone, and clams, is heavily dependent on natural seed from the plankton. Closing the lifecycle of species in hatcheries can secure independence from wild stocks and enables long-term genetic improvement of broodstock through selective breeding. Genomic techniques have the potential to revolutionize hatchery-based selective breeding by improving our understanding of the characteristics of mollusc genetics that can pose a challenge for intensive aquaculture and by providing a new suite of tools for genetic improvement. Here we review characteristics of the life history and genetics of molluscs including high fecundity, self-fertilization, high genetic diversity, genetic load, high incidence of deleterious mutations and segregation distortion, and critically assess their impact on the design and effectiveness of selective breeding strategies. A survey of the results of current breeding programs in the literature show that selective breeding with inbreeding control is likely the best strategy for genetic improvement of most molluscs, and on average growth rate can be improved by 10% per generation and disease resistance by 15% per generation across the major farmed species by implementing individual or family-based selection. Rapid advances in sequencing technology have resulted in a wealth of genomic resources for key species with the potential to greatly improve hatchery-based selective breeding of molluscs. In this review, we catalog the range of genomic resources currently available for molluscs of aquaculture interest and discuss the bottlenecks, including lack of high-quality reference genomes and the relatively high cost of genotyping, as well as opportunities for applying genomics-based selection.

These aren't the loci you'e looking for: Principles of effective SNP filtering for molecular ecologists.

Sequencing reduced-representation libraries of restriction site-associated DNA (RADseq) to identify single nucleotide polymorphisms (SNPs) is quickly becoming a standard methodology for molecular ecologists. Because of the scale of RADseq data sets, putative loci cannot be assessed individually, making the process of filtering noise and correctly identifying biologically meaningful signal more difficult. Artefacts introduced during library preparation and/or bioinformatic processing of SNP data can create patterns that are incorrectly interpreted as indicative of population structure or natural selection. Therefore, it is crucial to carefully consider types of errors that may be introduced during laboratory work and data processing, and how to minimize, detect and remove these errors. Here, we discuss issues inherent to RADseq methodologies that can result in artefacts during library preparation and locus reconstruction resulting in erroneous SNP calls and, ultimately, genotyping error. Further, we describe steps that can be implemented to create a rigorously filtered data set consisting of markers accurately representing independent loci and compare the effect of different combinations of filters on four RAD data sets. At last, we stress the importance of publishing raw sequence data along with final filtered data sets in addition to detailed documentation of filtering steps and quality control measures.

Genetic mapping and comparative genomics to inform restoration enhancement and culture of southern flounder, Paralichthys lethostigma.

BACKGROUND: Southern flounder, Paralichthys lethostigma, historically support a substantial fishery along the Atlantic and Gulf coasts of the southern United States. Low year-class strengths over the past few years in the western Gulf of Mexico have raised concern that spawning stocks may be overfished. Current management of the resource includes releasing hatchery-raised juveniles to restock bays and estuaries; additionally, there is a growing interest in the potential for commercial aquaculture of the species. Currently, genomic resources for southern flounder do not exist. Here, we used two hatchery-reared families and double-digest, restriction-site-associated DNA (ddRAD) sequencing to create a reduced-representation genomic library consisting of several thousand single nucleotide polymorphisms (SNPs) located throughout the genome. RESULTS: The relative position of each SNP-containing locus was determined to create a high-density genetic map spanning the 24 linkage groups of the southern flounder genome. The consensus map was used to identify regions of shared synteny between southern flounder and seven other fish species for which genome assemblies are available. Finally, syntenic blocks were used to localize genes identified from transcripts in European flounder as potentially being involved in ecotoxicological and osmoregulatory responses, as well as QTLs associated with growth and disease resistance in Japanese flounder, on the southern flounder linkage map. CONCLUSIONS: The information provided by the linkage map will enrich restoration efforts by providing a foundation for interpreting spatial genetic variation within the species, ultimately furthering an understanding of the adaptive potential and resilience of southern flounder to future changes in local environmental conditions. Further, the map will facilitate the use of genetic markers to enhance restoration and commercial aquaculture.

Linkage Mapping and Comparative Genomics of Red Drum (Sciaenops ocellatus) Using Next-Generation Sequencing.

Developments in next-generation sequencing allow genotyping of thousands of genetic markers across hundreds of individuals in a cost-effective manner. Because of this, it is now possible to rapidly produce dense genetic linkage maps for nonmodel species. Here, we report a dense genetic linkage map for red drum, a marine fish species of considerable economic importance in the southeastern United States and elsewhere. We used a prior microsatellite-based linkage map as a framework and incorporated 1794 haplotyped contigs derived from high-throughput, reduced representation DNA sequencing to produce a linkage map containing 1794 haplotyped restriction-site associated DNA (RAD) contigs, 437 anonymous microsatellites, and 44 expressed sequence-tag-linked microsatellites (EST-SSRs). A total of 274 candidate genes, identified from transcripts from a preliminary hydrocarbon exposure study, were localized to specific chromosomes, using a shared synteny approach. The linkage map will be a useful resource for red drum commercial and restoration aquaculture, and for better understanding and managing populations of red drum in the wild.

Haplotyping RAD loci: an efficient method to filter paralogs and account for physical linkage.

Next-generation sequencing of reduced-representation genomic libraries provides a powerful methodology for genotyping thousands of single-nucleotide polymorphisms (SNPs) among individuals of nonmodel species. Utilizing genotype data in the absence of a reference genome, however, presents a number of challenges. One major challenge is the trade-off between splitting alleles at a single locus into separate clusters (loci), creating inflated homozygosity, and lumping multiple loci into a single contig (locus), creating artefacts and inflated heterozygosity. This issue has been addressed primarily through the use of similarity cut-offs in sequence clustering. Here, two commonly employed, postclustering filtering methods (read depth and excess heterozygosity) used to identify incorrectly assembled loci are compared with haplotyping, another postclustering filtering approach. Simulated and empirical data sets were used to demonstrate that each of the three methods separately identified incorrectly assembled loci; more optimal results were achieved when the three methods were applied in combination. The results confirmed that including incorrectly assembled loci in population-genetic data sets inflates estimates of heterozygosity and deflates estimates of population divergence. Additionally, at low levels of population divergence, physical linkage between SNPs within a locus created artificial clustering in analyses that assume markers are independent. Haplotyping SNPs within a locus effectively neutralized the physical linkage issue without having to thin data to a single SNP per locus. We introduce a Perl script that haplotypes polymorphisms, using data from single or paired-end reads, and identifies potentially problematic loci.

Rapid genetic and morphologic divergence between captive and wild populations of the endangered Leon Springs pupfish, Cyprinodon bovinus.

The Leon Springs pupfish (Cyprinodon bovinus) is an endangered species currently restricted to a single desert spring and a separate captive habitat in southwestern North America. Following establishment of the captive population from wild stock in 1976, the wild population has undergone natural population size fluctuations, intentional culling to purge genetic contamination from an invasive congener (Cyprinodon variegatus) and augmentation/replacement of wild fish from the captive stock. A severe population decline following the most recent introduction of captive fish prompted us to examine whether the captive and wild populations have differentiated during the short time they have been isolated from one another. If so, the development of divergent genetic and/or morphologic traits between populations could contribute to a diminished ability of fish from one location to thrive in the other. Examination of genomewide single nucleotide polymorphisms and morphologic variation revealed no evidence of residual C. variegatus characteristics in contemporary C. bovinus samples. However, significant genetic and morphologic differentiation was detected between the wild and captive populations, some of which might reflect local adaptation. Our results indicate that genetic and physical characteristics can diverge rapidly between isolated subdivisions of managed populations, potentially compromising the value of captive stock for future supplementation efforts. In the case of C. bovinus, our findings underscore the need to periodically inoculate the captive population with wild genetic material to help mitigate genetic, and potentially morphologic, divergence between them and also highlight the utility of parallel morphologic and genomic evaluation to inform conservation management planning.

dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms.

Restriction-site associated DNA sequencing (RADseq) has become a powerful and useful approach for population genomics. Currently, no software exists that utilizes both paired-end reads from RADseq data to efficiently produce population-informative variant calls, especially for non-model organisms with large effective population sizes and high levels of genetic polymorphism. dDocent is an analysis pipeline with a user-friendly, command-line interface designed to process individually barcoded RADseq data (with double cut sites) into informative SNPs/Indels for population-level analyses. The pipeline, written in BASH, uses data reduction techniques and other stand-alone software packages to perform quality trimming and adapter removal, de novo assembly of RAD loci, read mapping, SNP and Indel calling, and baseline data filtering. Double-digest RAD data from population pairings of three different marine fishes were used to compare dDocent with Stacks, the first generally available, widely used pipeline for analysis of RADseq data. dDocent consistently identified more SNPs shared across greater numbers of individuals and with higher levels of coverage. This is due to the fact that dDocent quality trims instead of filtering, incorporates both forward and reverse reads (including reads with INDEL polymorphisms) in assembly, mapping, and SNP calling. The pipeline and a comprehensive user guide can be found at http://dDocent.wordpress.com.

A comparison of neutral and immune genetic variation in Atlantic salmon, Salmo salar L. in Chilean aquaculture facilities.

Genetic diversity was assessed in samples of cultured Atlantic salmon, Salmo salar L., obtained from facilities in Chile between 2005 and 2010, a period of time during which the infectious pathogens Infectious Salmon Anemia (ISA) virus, Caligus rogercresseyi (sea lice), and Piscirickettsia salmonis (salmon rickettsial syndrome) were common. Two panels of microsatellite markers were utilized: one with microsatellites with no known gene associations (neutral) and one featuring microsatellites linked to putative immune-related genes (immune-related). Allelic richness and gene diversity across samples were significantly greater in neutral loci as compared to immune-related loci. Both diversity measures were homogeneous among samples for immune-related loci and heterogeneous among samples for neutral loci. Immune-related loci were identified as F(ST) outliers in pairwise comparisons of samples at a 10-fold higher frequency than neutral loci. These results indicate that neutral and immune-related portions of the Atlantic salmon genome may have differed in response to the gauntlet of pathogens and that monitoring of specific, well characterized immune-related loci as well as neutral loci in cultured species could be useful when disease control and prevention is a goal.

Use of comparative genomics to develop EST-SSRs for red drum (Sciaenops ocellatus).

Microsatellites physically linked to expressed sequence tags (EST-SSRs) are an important resource for linkage mapping and comparative genomics, and data mining in publicly available EST databases is a common strategy for EST-SSR discovery. At present, many species lack species-specific EST sequence data needed for the efficient characterization of EST-SSRs. This paper describes the discovery and development of EST-SSRs for red drum (Sciaenops ocellatus), an estuarine-dependent sciaenid species of economic importance in the USA and elsewhere, using a phylogenetically informed, comparative genomics approach to primer design. The approach entailed comparing existing genomic resources from species closely allied phylogenetically to red drum, with resources from more distantly related outgroup species. By taking into account the degree to which flanking regions are conserved across taxa, the efficiency of PCR primer design was increased greatly. The amplification success rate for primers designed for red drum was 100 % when using EST libraries from confamilial species and 92 % when using an EST library from a species in the same suborder. The primers developed also amplified EST-SSRs in a wide range of perciform fishes, suggesting potential use in comparative genomics. This study demonstrates that EST-SSRs can be efficiently developed for an organism when limited species-specific data are available by exploiting genomic resources from well-studied species, even those at extended phylogenetic distances.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2011-31 January 2012.

This article documents the addition of 473 microsatellite marker loci and 71 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Barteria fistulosa, Bombus morio, Galaxias platei, Hematodinium perezi, Macrocentrus cingulum Brischke (a.k.a. M. abdominalis Fab., M. grandii Goidanich or M. gifuensis Ashmead), Micropogonias furnieri, Nerita melanotragus, Nilaparvata lugens Stål, Sciaenops ocellatus, Scomber scombrus, Spodoptera frugiperda and Turdus lherminieri. These loci were cross-tested on the following species: Barteria dewevrei, Barteria nigritana, Barteria solida, Cynoscion acoupa, Cynoscion jamaicensis, Cynoscion leiarchus, Cynoscion nebulosus, Cynoscion striatus, Cynoscion virescens, Macrodon ancylodon, Menticirrhus americanus, Nilaparvata muiri and Umbrina canosai. This article also documents the addition of 116 sequencing primer pairs for Dicentrarchus labrax.