Harnessing genomics to fast-track genetic improvement in aquaculture.

Aquaculture is the fastest-growing farmed food sector and will soon become the primary source of fish and shellfish for human diets. In contrast to crop and livestock production, aquaculture production is derived from numerous, exceptionally diverse species that are typically in the early stages of domestication. Genetic improvement of production traits via well-designed, managed breeding programmes has great potential to help meet the rising seafood demand driven by human population growth. Supported by continuous advances in sequencing and bioinformatics, genomics is increasingly being applied across the broad range of aquaculture species and at all stages of the domestication process to optimize selective breeding. In the future, combining genomic selection with biotechnological innovations, such as genome editing and surrogate broodstock technologies, may further expedite genetic improvement in aquaculture.

Conserved and divergent arms of the antiviral response in the duplicated genomes of salmonid fishes.

Antiviral innate immunity is orchestrated by the interferon system, which appeared in ancestors of jawed vertebrates. Interferon upregulation induces hundreds of interferon-stimulated-genes (ISGs) with effector or regulatory functions. Here we investigated the evolutionary diversification of ISG responses through comparison of two salmonid fishes, accounting for the impact of sequential whole genome duplications ancestral to teleosts and salmonids. We analysed the transcriptomic response of the IFN pathway in the head kidney of rainbow trout and Atlantic salmon, which separated 25-30 Mya. We identified a large set of ISGs conserved in both species and cross-referenced them with zebrafish and human ISGs. In contrast, around one-third of salmonid ISG lacked orthologs in human, mouse, chicken or frog, and often between rainbow trout and Atlantic salmon, revealing a fast-evolving, lineage-specific arm of the antiviral response. This study also provides a key resource for in-depth functional analysis of ISGs in salmonids of commercial significance.

Genome-Wide Reconstruction of Rediploidization Following Autopolyploidization across One Hundred Million Years of Salmonid Evolution.

The long-term evolutionary impacts of whole-genome duplication (WGD) are strongly influenced by the ensuing rediploidization process. Following autopolyploidization, rediploidization involves a transition from tetraploid to diploid meiotic pairing, allowing duplicated genes (ohnologs) to diverge genetically and functionally. Our understanding of autopolyploid rediploidization has been informed by a WGD event ancestral to salmonid fishes, where large genomic regions are characterized by temporally delayed rediploidization, allowing lineage-specific ohnolog sequence divergence in the major salmonid clades. Here, we investigate the long-term outcomes of autopolyploid rediploidization at genome-wide resolution, exploiting a recent "explosion" of salmonid genome assemblies, including a new genome sequence for the huchen (Hucho hucho). We developed a genome alignment approach to capture duplicated regions across multiple species, allowing us to create 121,864 phylogenetic trees describing genome-wide ohnolog divergence across salmonid evolution. Using molecular clock analysis, we show that 61% of the ancestral salmonid genome experienced an initial "wave" of rediploidization in the late Cretaceous (85-106 Ma). This was followed by a period of relative genomic stasis lasting 17-39 My, where much of the genome remained tetraploid. A second rediploidization wave began in the early Eocene and proceeded alongside species diversification, generating predictable patterns of lineage-specific ohnolog divergence, scaling in complexity with the number of speciation events. Using gene set enrichment, gene expression, and codon-based selection analyses, we provide insights into potential functional outcomes of delayed rediploidization. This study enhances our understanding of delayed autopolyploid rediploidization and has broad implications for future studies of WGD events.

SalMotifDB: a tool for analyzing putative transcription factor binding sites in salmonid genomes.

BACKGROUND: Recently developed genome resources in Salmonid fish provides tools for studying the genomics underlying a wide range of properties including life history trait variation in the wild, economically important traits in aquaculture and the evolutionary consequences of whole genome duplications. Although genome assemblies now exist for a number of salmonid species, the lack of regulatory annotations are holding back our mechanistic understanding of how genetic variation in non-coding regulatory regions affect gene expression and the downstream phenotypic effects. RESULTS: We present SalMotifDB, a database and associated web and R interface for the analysis of transcription factors (TFs) and their cis-regulatory binding sites in five salmonid genomes. SalMotifDB integrates TF-binding site information for 3072 non-redundant DNA patterns (motifs) assembled from a large number of metazoan motif databases. Through motif matching and TF prediction, we have used these multi-species databases to construct putative regulatory networks in salmonid species. The utility of SalMotifDB is demonstrated by showing that key lipid metabolism regulators are predicted to regulate a set of genes affected by different lipid and fatty acid content in the feed, and by showing that our motif database explains a significant proportion of gene expression divergence in gene duplicates originating from the salmonid specific whole genome duplication. CONCLUSIONS: SalMotifDB is an effective tool for analyzing transcription factors, their binding sites and the resulting gene regulatory networks in salmonid species, and will be an important tool for gaining a better mechanistic understanding of gene regulation and the associated phenotypes in salmonids. SalMotifDB is available at https://salmobase.org/apps/SalMotifDB .

Independent rediploidization masks shared whole genome duplication in the sturgeon-paddlefish ancestor.

Whole genome duplication (WGD) is a dramatic evolutionary event generating many new genes and which may play a role in survival through mass extinctions. Paddlefish and sturgeon are sister lineages that both show genomic evidence for ancient WGD. Until now this has been interpreted as two independent WGD events due to a preponderance of duplicate genes with independent histories. Here we show that although there is indeed a plurality of apparently independent gene duplications, these derive from a shared genome duplication event occurring well over 200 million years ago, likely close to the Permian-Triassic mass extinction period. This was followed by a prolonged process of reversion to stable diploid inheritance (rediploidization), that may have promoted survival during the Triassic-Jurassic mass extinction. We show that the sharing of this WGD is masked by the fact that paddlefish and sturgeon lineage divergence occurred before rediploidization had proceeded even half-way. Thus, for most genes the resolution to diploidy was lineage-specific. Because genes are only truly duplicated once diploid inheritance is established, the paddlefish and sturgeon genomes are thus a mosaic of shared and non-shared gene duplications resulting from a shared genome duplication event.

Exploration of the Nurse Shark (Ginglymostoma cirratum) Plasma Immunoproteome Using High-Resolution LC-MS/MS.

Many animals of scientific importance lack species-specific reagents (e.g., monoclonal antibodies) for in-depth studies of immune proteins. Mass spectrometry (MS)-based proteomics has emerged as a useful method for monitoring changes in protein abundance and modifications in non-model species. It can be used to quantify hundreds of candidate immune molecules simultaneously without the generation of new reagents. Here, we used MS-based proteomics to identify and quantify candidate immune proteins in the plasma of the nurse shark (Ginglymostoma cirratum), a cartilaginous fish and representative of the most basal extant vertebrate lineage with an immunoglobulin-based immune system. Mass spectrometry-based LC-MS/MS was performed on the blood plasma of nurse sharks immunized with human serum albumin (n=4) or sham immunized (n=1), and sampled at days 0 (baseline control), 1, 2, 3, 5, 7, 14, 21, 28, 25, 42 and 49. An antigen-specific antibody response was experimentally confirmed post-immunization. To provide a high-quality reference to identify proteins, we assembled and annotated a multi-tissue de novo transcriptome integrating long- and short-read sequence data. This comprised 62,682 contigs containing open reading frames (ORFs) with a length >80 amino acids. Using this transcriptome, we reliably identified 626 plasma proteins which were broadly categorized into coagulation, immune, and metabolic functional groups. To assess the feasibility of performing LC-MS/MS proteomics in nurse shark in the absence of species-specific protein annotations, we compared the results to an alternative strategy, mapping peptides to proteins predicted in the genome assembly of a related species, the whale shark (Rhincodon typus). This approach reliably identified 297 proteins, indicating that useful data on the plasma proteome may be obtained in many instances despite the absence of a species-specific reference protein database. Among the plasma proteins defined against the nurse shark transcriptome, fifteen showed consistent changes in abundance across the immunized shark individuals, indicating a role in the immune response. These included alpha-2-macroglobulin (A2M) and a novel protein yet to be characterized in diverse vertebrate lineages. Overall, this study enhances genetic and protein-level resources for nurse shark research and vastly improves our understanding of the elasmobranch plasma proteome, including its remodelling following immune stimulation.

Zebrafish as a Model to Unveil the Pro-Osteogenic Effects of Boron-Vitamin D3 Synergism.

The micronutrient boron (B) plays a key role during the ossification process as suggested by various in vitro and in vivo studies. To deepen our understanding of the molecular mechanism involved in the osteogenicity of B and its possible interaction with vitamin D3 (VD), wild-type AB zebrafish (Danio rerio) were used for morphometric analysis and transcriptomic analysis in addition to taking advantage of the availability of specific zebrafish osteoblast reporter lines. First, osteoactive concentrations of B, VD, and their combinations were established by morphometric analysis of the opercular bone in alizarin red-stained zebrafish larvae exposed to two selected concentrations of B (10 and 100 ng/ml), one concentration of VD (10 pg/ml), and their respective combinations. Bone formation, as measured by opercular bone growth, was significantly increased in the two combination treatments than VD alone. Subsequently, a transcriptomic approach was adopted to unveil the molecular key regulators involved in the synergy. Clustering of differentially expressed genes revealed enrichment toward bone and skeletal functions in the groups co-treated with B and VD. Downstream analysis confirmed mitogen-activated protein kinase as the most regulated pathway by the synergy groups in addition to transforming growth factor-ß signaling, focal adhesion, and calcium signaling. The best-performing synergistic treatment, B at 10 ng/ml and VD at 10 pg/ml, was applied to two zebrafish transgenic lines, Tg(sp7:mCherry) and Tg(bglap:EGFP), at multiple time points to further explore the results of the transcriptomic analysis. The synergistic treatment with B and VD induced enrichment of intermediate (sp7+) osteoblast at 6 and 9 days post fertilization (dpf) and of mature (bglap +) osteoblasts at 15 dpf. The results obtained validate the role of B in VD-dependent control over bone mineralization and can help to widen the spectrum of therapeutic approaches to alleviate pathological conditions caused by VD deficiency by using low concentrations of B as a nutritional additive.

Two parallel chromosome-level reference genomes to support restoration and aquaculture of European flat oyster Ostrea edulis.

This volume of Evolutionary Applications sees the publication of two genomes for the European native flat oyster Ostrea edulis, a species of significant evolutionary, ecological and commercial value. Each is a highly contiguous chromosome-level assembly from individuals of different genetic backgrounds, which have been benchmarked against one another. This situation has resulted from the serendipitous discovery that two independent research groups were both deep into the process of building, annotating and investigating separately produced assemblies. Due to constraints with funder requirements and the need to recognize early career researchers for their work, alongside the technical challenge of integrating assemblies from two very different genomes, there was limited capacity to merge the sequences into one publication at the stage of discovery. This issue is likely to become very common over the next few years until the technologies for working with multiple genomes at once, for example, graph genomes, become commonplace in nonmodel species. Consequently, both of our teams have decided to collaborate rather than compete, recognizing the benefit to copublishing two separate genome resources for the research community, each with distinct scientific investigations, and working collaboratively to benchmark the assemblies.

Chromosome-level reference genome for European flat oyster (Ostrea edulis L.).

The European flat oyster (Ostrea edulis L.) is a bivalve naturally distributed across Europe, which was an integral part of human diets for centuries, until anthropogenic activities and disease outbreaks severely reduced wild populations. Despite a growing interest in genetic applications to support population management and aquaculture, a reference genome for this species is lacking to date. Here, we report a chromosome-level assembly and annotation for the European Flat oyster genome, generated using Oxford Nanopore, Illumina, Dovetail OmniC™ proximity ligation and RNA sequencing. A contig assembly (N50: 2.38 Mb) was scaffolded into the expected karyotype of 10 pseudochromosomes. The final assembly is 935.13 Mb, with a scaffold-N50 of 95.56 Mb, with a predicted repeat landscape dominated by unclassified elements specific to O. edulis. The assembly was verified for accuracy and completeness using multiple approaches, including a novel linkage map built with ddRAD-Seq technology, comprising 4016 SNPs from four full-sib families (eight parents and 163 F1 offspring). Annotation of the genome integrating multitissue transcriptome data, comparative protein evidence and ab-initio gene prediction identified 35,699 protein-coding genes. Chromosome-level synteny was demonstrated against multiple high-quality bivalve genome assemblies, including an O. edulis genome generated independently for a French O. edulis individual. Comparative genomics was used to characterize gene family expansions during Ostrea evolution that potentially facilitated adaptation. This new reference genome for European flat oyster will enable high-resolution genomics in support of conservation and aquaculture initiatives, and improves our understanding of bivalve genome evolution.

Genome-Wide Association and Genomic Prediction of Growth Traits in the European Flat Oyster (Ostrea edulis).

The European flat oyster (Ostrea edulis) is a bivalve mollusc that was once widely distributed across Europe and represented an important food resource for humans for centuries. Populations of O. edulis experienced a severe decline across their biogeographic range mainly due to overexploitation and disease outbreaks. To restore the economic and ecological benefits of European flat oyster populations, extensive protection and restoration efforts are in place within Europe. In line with the increasing interest in supporting restoration and oyster farming through the breeding of stocks with enhanced performance, the present study aimed to evaluate the potential of genomic selection for improving growth traits in a European flat oyster population obtained from successive mass-spawning events. Four growth-related traits were evaluated: total weight (TW), shell height (SH), shell width (SW) and shell length (SL). The heritability of the growth traits was in the low-moderate range, with estimates of 0.45, 0.37, 0.22, and 0.32 for TW, SH, SW and SL, respectively. A genome-wide association analysis revealed a largely polygenic architecture for the four growth traits, with two distinct QTLs detected on chromosome 4. To investigate whether genomic selection can be implemented in flat oyster breeding at a reduced cost, the utility of low-density SNP panels was assessed. Genomic prediction accuracies using the full density panel were high (> 0.83 for all traits). The evaluation of the effect of reducing the number of markers used to predict genomic breeding values revealed that similar selection accuracies could be achieved for all traits with 2K SNPs as for a full panel containing 4,577 SNPs. Only slight reductions in accuracies were observed at the lowest SNP density tested (i.e., 100 SNPs), likely due to a high relatedness between individuals being included in the training and validation sets during cross-validation. Overall, our results suggest that the genetic improvement of growth traits in oysters is feasible. Nevertheless, and although low-density SNP panels appear as a promising strategy for applying GS at a reduced cost, additional populations with different degrees of genetic relatedness should be assessed to derive estimates of prediction accuracies to be expected in practical breeding programmes.

Genomic Epidemiology of Salmonid Alphavirus in Norwegian Aquaculture Reveals Recent Subtype-2 Transmission Dynamics and Novel Subtype-3 Lineages.

Viral disease poses a major barrier to sustainable aquaculture, with outbreaks causing large economic losses and growing concerns for fish welfare. Genomic epidemiology can support disease control by providing rapid inferences on viral evolution and disease transmission. In this study, genomic epidemiology was used to investigate salmonid alphavirus (SAV), the causative agent of pancreas disease (PD) in Atlantic salmon. Our aim was to reconstruct SAV subtype-2 (SAV2) diversity and transmission dynamics in recent Norwegian aquaculture, including the origin of SAV2 in regions where this subtype is not tolerated under current legislation. Using nanopore sequencing, we captured ~90% of the SAV2 genome for n = 68 field isolates from 10 aquaculture production regions sampled between 2018 and 2020. Using time-calibrated phylogenetics, we infer that, following its introduction to Norway around 2010, SAV2 split into two clades (SAV2a and 2b) around 2013. While co-present at the same sites near the boundary of Møre og Romsdal and Trøndelag, SAV2a and 2b were generally detected in non-overlapping locations at more Southern and Northern latitudes, respectively. We provide evidence for recent SAV2 transmission over large distances, revealing a strong connection between Møre og Romsdal and SAV2 detected in 2019/20 in Rogaland. We also demonstrate separate introductions of SAV2a and 2b outside the SAV2 zone in Sognefjorden (Vestland), connected to samples from Møre og Romsdal and Trøndelag, respectively, and a likely 100 km Northward transmission of SAV2b within Trøndelag. Finally, we recovered genomes of SAV2a and SAV3 co-infecting single fish in Rogaland, involving novel SAV3 lineages that diverged from previously characterized strains >25 years ago. Overall, this study demonstrates useful applications of genomic epidemiology for tracking viral disease spread in aquaculture.

Comparative regulomics supports pervasive selection on gene dosage following whole genome duplication.

BACKGROUND: Whole genome duplication (WGD) events have played a major role in eukaryotic genome evolution, but the consequence of these extreme events in adaptive genome evolution is still not well understood. To address this knowledge gap, we used a comparative phylogenetic model and transcriptomic data from seven species to infer selection on gene expression in duplicated genes (ohnologs) following the salmonid WGD 80-100 million years ago. RESULTS: We find rare cases of tissue-specific expression evolution but pervasive expression evolution affecting many tissues, reflecting strong selection on maintenance of genome stability following genome doubling. Ohnolog expression levels have evolved mostly asymmetrically, by diverting one ohnolog copy down a path towards lower expression and possible pseudogenization. Loss of expression in one ohnolog is significantly associated with transposable element insertions in promoters and likely driven by selection on gene dosage including selection on stoichiometric balance. We also find symmetric expression shifts, and these are associated with genes under strong evolutionary constraints such as ribosome subunit genes. This possibly reflects selection operating to achieve a gene dose reduction while avoiding accumulation of "toxic mutations". Mechanistically, ohnolog regulatory divergence is dictated by the number of bound transcription factors in promoters, with transposable elements being one likely source of novel binding sites driving tissue-specific gains in expression. CONCLUSIONS: Our results imply pervasive adaptive expression evolution following WGD to overcome the immediate challenges posed by genome doubling and to exploit the long-term genetic opportunities for novel phenotype evolution.

The structural variation landscape in 492 Atlantic salmon genomes.

Structural variants (SVs) are a major source of genetic and phenotypic variation, but remain challenging to accurately type and are hence poorly characterized in most species. We present an approach for reliable SV discovery in non-model species using whole genome sequencing and report 15,483 high-confidence SVs in 492 Atlantic salmon (Salmo salar L.) sampled from a broad phylogeographic distribution. These SVs recover population genetic structure with high resolution, include an active DNA transposon, widely affect functional features, and overlap more duplicated genes retained from an ancestral salmonid autotetraploidization event than expected. Changes in SV allele frequency between wild and farmed fish indicate polygenic selection on behavioural traits during domestication, targeting brain-expressed synaptic networks linked to neurological disorders in humans. This study offers novel insights into the role of SVs in genome evolution and the genetic architecture of domestication traits, along with resources supporting reliable SV discovery in non-model species.

Lineage-specific rediploidization is a mechanism to explain time-lags between genome duplication and evolutionary diversification.

BACKGROUND: The functional divergence of duplicate genes (ohnologues) retained from whole genome duplication (WGD) is thought to promote evolutionary diversification. However, species radiation and phenotypic diversification are often temporally separated from WGD. Salmonid fish, whose ancestor underwent WGD by autotetraploidization ~95 million years ago, fit such a 'time-lag' model of post-WGD radiation, which occurred alongside a major delay in the rediploidization process. Here we propose a model, 'lineage-specific ohnologue resolution' (LORe), to address the consequences of delayed rediploidization. Under LORe, speciation precedes rediploidization, allowing independent ohnologue divergence in sister lineages sharing an ancestral WGD event. RESULTS: Using cross-species sequence capture, phylogenomics and genome-wide analyses of ohnologue expression divergence, we demonstrate the major impact of LORe on salmonid evolution. One-quarter of each salmonid genome, harbouring at least 4550 ohnologues, has evolved under LORe, with rediploidization and functional divergence occurring on multiple independent occasions >50 million years post-WGD. We demonstrate the existence and regulatory divergence of many LORe ohnologues with functions in lineage-specific physiological adaptations that potentially facilitated salmonid species radiation. We show that LORe ohnologues are enriched for different functions than 'older' ohnologues that began diverging in the salmonid ancestor. CONCLUSIONS: LORe has unappreciated significance as a nested component of post-WGD divergence that impacts the functional properties of genes, whilst providing ohnologues available solely for lineage-specific adaptation. Under LORe, which is predicted following many WGD events, the functional outcomes of WGD need not appear 'explosively', but can arise gradually over tens of millions of years, promoting lineage-specific diversification regimes under prevailing ecological pressures.