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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 anadromous Atlantic salmon undergo a preparatory physiological transformation before seawater entry, referred to as smoltification. Key molecular developmental processes involved in this life stage transition, such as remodeling of gill functions, are known to be synchronized and modulated by environmental cues like photoperiod. However, little is known about the photoperiod influence and genome regulatory processes driving other canonical aspects of smoltification such as the large-scale changes in lipid metabolism and energy homeostasis in the developing smolt liver. Here we generate transcriptome, DNA methylation, and chromatin accessibility data from salmon livers across smoltification under different photoperiod regimes. We find a systematic reduction of expression levels of genes with a metabolic function, such as lipid metabolism, and increased expression of energy related genes such as oxidative phosphorylation, during smolt development in freshwater. However, in contrast to similar studies of the gill, smolt liver gene expression prior to seawater transfer was not impacted by photoperiodic history. Integrated analyses of gene expression, chromatin accessibility, and transcription factor (TF) binding signatures highlight chromatin remodeling and TF dynamics underlying smolt gene regulatory changes. Differential peak accessibility patterns largely matched differential gene expression patterns during smoltification and we infer that ZNF682, KLFs, and NFY TFs are important in driving a liver metabolic shift from synthesis to break down of organic compounds in freshwater. Overall, chromatin accessibility and TFBS occupancy were highly correlated to changes in gene expression. On the other hand, we identified numerous differential methylation patterns across the genome, but associated genes were not functionally enriched or correlated to observed gene expression changes across smolt development. Taken together, this work highlights the relative importance of chromatin remodeling during smoltification and demonstrates that metabolic remodeling occurs as a preadaptation to life at sea that is not to a large extent driven by photoperiod history.

Transposable elements (TEs) are hypothesized to play important roles in shaping genome evolution following whole-genome duplications (WGDs), including rewiring of gene regulation. In a recent analysis, duplicate gene copies that had evolved higher expression in liver following the salmonid WGD ∼100 million years ago were associated with higher numbers of predicted TE-derived cis-regulatory elements (TE-CREs). Yet, the ability of these TE-CREs to recruit transcription factors (TFs) in vivo and impact gene expression remains unknown. Here, we evaluated the gene-regulatory functions of 11 TEs using luciferase promoter reporter assays in Atlantic salmon (Salmo salar) primary liver cells. Canonical Tc1-Mariner elements from intronic regions showed no or small repressive effects on transcription. However, other TE-CREs upstream of transcriptional start sites increased expression significantly. Our results question the hypothesis that TEs in the Tc1-Mariner superfamily, which were extremely active following WGD in salmonids, had a major impact on regulatory rewiring of gene duplicates, but highlights the potential of other TEs in post-WGD rewiring of gene regulation in the Atlantic salmon genome.

Two of the most potent drivers of genome evolution in eukaryotes are whole genome duplications (WGD) and transposable element (TE) activity. These two mutational forces can also play synergistic roles; WGDs result in both cellular stress and functional redundancy, which would allow TEs to escape host-silencing mechanisms and effectively spread with reduced impact on fitness. As TEs can function as, or evolve into, TE-derived cis-regulatory elements (TE-CREs), bursts of TE-activity following WGD are likely to impact evolution of gene regulation. However, the role of TEs in genome regulatory remodelling after WGDs is unclear. Here we used the genome of Atlantic salmon, which is known to have experienced massive expansion of TEs after a WGD ~100 Mya, as a model system to explore the synergistic roles of TEs and WGDs on genome regulatory evolution. We identified 55,080 putative TE-CREs in Atlantic salmon using chromatin accessibility data from brain and liver. Of these, 80% were tissue specific to liver (43%) or brain (37%) and TE-CREs originating from retroelements were twice as common as those originating from DNA elements. Signatures of selection shaping TE-CRE evolution were evident from depletion of TEs in open chromatin, a bias in tissue-shared TE-CREs towards older TE-insertions, as well as tissue-specific processes shaping the TE-CRE repertoire. A minority of TE-families (16%) accounted for the origin of 46% of all TE-CREs, but the transposition activity of these CRE-superspreader families happened mostly prior to the WGD. Analyses of individual TE-CREs do however support a significantly higher rate of TE-CRE evolution from insertions happening around the time of the salmonid WGD. This pattern was particularly striking for the DTT elements, despite having generally low propensity to evolve into TE-CREs and impact transcription. Furthermore, co-expression based analyses supported the presence of TE-driven gene regulatory network evolution, including DTT elements active at the time of WGD. In conclusion, we find a strong association between TE insertions at the time of WGD and TE-CRE evolution. This association was not driven by particular TE-families with high capability to evolve into TE-CREs but likely a consequence of the concurrent surge of novel TE insertions, mostly from DTT elements, in combination with a shift in selective pressure on genome regulation following the WGD.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We have implemented a new method to identify TEs enriched in open chromatin that takes genomic context into account. We reran all our analyses with improved TE-divergence estimates (CpG normalised Kimura divergence) When implementing the use of Kimura distances instead of %-divergence we also had to come up with a new and empirically derived cutoff for how to define TE-activity relative to the WGD. We have added a new analysis of temporal co-occurrence between TE-CREs and WGD (Figures 4 G-H) which revealed patterns we overlooked in the first version. We have revised the text to address confusions and lack of clarity in some parts of the manuscript. The implementation of the new enrichment tests did not change the overall results. Neither did the use of Kimura distances and the new approach to classify temporal activity relative to the WGD for entire TE-families. However, our new analysis that take the full heterogeneity of TE-CRE ages into account did support a strong enrichment of TE-CREs originating from the time around the WGD.

The anadromous Atlantic salmon undergo a preparatory physiological transformation before seawater entry, referred to as smoltification. Key molecular developmental processes involved in this life stage transition, such as remodeling of gill functions, are known to be synchronized and modulated by environmental cues like photoperiod. However, little is known about the photoperiod influence and genome regulatory processes driving other canonical aspects of smoltification such as the large-scale changes in lipid metabolism and energy homeostasis in the developing smolt liver. Here we generate transcriptome, DNA methylation, and chromatin accessibility data from salmon livers across smoltification under different photoperiod regimes. We find a systematic reduction of expression levels of genes with a metabolic function, such as lipid metabolism, and increased expression of energy related genes such as oxidative phosphorylation, during smolt development in freshwater. However, in contrast to similar studies of the gill, smolt liver gene expression prior to seawater transfer was not impacted by photoperiodic history. Integrated analyses of gene expression and transcription factor (TF) binding signatures highlight likely important TF dynamics underlying smolt gene regulatory changes. We infer that ZNF682, KLFs, and NFY TFs are important in driving a liver metabolic shift from synthesis to break down of organic compounds in freshwater. Moreover, the increased expression of ribosomal associated genes after smolts were transferred to seawater was associated with increased occupancy of NFIX and JUN/FOS TFs proximal to transcription start sites, which could be the molecular consequence of rising levels of circulating growth hormones after seawater transition. We also identified differential methylation patterns across the genome, but associated genes were not functionally enriched or correlated to observed gene expression changes across smolt development. This contrasts with changes in TF binding which were highly correlated to gene expression, underscoring the relative importance of chromatin accessibility and transcription factor regulation in smoltification.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Added funding statement.

Transposable elements (TEs) are hypothesized to play important roles in shaping genome evolution following whole genome duplications (WGD), including rewiring of gene regulation. In a recent analysis, duplicate gene copies that had evolved higher expression in liver following the salmonid WGD ~100 million years ago were associated with higher numbers of predicted TE-derived cis-regulatory elements (TE-CREs). Yet, the ability of these TE-CREs to recruit transcription factors (TFs) in vivo and impact gene expression remains unknown. Here, we evaluated the gene regulatory functions of 11 TEs using luciferase promoter reporter assays in Atlantic salmon (Salmo salar) primary liver cells. Canonical Tc1-Mariner elements from intronic regions showed no or small repressive effects on transcription. However, other TE-derived cis-regulatory elements upstream of transcriptional start sites increased expression significantly. Our results question the hypothesis that TEs in the Tc1-Mariner superfamily, which were extremely active following WGD in salmonids, had a major impact on regulatory rewiring of gene duplicates, but highlights the potential of other TEs in post-WGD rewiring of gene regulation in the Atlantic salmon genome.

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. 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. Although ohnolog expression levels have evolved mostly asymmetrically, by diverting one ohnolog copy down a path towards pseudogenization, strong evolutionary constraints have frequently also favoured symmetric shifts in gene dosage of both copies, likely to achieve 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 source of novel binding sites driving tissue-specific gains expression. 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. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://gitlab.com/sandve-lab/gillard-groenvold

Melanised focal changes (black spots) are common findings in the white skeletal muscle of seawater-farmed Atlantic salmon ( Salmo salar ). Fillets with melanised focal changes are considered as lower quality and cause large economic losses. It has been suggested that red focal changes (red spots) precede the melanised focal changes. In the present work, we examined different populations of captive and wild salmon for the occurrence of both types of changes, which were investigated for the presence of different viruses by immunohistochemistry and RT-qPCR. The occurrence of red or melanised foci varied significantly between the populations, from none in wild fish control group, low prevalence of small foci in fish kept in in-house tanks, to high prevalence of large foci in farm-raised salmon. Large amounts of Piscine orthoreovirus (PRV) antigen were detected in all foci. No other viruses were detected. Red focal changes contained significantly higher levels of PRV RNA than apparently non-affected areas in white muscle of the same individuals. Some changes displayed a transient form between a red and melanised pathotype, indicating a progression from an acute to a chronic manifestation. We conclude that PRV is associated with the focal pathological changes in the white muscle of farmed Atlantic salmon and is a premise for the development of focal melanised changes.