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.

Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable transformation and expression changes in plant cells. However, the production of stable transformants can be slow and laborious with no guarantee of success. Furthermore, transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. Therefore, fast, efficient, high-throughput methods for assaying TF function are needed. Agroinfiltration is a simple plant biology method that allows transient gene expression. It is a rapid and powerful tool for the functional characterisation of TF genes in planta. High throughput RNA sequencing is now a widely used method for analysing gene expression profiles (transcriptomes). By coupling TF agroinfiltration with RNA sequencing (named here as Infiltration-RNAseq), gene expression networks and gene function can be identified within a few weeks rather than many months. As a proof of concept, we agroinfiltrated Medicago truncatula leaves with M. truncatula LEGUME ANTHOCYANIN PRODUCITION 1 (MtLAP1), a MYB transcription factor involved in the regulation of the anthocyanin pathway, and assessed the resulting transcriptome. Leaves infiltrated with MtLAP1 turned red indicating the production of anthocyanin pigment. Consistent with this, genes encoding enzymes in the anthocyanin biosynthetic pathway, and known transcriptional activators and repressors of the anthocyanin biosynthetic pathway, were upregulated. A novel observation was the induction of a R3-MYB transcriptional repressor that likely provides transcriptional feedback inhibition to prevent the deleterious effects of excess anthocyanins on photosynthesis. Infiltration-RNAseq is a fast and convenient method for profiling TF-mediated gene expression changes. We utilised this method to identify TF-mediated transcriptional changes and TF target genes in M. truncatula and Nicotiana benthamiana. This included the identification of target genes of a TF not normally expressed in leaves, and targets of TFs from other plant species. Infiltration-RNAseq can be easily adapted to other plant species where agroinfiltration protocols have been optimised. The ability to identify downstream genes, including positive and negative transcriptional regulators, will result in a greater understanding of TF function.

Salmon is a rich source of health-promoting omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The LC-PUFA biosynthetic pathway in Atlantic salmon is one of the most studied compared to other teleosts. This has largely been due to the massive replacement of LC-PUFA-rich ingredients in aquafeeds with terrestrial plant oils devoid of these essential fatty acids (EFA) which ultimately pushed dietary content towards the minimal requirement of EFA. The practice would also reduce tissue content of n-3 LC-PUFA compromising the nutritional value of salmon to the human consumer. These necessitated detailed studies of endogenous biosynthetic capability as a contributor to these EFA. This review seeks to provide a comprehensive and concise overview of the current knowledge about the molecular genetics of PUFA biosynthesis in Atlantic salmon, highlighting the enzymology and nutritional regulation as well as transcriptional control networks. Furthermore, we discuss the impact of genome duplication on the complexity of salmon LC-PUFA pathway and highlight probable implications on endogenous biosynthetic capabilities. Finally, we have also compiled and made available a large RNAseq dataset from 316 salmon liver samples together with an R-script visualization resource to aid in explorative and hypothesis-driven research into salmon lipid metabolism.

It has been suggested that the high phospholipid (PL) requirement in Atlantic salmon (Salmo salar) fry is due to insufficient intestinal de-novo synthesis causing low lipoprotein (LP) production and reduced transport capacity of dietary lipids. However, in-depth ontogenetic analysis of intestinal PL and LP synthesis with the development of salmon has yet to be performed. Therefore, in this paper we used RNA-Seq technology to investigate the expression of genes involved in PL synthesis and LP formation throughout early developmental stages and associate insufficient expression of synthesis pathways in salmon fry with its higher dietary PL requirement. There was a special focus on the understanding homologous genes, especially those from salmonid-specific fourth vertebrate whole-genome duplication (Ss4R), and their contribution to salmonid specific features of regulation of PL metabolic pathways. Salmon fry were sampled at 0.16 g (1 day before first-feeding), 2.5 and 10 g stages of development and transcriptomic analysis was applied separately on stomach, pyloric caeca and hindgut of the fish. In general, we found up-regulated pathways involved in synthesis of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and LP in pyloric caeca of salmon between 0.16 and 10 g. Thirteen differentially expressed genes (q < 0.05) in these pathways were highly up-regulated in 2.5 g salmon compared to 0.16 g, while only five more differentially expressed (q < 0.05) genes were found when the fish grew up to 10 g. Different homologous genes were found dominating in stomach, pyloric caeca and hindgut. However, the expression of dominating genes in pathways of PL and LP synthesis were much higher in pyloric caeca than stomach and hindgut. Salmon-specific homologous genes (Ss4R) had similar expression during development, while other homologs had more diverged expression. The up-regulation of the de-novo PtdCho and PtdEtn pathways confirm that salmon have decreasing requirement for dietary PL as the fish develops. The similar expressions between Ss4R homologous genes suggest that the functional divergence of these genes was incomplete compared to homologs derived from other genome duplication. The results of the present study have provided new information on the molecular mechanisms of phospholipid synthesis and lipoprotein formation in fish.

Whole genome duplication (WGD) has been a major evolutionary driver of increased genomic complexity in vertebrates. One such event occurred in the salmonid family ~80 million years ago (Ss4R) giving rise to a plethora of structural and regulatory duplicate-driven divergence, making salmonids an exemplary system to investigate the evolutionary consequences of WGD. Here, we present a draft genome assembly of European grayling (Thymallus thymallus) and use this in a comparative framework to study evolution of gene regulation following WGD. Among the Ss4R duplicates identified in European grayling and Atlantic salmon (Salmo salar) , one third reflect non-neutral tissue expression evolution, with strong purifying selection, maintained over ~50 million years. Of these, the majority reflect conserved tissue regulation under strong selective constraints related to brain and neural-related functions, as well as higher-order protein-protein interactions. A small subset of the duplicates has evolved tissue regulatory expression divergence in a common ancestor, which have been subsequently conserved in both lineages, suggestive of adaptive divergence following WGD. These candidates for adaptive tissue expression divergence have elevated rates of protein coding- and promoter-sequence evolution and are enriched for immune- and lipid metabolism ontology terms. Lastly, lineage-specific duplicate divergence points towards underlying differences in adaptive pressures on expression regulation in the non-anadromous grayling versus the anadromous Atlantic salmon. Our findings enhance our understanding of the role of WGD in genome evolution and highlights cases of regulatory divergence of Ss4R duplicates, possibly related to a niche shift in early salmonid evolution.