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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.

In salmon farming, the scarcity of fish oil has driven a shift towards the use of plant-based oil from vegetable or seed, leading to fish feed low in long-chain PUFA (LC-PUFA) and cholesterol. Atlantic salmon has the capacity to synthesise both LC-PUFA and cholesterol, but little is known about the regulation of synthesis and how it varies throughout salmon life span. Here, we present a systemic view of lipid metabolism pathways based on lipid analyses and transcriptomic data from salmon fed contrasting diets of plant or fish oil from first feeding. We analysed four tissues (stomach, pyloric caeca, hindgut and liver) at three life stages (initial feeding 0·16 g, 2·5 g fingerlings and 10 g juveniles). The strongest response to diets higher in plant oil was seen in pyloric caeca of fingerlings, with up-regulation of thirty genes in pathways for cholesterol uptake, transport and biosynthesis. In juveniles, only eleven genes showed differential expression in pyloric caeca. This indicates a higher requirement of dietary cholesterol in fingerlings, which could result in a more sensitive response to plant oil. The LC-PUFA elongation and desaturation pathway was down-regulated in pyloric caeca, probably regulated by srebp1 genes. In liver, cholesterol metabolism and elongation and desaturation genes were both higher on plant oil. Stomach and hindgut were not notably affected by dietary treatment. Plant oil also had a higher impact on fatty acid composition of fingerlings compared with juveniles, suggesting that fingerlings have less metabolic regulatory control when primed with plant oil diet compared with juveniles.

Background Sea urchins are studied as model organisms for developmental and systems biology and also produce highly valued food products. Evechinus chloroticus (Kina) is a sea urchin species that is indigenous to New Zealand. It is the type member of the Evechinus genus based on its morphological characteristics. Previous research has focused on identifying physical factors affecting commercial roe quality of E. chloroticus , but there is almost no genetic information available for E. chloroticus. E. chloroticus is the only species in its genus and has yet to be subject to molecular phylogenetic analysis. Results In this study we performed a de novo transcriptome assembly of Illumina sequencing data. A total of 123 million 100 base length paired-end reads were generated using RNA-Seq libraries from a range of E. chloroticus tissues from two individuals obtained from Fiordland, New Zealand. The assembly resulted in a set of 75,002 transcripts with an accepted read coverage and length, of which 24,655 transcripts could be functionally annotated using protein similarity. Transcripts could be further annotated with Gene Ontology, KEGG Orthology and InterPro terms. With this sequence data we could perform the first phylogenetic analysis of E. chloroticus to other species of its family using multiple genes. When sequences for the mitochondrial nitrogen dehydrogenase genes were compared, E. chloroticus remained outside of a family level clade, which indicated E. chloroticus is indeed a genetically distinct genus within its family. Conclusions This study has produced a large set of E. chloroticus transcripts/proteins along with functional annotations, vastly increasing the amount of genomic data available for this species. This provides a resource for current and future studies on E. chloroticus , either to increase its commercial value, or its use as a model organism. The phylogenetic results provide a basis for further analysis of relationships between E. chloroticus , its family members, and its evolutionary history.

Background 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. Results 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. Conclusions 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.

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.

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

Risk assessment and protection of plant communities in contaminated ecosystems require in-depth understanding of differential sensitivity to chronic ionising radiation in plants. However, the contributing molecular factors to differential radiosensitivity among plant species are poorly understood. To shed light on this, we compared early events associated with protection, repair, and stress responses in gamma-irradiated (1-290 mGy h−1) seedlings of the radiosensitive conifer Norway spruce (Picea abies) and the radiotolerant Arabidopsis thaliana, by analysing growth, organelle and DNA damage, transcriptomes and the dynamics of antioxidant activities and expression of relevant genes. After 48 h of gamma radiation exposure, Norway spruce showed significantly reduced growth at 100-290 mGy h−1 and organelle damage, especially in mitochondria, at ≥ 1 mGy h−1 whereas A. thaliana showed normal vegetative growth at all dose rates, transiently delayed reproductive development at 290 mGy h−1 only, minor organelle damage only at ≥ 100 mGy h−1 and significantly less DNA damage than in Norway spruce at all dose rates. Comparative transcriptomics revealed that A. thaliana showed massive activation of genes related to DNA damage repair, antioxidants, and other stress responses at ≥ 1 mGy h−1 while Norway spruce mobilized transcription of such pathways only at ≥ 40 mGy h−1. The transcriptional activation of repair and protection responses at higher gamma dose-rates only and its absence in lower dose-rates, correlates with high radiosensitivity of Norway spruce, compared to the massive transcriptional activation from low dose-rates in the radiotolerant A. thaliana.

Conifers are among the most radiosensitive plant species. Elevated, sublethal levels of ionising radiation result in reduced apical dominance in conifers, indicating a negative effect on shoot apical meristems (SAMs). The SAMs, harbouring the pluripotent stem cells, generate all the cells of the shoot, enabling growth and reproduction. However, knowledge on the effects of ionising radiation on such stem cells is scarce, but important for risk assessment and radioprotection of plants in contaminated ecosystems. Here, we assessed the sensitivity of in vitro-grown stem cells of Norway spruce to 144-h of gamma irradiation at 1-100 mGy h-1, using such cells as a model for molecular toxicity of gamma radiation in conifers. Although there were no visible effects of the gamma irradiation on cell proliferation and subsequent embryo formation, dose rate-dependent DNA damage was observed at ≥10 mGy h-1, and comprehensive organelle damage at all dose rates. Massive dose rate-dependent transcriptome changes occurred, with downregulation of a range of genes related to cell division, DNA repair and protein folding but upregulation of stress-related hormonal pathways and several antioxidant-related genes. The upregulation of such genes, survival and continued proliferation of at least a subset of cells and the post-irradiation normalisation of expression of DNA repair and protein-folding genes together with somatic embryo formation suggest that stem cells are able to recover from gamma-irradiation-induced stress. Collectively, regardless of cellular abnormalities after gamma irradiation, and huge transcriptomic shifts towards stress management pathways, the pluripotent stem cell cultures were able to retain their stemness. Norway spruce stem cells retained stemness after gamma irradiation (1-100 mGy h-1). DNA damage was observed at ≥ 10 mGy h-1 and organelle damage at all dose rates. Huge dose rate-dependent transcriptomic changes occurred after 144-h irradiation. Downregulated key DNA repair-genes recovered post-irradiation. Induction of stress-management and antioxidant genes may aid stem cell maintenance.