Periodic Reporting for period 4 - EPIFISH (INNOVATIVE EPIGENETIC MARKERS FOR FISH DOMESTICATION) Periodo di rendicontazione: 2021-01-01 al 2022-06-30 Sintesi del contesto e degli obiettivi generali del progetto Farming of domesticated fish species is one of the fastest growing food production sectors worldwide, since fisheries are unable to meet the increasing demand for fish protein to feed a growing global population. In order to ensure sustainability of the aquaculture sector it is critical to selectively improve the major commercial fish species. To date, phenotypic and genetic selection for traits of interest, such as growth, account only for a fraction of the observed variation. Moreover, the rapid changes that occur during adaptation to new environmental conditions in fish undergoing domestication raises the original hypothesis that epigenetic mechanisms are involved in this process. Hence, the overarching aim of EPIFISH is to ascertain the importance of epigenetics in fish domestication using the Nile tilapia (Oreochromis niloticus) as model species, since tilapias are the second most important group of farmed fish. Specific objectives are:1. To determine how selection for growth affects the epigenetic landscape (DNA methylation and hydroxymethylation) and the expression of non-coding RNAs (miRNAs and circRNAs) during domestication.2. To characterize miRNA variants and epigenetic alleles associated with improved growth.3. To validate the potential epigenetic markers of growth for future selective breeding programmes.Our data revealed that DNA methylation and hydroxymethylation in muscle change dramatically within the first generation of domestication, along with the non-coding RNA (miRNA, circRNA) transcriptomes. These modifications are present in genes involved in immune-, growth- and neuronal-related pathways, which help us to understand how animals can adapt so rapidly to captivity. In addition to providing novel mechanistic insights into the role of epigenetics in fish domestication, the growth-related non-coding RNAs and epialleles identified in EPIFISH can have a major impact on aquaculture biotechnology, since they provide a basis for the implementation of epigenomic selection as a new feature in future selective breeding programmes. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti The EPIFISH project started with a field trip to Alexandria and Luxor in Egypt. The team spent 7 weeks with local fisherman capturing live Nile tilapia using traditional methods, such as traps (Figure 1). Fertilised eggs collected from the mouth of several females were transported to the research station at Nord University in Bodø (Norway), where they were reared in a state-of-the-art recirculating aquaculture system for three generations. The fish were separated in two groups according to their weight: a control line comprising individuals of average weight and a selected line of fish that were at least 10% larger than average. Using next-generation sequencing to compare gene expression levels in muscle, we found over 2,700 protein-coding genes that were differentially expressed between wild tilapia females and their progeny reared in captivity. There was an up-regulation of genes related to metabolic processes and muscle development, which may account for the improved growth of domesticated fish. A large genome-wide association study identified outliers that explain to what extent genetics contributes to growth changes during domestication.We have identified several circRNAs, miRNAs and isomiRs that target genes involved in metabolic pathways and muscle development. Several of these non-coding RNAs are expressed at different levels in muscle and blood serum expressed between slow- and fast-growing Nile tilapia. Hence, they are promising candidates for epigenetic markers (epimarkers) of growth, since their expression is strongly correlated to fish weight. Using our newly developed pipeline for in silico prediction of circRNAs, we have discovered novel circRNAs associated with domestication. After optimising a protocol to cover a larger proportion of methylated cytosines in the genome, we obtained a high-resolution, genome-wide methylation map in fast muscle of Nile tilapia. The mitochondrial methylome in fast muscle was determined by whole genome bisulfite sequencing. Another study comparing wild and domesticated fish revealed that differentially methylated genes were mainly associated with muscle growth, epigenetic mechanisms, immunity and diet (Figure 2). We also investigated a closely related DNA modification termed hydroxymethylation, since there is increasing evidence that it is a stable epigenetic mark. Our genome-wide profiling revealed that hydroxymethylation is a ubiquitous DNA modification throughout the nuclear and mitochondrial genomes both in muscle and liver. Nile tilapia domestication was associated with a generalised decrease in muscle hydroxymethylation. An artificial breeding protocol was developed to obtain fertilized eggs for the functional assays. After optimizing the microinjection and egg incubation methods, we microinjected sgRNA and Cas9mRNA into hormone-induced at the single-cell stage. Finally, high-resolution melting assays were developed to determine differences in levels of the candidate 5mC and 5hmC epimarkers in muscle from slow- and fast-growing fish, and primer sets for each miRNA target were optimized for specific quantification. Their potential as markers of growth in Nile tilapia has been assessed in muscle and blood serum with the aim of developing a reference panel of epimarkers.The 21 publications from EPIFISH, including MSc and PhD theses, have been published as Gold Open Access or are registered in public databases, and next-generation sequencing data have been submitted to public repositories. The results were presented to the academic community in 47 communications, as well as in workshops, trade magazines, newspaper articles and open science events for the general public. We have worked closely with one of the major Nile tilapia producers (Genomar) and Nord Innovasjon to identify intellectual property assets during the project. A patent has been prepared for a multi-panel of epigenetic markers (non-coding RNAs and DNA methylation/ hydroxymethylation) of improved growth. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The EPIFISH project has made a major contribution to our current understanding of the epigenetic mechanisms associated with animal domestication and improved growth (Figure 3). The main results beyond the state of the art are the following:1. The identification of differentially hydroxymethylated cytosines in muscle of Nile tilapia after a single generation in captivity provided the first evidence that hydroxymethylation is involved in domestication.2. The pioneer single-cytosine resolution studies of methylation in fast muscle of Nile tilapia in the context of growth and the early domestication revealed that the epigenetic regulation of growth in Nile tilapia involves different gene networks in males and females. Our results also highlighted the potential impact of diet on the epigenome at the very early stages of domestication.3. The discovery of miRNAs and circRNAs differentially expressed between wild fish and their offspring after just one generation in captivity adds a novel dimension to the complex molecular network underlying domestication.In addition to representing a leap forward from the state of the art in understanding the molecular basis of fish domestication and selective breeding, these results open new horizons for future frontier research in epigenetics, namely within the fields of evolutionary biology, transgenerational inheritance and nutritional epigenetics. 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