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Abstract Increased knowledge of heritable traits in Atlantic Salmon (Salmo salar) is important to overcome bottlenecks in salmonid aquaculture. Atlantic salmonid populations, both landlocked and anadromous, represent an interesting model to gain insight into anadromy related traits, most notably, the probability to smoltify. While a previous study has identified several genomic regions diverging between anadromous and landlocked populations across the species range, the present study explores these data further with the aim to uncover if some of these genomic regions are linked to beneficial genetic traits associated with smoltification. In this study 17 of these loci were monitored in 669 anadromous salmon originating from 36 full-sibling families that had been reared under common garden conditions. The Smolt Index was calculated, using multiple visual markers, and provided a means of assessing smoltification stage. One SNP, located in Ssa04, showed a significant association with probability to smoltify, where individuals homozygous for the landlocked variant (LL) displayed a decrease in probability of smoltifying after one winter when compared with the homozygous for the anadromous variant (AA). This effect was independent of individual fish size. A separate common garden study comprising 200 individuals from either anadromous or landlocked strains showed that expression levels of ncor1, a thyroid mediator hormone located on the same chromosomal region (Ssa04), were significantly reduced in landlocked individuals post smoltification but remained constant in their anadromous counterparts. This study therefore suggests that while size is still the most important trigger for the induction of smoltification, there may also be an additional genetic component or trigger that has been ‘lost’ during the years deprived of SW transfer. In conclusion, the LL genotype identified here could potentially be used by the industry to delay smoltification and may also represent one of the first clues to the genetic regulation of smoltification in Atlantic salmon.

Abstract Background Tandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels. Results A total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs. Conclusion The results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.

Genetic introgression of farmed salmon into wild populations can damage the genetic integrity of wild stocks and is therefore considered as an environmental threat. One possible solution is to induce sterility in farmed salmon. We have searched for proteins potentially essential for germline survival in Atlantic salmon. One of these is the argonaute protein Piwil1, known to be required for germ cell survival. To examine Piwil1 function in salmon, we induced indels in the N domain by CRISPR-Cas9. The encoded domain is present in all vertebrate Piwi proteins and has been linked to Tdrd1 protein interaction and PAZ lobe structure. The F0 founder generation of piwil1 crispant males and females displayed a mosaic pattern of piwil1 mutations, exhibiting highly mutated alleles (53%–97%) in their fin gDNA samples. In general, piwil1 crispants carried germ cells, went through puberty and became fertile, although a transient and partial germ cell loss and delays during the spermatogenic process were observed in many male crispants, suggesting that Piwil1 functions during salmon spermatogenesis. By crossing highly mutated F0 founders, we produced F1 fish with a mixture of: loss-of-function alleles (−); functional in frame mutated alleles (+) and wt alleles (+). In F1, all piwil1−/− fish lacked germ cells, while piwil1+/+ siblings showed normal ovaries and testes. Yet, most juvenile F1 piwil1+/−males and females displayed an intermediate phenotype with a higher somatic/germ cell ratio without an increase in germ cell apoptosis, suggestive of a gene dose effect on the number of germ cells and/or insufficient replacement of lost germ cells in heterozygous fish. Interestingly, the two longest in-frame indels in the N domain also ensured germ cell loss. Hence, the loss of 4–6 aa in this region Phe130-Ser136 may result in crucial changes of the protein structure, potentially affecting piRNA binding of the PAZ lobe, and/or affecting the binding of Piwil1 interacting proteins such as Tdrd protein, with critical consequences for the survival of primordial germ cells. In conclusion, we show that loss of piwil1 leads to loss of germ cells in salmon and that part of the N domain of Piwil1 is crucial for its function.

Abstract Background New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon. Results Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively). Conclusions In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.

Abstract Most Atlantic salmon (Salmo salar L.) populations follow an anadromous life cycle, spending early life in freshwater, migrating to the sea for feeding, and returning to rivers to spawn. At the end of the last ice age ~10,000 years ago, several populations of Atlantic salmon became landlocked. Comparing their genomes to their anadromous counterparts can help identify genetic variation related to either freshwater residency or anadromy. The objective of this study was to identify consistently divergent loci between anadromous and landlocked Atlantic salmon strains throughout their geographical distribution, with the long‐term aim of identifying traits relevant for salmon aquaculture, including fresh and seawater growth, omega‐3 metabolism, smoltification, and disease resistance. We used a Pool‐seq approach (n = 10–40 individuals per population) to sequence the genomes of twelve anadromous and six landlocked Atlantic salmon populations covering a large part of the Northern Hemisphere and conducted a genomewide association study to identify genomic regions having been under different selection pressure in landlocked and anadromous strains. A total of 28 genomic regions were identified and included cadm1 on Chr 13 and ppargc1a on Chr 18. Seven of the regions additionally displayed consistently reduced heterozygosity in fish obtained from landlocked populations, including the genes gpr132, cdca4, and sertad2 on Chr 15. We also found 16 regions, including igf1 on Chr 17, which consistently display reduced heterozygosity in the anadromous populations compared to the freshwater populations, indicating relaxed selection on traits associated with anadromy in landlocked salmon. In conclusion, we have identified 37 regions which may harbor genetic variation relevant for improving fish welfare and quality in the salmon farming industry and for understanding life‐history traits in fish.

Abstract Background With declining wild fish populations, farmed salmon has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA). However, the introduction of plant oil in farmed salmon feeds has reduced the content of these beneficial LC-HUFA. The synthetic capability for LC-HUFAs depends upon the dietary precursor fatty acids and the genetic potential, thus there is a need for in-depth understanding of LC-HUFA synthetic genes and their interactions with other genes involved in lipid metabolism. Several key genes of LC-HUFA synthesis in salmon belong to the fatty acid desaturases 2 (fads2) family. The present study applied whole transcriptome analysis on two CRISPR-mutated salmon strains (crispants), 1) Δ6abc/5 Mt with mutations in Δ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-c genes, and 2) Δ6bc Mt with mutations in Δ6fads2-b and Δ6fads2-c genes. Our purpose is to evaluate the genetic effect fads2 mutations have on other lipid metabolism pathways in fish, as well as to investigate mosaicism in a commercial species with a very long embryonal period. Results Both Δ6abc/5 Mt and Δ6bc Mt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. The Δ6abc/5 Mt fish displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation of srebp1 genes which are known key transcription regulators of lipid metabolism as well as a number of down-stream genes involved in fatty acid de-novo synthesis, fatty acid β-oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of Δ6bc Mt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability. Conclusions CRISPR-Cas9 can efficiently mutate multiple fads2 genes simultaneously in salmon. The results of the present study have provided new information on the transcriptional regulations of lipid metabolism genes after reduction of LC-HUFA synthesis pathways in salmon.

Abstract Background Farmed Atlantic salmon are one of the most economically significant global aquaculture products. Early sexual maturation of farmed males represents a significant challenge to this industry and has been linked with the vgll3 genotype. However, tools to aid research of this topic, such as all-male and clonal fish, are still lacking. The present 6-year study examined if all-male production is possible in Atlantic salmon, a species with heteromorphic sex chromosomes (males being XY, females XX), and if all-male fish can be applied to further explore the vgll3 contribution on the likelihood of early maturation. Results Estrogen treatment of mixed sex yolk sac larvae gave rise to one sexually mature hermaphrodite with a male genotype (XY) that was used to produce both self-fertilized offspring and androgenetic double haploid (dh) offspring following egg activation with UV treated sperm and pressure shock to block the first mitotic division. There were YY supermales among both offspring types, which were crossed with dh females. Between 1 and 8% of the putative all-male offspring from the eight crosses with self-fertilized supermales were found to have ovaries, and 95% of these phenotypic females were also genetically female. None of the offspring from the one dh supermale cross had ovaries. When assessing the general contribution of the vgll3 locus on the likelihood of early post-smolt sexual maturation (jacking) in the all-male populations we found individuals that were homozygous for the early maturing genotype (97%) were more likely to enter puberty than individuals that were homozygous for the late maturing genotype (26%). However, the likelihood of jacking within individuals with an early/late heterozygous genotype was higher when the early allele came from the dam (94%) compared to the sire (45%). Conclusions The present results show that supermale Atlantic salmon are viable and fertile and can be used as a research tool to study important aspects of sexual maturation, such as to further explore the sex dependent parental genetic contribution to age at puberty in Atlantic salmon. In addition, we report the production of viable double haploid supermale fish.

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17β-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.

Abstract Background Sustainability challenges are currently hampering an increase in salmon production. Using sterile salmon can solve problems with precocious puberty and genetic introgression from farmed escapees to wild populations. Recently sterile salmon was produced by knocking out the germ cell-specific dead end (dnd). Several approaches may be applied to inhibit Dnd function, including gene knockout, knockdown or immunization. Since it is challenging to develop a successful treatment against a gene product already existing in the body, alternative targets are being explored. Germ cells are surrounded by, and dependent on, gonadal somatic cells. Targeting genes essential for the survival of gonadal somatic cells may be good alternative targets for sterility treatments. Our aim was to identify and characterize novel germ cell and gonadal somatic factors in Atlantic salmon. Results We have for the first time analysed RNA-sequencing data from germ cell-free (GCF)/dnd knockout and wild type (WT) salmon testis and searched for genes preferentially expressed in either germ cells or gonadal somatic cells. To exclude genes with extra-gonadal expression, our dataset was merged with available multi-tissue transcriptome data. We identified 389 gonad specific genes, of which 194 were preferentially expressed within germ cells, and 11 were confined to gonadal somatic cells. Interestingly, 5 of the 11 gonadal somatic transcripts represented genes encoding secreted TGF-β factors; gsdf, inha, nodal and two bmp6-like genes, all representative vaccine targets. Of these, gsdf and inha had the highest transcript levels. Expression of gsdf and inha was further confirmed to be gonad specific, and their spatial expression was restricted to granulosa and Sertoli cells of the ovary and testis, respectively. Finally, we show that inha expression increases with puberty in both ovary and testis tissue, while gsdf expression does not change or decreases during puberty in ovary and testis tissue, respectively. Conclusions This study contributes with transcriptome data on salmon testis tissue with and without germ cells. We provide a list of novel and known germ cell- and gonad somatic specific transcripts, and show that the expression of two highly active gonadal somatic secreted TGF-β factors, gsdf and inha, are located within granulosa and Sertoli cells.

Abstract Background In Atlantic salmon in the wild, age at maturity is strongly influenced by the vgll3 locus. Under farming conditions, light, temperature and feeding regimes are known significantly advance or delay age at maturity. However, the potential influence of the vgll3 locus on the maturation of salmon reared under farming conditions has been rarely investigated, especially in females. Results Here, we reared domesticated salmon (mowi strain) with different vgll3 genotypes under standard farming conditions until they matured at either one, two or more than two sea winters. Interestingly, and in contrast to previous findings in the wild, we were not able to identify a link between vgll3 and age at maturity in females when reared under farming conditions. For males however, we found that the probability of delaying maturation from one to two sea winters was significantly lower in fish homozygous for the early allele compared to homozygous fish for the late allele, while the probability for heterozygous fish was intermediate. These data also contrast to previous findings in the wild where the early allele has been reported as dominant. However, we found that the probability of males delaying maturation from two to three sea winters was regulated in the same manner as the wild. Conclusions Collectively, our data suggest that increased growth rates in mowi salmon, caused by high feed intake and artificial light and temperature regimes together with other possible genetic/epigenetic components, may significantly influence the impact that the vgll3 locus has on age at maturity, especially in females. In turn, our results show that the vgll3 locus can only to a large extent be used in selective breeding to control age at maturation in mowi males. In summary, we here show that in contrast to the situation in wild salmon, under farming conditions vgll3 does not seem to influence age at maturity in mowi females whereas in mowi males, maturing as one or two sea winters it alters the early allele effect from dominant to intermediate.

Entering meiosis strictly depends on stimulated by retinoic acid 8 (Stra8) gene function in mammals. This gene is missing in a number of fish species, including medaka and zebrafish, but is present in the majority of fishes, including Atlantic salmon. Here, we have examined the effects of removing stra8 on male fertility in Atlantic salmon. As in mammals, stra8 expression was restricted to germ cells in the testis, transcript levels increased during the start of puberty, and decreased when blocking the production of retinoic acid. We targeted the salmon stra8 gene with two gRNAs one of these were highly effective and produced numerous mutations in stra8, which led to a loss of wild-type (WT) stra8 expression in F0 salmon testis. In maturing stra8 crispants, the spermatogenetic tubuli were partially disorganized and displayed a sevenfold increase in germ cell apoptosis, in particular among type B spermatogonia and spermatocytes. The production of spermatogenic cysts, on the other hand, increased in maturing stra8 crispants. Gene expression analysis revealed unchanged (lin28a, ret) or reduced levels (egr1, dusp4) of transcripts associated with undifferentiated spermatogonia. Decreased expression was recorded for some genes expressed in differentiating spermatogonia including dmrt1 and ccnd2 or in spermatocytes, such as ccna1. Different from Stra8-deficient mammals, a large number of germ cells completed spermatogenesis, sperm was produced and fertilization rates were similar in WT and crispant males. While loss of stra8 increased germ cell apoptosis during salmon spermatogenesis, crispants compensated this cell loss by an elevated production of spermatogenic cysts, and were able to produce functional sperm. It appears that also in a fish species with a stra8 gene in the genome, the critical relevance this gene has attained for mammalian spermatogenesis is not yet given, although detrimental effects of the loss of stra8 were clearly visible during maturation.

Despite the key role that sex-determination plays in evolutionary processes, it is still poorly understood in many species. In salmonids, which are among the best studied fishes, the master sex-determining gene sexually dimorphic on the Y-chromosome (sdY) has been identified. However, sdY displays unexplained discordance to the phenotypic sex, with a variable frequency of phenotypic females being reported as genetic males. Multiple sex determining loci in Atlantic salmon have also been reported, possibly as a result of recent transposition events in this species. We hypothesized the existence of an autosomal copy of sdY, causing apparent discordance between phenotypic and genetic sex, that is transmitted in accordance with autosomal inheritance. To test this, we developed a qPCR methodology to detect the total number of sdY copies present in the genome. Based on the observed phenotype/genotype frequencies and linkage analysis among 2,025 offspring from 64 pedigree-controlled families of accurately phenotyped Atlantic salmon, we identified both males and females carrying one or two autosomal copies of sdY in addition to the Y-specific copy present in males. Patterns across families were highly consistent with autosomal inheritance. These autosomal sdY copies appear to have lost the ability to function as a sex determining gene and were only occasionally assigned to the actual sex chromosome in any of the affected families.

Abstract Background Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Results Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. Conclusions This study has - for the first time - linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis.

Abstract We describe an emerging initiative - the ‘Functional Annotation of All Salmonid Genomes’ (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

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Wild and domesticated Atlantic salmon males display large variation for sea age at sexual maturation, which varies between 1-5 years. Previous studies have uncovered a genetic predisposition for variation of age at maturity with moderate heritability, thus suggesting a polygenic or complex nature of this trait. The aim of this study was to identify associated genetic loci, genes and ultimately specific sequence variants conferring sea age at maturity in salmon. We performed a genome wide association study (GWAS) using a pool sequencing approach (20 individuals per river and phenotype) of male salmon returning to rivers as sexually mature either after one sea winter (2009) or three sea winters (2011) in six rivers in Norway. The study revealed one major selective sweep, which covered 76 significant SNPs in which 74 were found in a 370 kb region of chromosome 25. Genotyping other smolt year classes of wild and domesticated salmon confirmed this finding. Genotyping domesticated fish narrowed the haplotype region to four SNPs covering 2386 bp, containing the vgll3 gene, including two missense mutations explaining 33-36% phenotypic variation. A single locus was found to have a highly significant role in governing sea age at maturation in this species. The SNPs identified may be both used as markers to guide breeding for late maturity in salmon aquaculture and in monitoring programs of wild salmon. Interestingly, a SNP in proximity of the VGLL3 gene in humans (Homo sapiens), has previously been linked to age at puberty suggesting a conserved mechanism for timing of puberty in vertebrates.