A genetic method to infer ploidy and aberrant inheritance in triploid organisms.

Polyploidy occurs naturally across eukaryotic lineages and has been harnessed in the domestication of many crops and vertebrates. In aquaculture, triploidy can be induced as a biocontainment strategy, as it creates a reproductive barrier preventing farm-to-wild introgression, which is currently a major conservation issue for the industry. However, recent work suggests that triploidisation protocols may, on occasion, produce 'failed triploids' displaying diploidy, aneuploidy and aberrant inheritance. The potentially negative consequences for conservation and animal welfare motivate the need for methods to evaluate the success of ploidy-manipulation protocols early in the production process. We developed a semi-automated version of the MAC-PR (microsatellite DNA allele counting - peak ratios) method to resolve the allelic configuration of large numbers of individuals across a panel of microsatellite markers that can be used to infer ploidy, pedigree and inheritance aberrations. We demonstrate an application of the approach using material from a series of Atlantic salmon (Salmo salar) breeding experiments where ploidy was manipulated using a hydrostatic pressure treatment. We validated the approach to infer ploidy against blood smears, finding a > 99% agreement between these methods, and demonstrate its potential utility to infer ploidy as early as the embryonic stage. Furthermore, we present tools to assign diploid and triploid progeny to families and to detect aberrant inheritance, which may be useful for breeding programmes that utilise ploidy manipulation techniques. The approach adds to the ploidy verification toolbox. The increased precision in detecting ploidy and inheritance aberrations will facilitate the ability of triploidisation programmes to prevent farm-to-wild introgression.

Estimating blue mussel (Mytilus edulis) connectivity and settlement capacity in mid-latitude fjord regions.

The mussel industry faces challenges such as low and inconsistent levels of larvae settlement and poor-quality spat, leading to variable production. However, mussel farming remains a vital sustainable and environmentally responsible method for producing protein, fostering ecological responsibility in the aquaculture sector. We investigate the population connectivity and larval dispersion of blue mussels (Mytilus edulis) in Scottish waters, as a case study, using a multidisciplinary approach that combined genetic data and particle modelling. This research allows us to develop a thorough understanding of blue mussel population dynamics in mid-latitude fjord regions, to infer gene-flow patterns, and to estimate population divergence. Our findings reveal a primary south-to-north particle transport direction and the presence of five genetic clusters. We discover a significant and continuous genetic material exchange among populations within the study area, with our biophysical model's outcomes aligning with our genetic observations. Additionally, our model reveals a robust connection between the southwest coast and the rest of the west coast. This study will guide the preservation of mussel farming regions, ensuring sustainable populations that contribute to marine ecosystem health and resilience.

DNA extraction from recently fertilised Atlantic salmon embryos for use in microsatellite validation of triploidy.

The current methods used for producing triploid Atlantic salmon are generally reliable but not infallible, and each batch of triploids must be validated to ensure consumer trust and licensing compliance. Microsatellites have recently been shown to offer a cheaper and more convenient alternative to traditional flow cytometry for triploidy validation in a commercial setting. However, incubating eggs to at least the eyed stage for microsatellite validation poses challenges, such as reduced quality and performance of triploids produced from later eggs in the stripping season. To address these issues, we propose another option: extracting DNA from recently fertilised eggs for use in conjunction with microsatellite validation. To achieve this, we have developed an optimized protocol for HotSHOT extraction that can rapidly and cheaply extract DNA from Atlantic salmon eggs, which can then be used for triploidy validation through microsatellites. Our approach offers a simpler and more cost-effective way to validate triploidy, without the need for skilled dissection or expensive kits.

Development of genomic markers associated to growth-related traits and sex determination in lumpfish (Cyclopterus lumpus).

Cleaner fish species have gained great importance in the control of sea lice, among them, lumpfish (Cyclopterus lumpus) has become one of the most popular. Lumpfish life cycle has been closed, and hatchery reproduction is now possible, however, current production is reliant on wild caught broodstock to meet the increasing demand. Selective breeding practices are called to play an important role in the successful breeding of most aquaculture species, including lumpfish. In this study we analysed a lumpfish population for the identification of genomic markers linked to production traits. Sequencing of RAD libraries allowed us to identify, 7193 informative markers within the sampled individuals. Genome wide association analysis for sex, weight, condition factor and standard length was performed. One single major QTL region was identified for sex, while nine QTL regions were detected for weight, and three QTL regions for standard length. A total of 177 SNP markers of interest (from QTL regions) and 399 high Fst SNP markers were combined in a low-density panel, useful to obtain relevant genetic information from lumpfish populations. Moreover, a robust combined subset of 29 SNP markers (10 associated to sex, 14 to weight and 18 to standard length) provided over 90% accuracy in predicting the animal's phenotype by machine learning. Overall, our findings provide significant insights into the genetic control of important traits in lumpfish and deliver important genomic resources that will facilitate the establishment of selective breeding programmes in lumpfish.

DNA polymorphism underlying allozyme variation at a malic enzyme locus (mMEP-2*) in Atlantic salmon (Salmo salar L.).

A non-synonymous single nucleotide polymorphism (SNP) underlies a diallelic allozyme polymorphism at the mitochondrial NADP-dependent mMEP-2* locus in Atlantic salmon (Salmo salar L.). The resultant amino acid substitution, which alters the charge of the allelic products, matches the differential mobility of the two allozyme alleles, whereas allozyme and SNP assays revealed genotyping concordance in 257 of 258 individuals. A single mismatch, homozygous allozyme vs. heterozygote SNP, suggests the presence of a second, less common null allele.

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon.

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.

Community Parameters and Genome-Wide RAD-Seq Loci of Ceratothoa oestroides Imply Its Transfer between Farmed European Sea Bass and Wild Farm-Aggregating Fish.

Wild fish assemblages that aggregate within commercial marine aquaculture sites for feeding and shelter have been considered as a primary source of pathogenic parasites vectored to farmed fish maintained in net pens at an elevated density. In order to evaluate whether Ceratothoa oestroides (Isopoda, Cymothoidae), a generalist and pestilent isopod that is frequently found in Adriatic and Greek stocks of farmed European sea bass (Dicentrarchus labrax), transfers between wild and farmed fish, a RAD-Seq (restriction-site-associated DNA sequencing)-mediated genetic screening approach was employed. The double-digest RAD-Seq of 310 C. oestroides specimens collected from farmed European sea bass (138) and different wild farm-aggregating fish (172) identified 313 robust SNPs that evidenced a close genetic relatedness between the "wild" and "farmed" genotypes. ddRAD-Seq proved to be an effective method for detecting the discrete genetic structuring of C. oestroides and genotype intermixing between two populations. The parasite prevalence in the farmed sea bass was 1.02%, with a mean intensity of 2.0 and mean abundance of 0.02, while in the wild fish, the prevalence was 8.1%; the mean intensity, 1.81; and the mean abundance, 0.15. Such differences are likely a consequence of human interventions during the farmed fish's rearing cycle that, nevertheless, did not affect the transfer of C. oestroides.

Genetic analysis redraws the management boundaries for the European sprat.

Sustainable fisheries management requires detailed knowledge of population genetic structure. The European sprat is an important commercial fish distributed from Morocco to the Arctic circle, Baltic, Mediterranean, and Black seas. Prior to 2018, annual catch advice on sprat from the International Council for the Exploration of the Sea (ICES) was based on five putative stocks: (a) North Sea, (b) Kattegat-Skagerrak and Norwegian fjords, (c) Baltic Sea, (d) West of Scotland-southern Celtic Seas, and (e) English Channel. However, there were concerns that the sprat advice on stock size estimates management plan inadequately reflected the underlying biological units. Here, we used ddRAD sequencing to develop 91 SNPs that were thereafter used to genotype approximately 2,500 fish from 40 locations. Three highly distinct and relatively homogenous genetic groups were identified: (a) Norwegian fjords; (b) Northeast Atlantic including the North Sea, Kattegat-Skagerrak, Celtic Sea, and Bay of Biscay; and (c) Baltic Sea. Evidence of genetic admixture and possibly physical mixing was detected in samples collected from the transition zone between the North and Baltic seas, but not between any of the other groups. These results have already been implemented by ICES with the decision to merge the North Sea and the Kattegat-Skagerrak sprat to be assessed as a single unit, thus demonstrating that genetic data can be rapidly absorbed to align harvest regimes and biological units.

Sex determination in the GIFT strain of tilapia is controlled by a locus in linkage group 23.

BACKGROUND: Tilapias (Family Cichlidae) are the second most important group of aquaculture species in the world. They have been the subject of much research on sex determination due to problems caused by early maturation in culture and their complex sex-determining systems. Different sex-determining loci (linkage group 1, 20 and 23) have been detected in various tilapia stocks. The 'genetically improved farmed tilapia' (GIFT) stock, founded from multiple Nile tilapia (Oreochromis niloticus) populations, with some likely to have been introgressed with O. mossambicus, is a key resource for tilapia aquaculture. The sex-determining mechanism in the GIFT stock was unknown, but potentially complicated due to its multiple origins. RESULTS: A bulk segregant analysis (BSA) version of double-digest restriction-site associated DNA sequencing (BSA-ddRADseq) was developed and used to detect and position sex-linked single nucleotide polymorphism (SNP) markers in 19 families from the GIFT strain breeding nucleus and two Stirling families as controls (a single XY locus had been previously mapped to LG1 in the latter). About 1500 SNPs per family were detected across the genome. Phenotypic sex in Stirling families showed strong association with LG1, whereas only SNPs located in LG23 showed clear association with sex in the majority of the GIFT families. No other genomic regions linked to sex determination were apparent. This region was validated using a series of LG23-specific DNA markers (five SNPs with highest association to sex from this study, the LG23 sex-associated microsatellite UNH898 and ARO172, and the recently isolated amhy marker for individual fish (n = 284). CONCLUSIONS: Perhaps surprisingly given its multiple origins, sex determination in the GIFT strain breeding nucleus was associated only with a locus in LG23. BSA-ddRADseq allowed cost-effective analysis of multiple families, strengthening this conclusion. This technique has potential to be applied to other complex traits. The sex-linked SNP markers identified will be useful for potential marker-assisted selection (MAS) to control sex-ratio in GIFT tilapia to suppress unwanted reproduction during growout.

Transcriptomic comparison of communally reared wild, domesticated and hybrid Atlantic salmon fry under stress and control conditions.

BACKGROUND: Domestication is the process by which organisms become adapted to the human-controlled environment. Since the selection pressures that act upon cultured and natural populations differ, adaptations that favour life in the domesticated environment are unlikely to be advantageous in the wild. Elucidation of the differences between wild and domesticated Atlantic salmon may provide insights into some of the genomic changes occurring during domestication, and, help to predict the evolutionary consequences of farmed salmon escapees interbreeding with wild conspecifics. In this study the transcriptome of the offspring of wild and domesticated Atlantic salmon were compared using a common-garden experiment under standard hatchery conditions and in response to an applied crowding stressor. RESULTS: Transcriptomic differences between wild and domesticated crosses were largely consistent between the control and stress conditions, and included down-regulation of environmental information processing, immune and nervous system pathways and up-regulation of genetic information processing, carbohydrate metabolism, lipid metabolism and digestive and endocrine system pathways in the domesticated fish relative to their wild counterparts, likely reflective of different selection pressures acting in wild and cultured populations. Many stress responsive functions were also shared between crosses and included down-regulation of cellular processes and genetic information processing and up-regulation of some metabolic pathways, lipid and energy in particular. The latter may be indicative of mobilization and reallocation of energy resources in response to stress. However, functional analysis indicated that a number of pathways behave differently between domesticated and wild salmon in response to stress. Reciprocal F1 hybrids permitted investigation of inheritance patterns that govern transcriptomic differences between these genetically divergent crosses. Additivity and maternal dominance accounted for approximately 42 and 25% of all differences under control conditions for both hybrids respectively. However, the inheritance of genes differentially expressed between crosses under stress was less consistent between reciprocal hybrids, potentially reflecting maternal environmental effects. CONCLUSION: We conclude that there are transcriptomic differences between the domesticated and wild salmon strains studied here, reflecting the different selection pressures operating on them. Our results indicate that stress may affect certain biological functions differently in wild, domesticated and hybrid crosses and these should be further investigated.

Species-Specific Marker Discovery in Tilapia.

Tilapias (family Cichlidae) are of importance in aquaculture and fisheries. Hybridisation and introgression are common within tilapia genera but are difficult to analyse due to limited numbers of species-specific genetic markers. We tested the potential of double digested restriction-site associated DNA (ddRAD) sequencing for discovering single nucleotide polymorphism (SNP) markers to distinguish between 10 tilapia species. Analysis of ddRAD data revealed 1,371 shared SNPs in the de novo-based analysis and 1,204 SNPs in the reference-based analysis. Phylogenetic trees based on these two analyses were very similar. A total of 57 species-specific SNP markers were found among the samples analysed of the 10 tilapia species. Another set of 62 species-specific SNP markers was identified from a subset of four species which have often been involved in hybridisation in aquaculture: 13 for Oreochromis niloticus, 23 for O. aureus, 12 for O. mossambicus and 14 for O. u. hornorum. A panel of 24 SNPs was selected to distinguish among these four species and validated using 91 individuals. Larger numbers of SNP markers were found that could distinguish between the pairs of species within this subset. This technique offers potential for the investigation of hybridisation and introgression among tilapia species in aquaculture and in wild populations.

Considering adaptive genetic variation in climate change vulnerability assessment reduces species range loss projections.

Local adaptations can determine the potential of populations to respond to environmental changes, yet adaptive genetic variation is commonly ignored in models forecasting species vulnerability and biogeographical shifts under future climate change. Here we integrate genomic and ecological modeling approaches to identify genetic adaptations associated with climate in two cryptic forest bats. We then incorporate this information directly into forecasts of range changes under future climate change and assessment of population persistence through the spread of climate-adaptive genetic variation (evolutionary rescue potential). Considering climate-adaptive potential reduced range loss projections, suggesting that failure to account for intraspecific variability can result in overestimation of future losses. On the other hand, range overlap between species was projected to increase, indicating that interspecific competition is likely to play an important role in limiting species' future ranges. We show that although evolutionary rescue is possible, it depends on a population's adaptive capacity and connectivity. Hence, we stress the importance of incorporating genomic data and landscape connectivity in climate change vulnerability assessments and conservation management.

Expanding the miRNA Repertoire in Atlantic Salmon; Discovery of IsomiRs and miRNAs Highly Expressed in Different Tissues and Developmental Stages.

MicroRNAs (miRNAs) are important post-transcriptional gene expression regulators. Here, 448 different miRNA genes, including 17 novel miRNAs, encoding for 589 mature Atlantic salmon miRNAs were identified after sequencing 111 samples (fry, pathogen challenged fry, various developmental and adult tissues). This increased the reference miRNAome with almost one hundred genes. Prior to isomiR characterization (mature miRNA variants), the proportion of erroneous sequence variants (ESVs) arising in the analysis pipeline was assessed. The ESVs were biased towards 5' and 3' end of reads in unexpectedly high proportions indicating that measurements of ESVs rather than Phred score should be used to avoid misinterpreting ESVs as isomiRs. Forty-three isomiRs were subsequently discovered. The biological effect of the isomiRs measured as increases in target diversity was small (<3%). Five miRNA genes showed allelic variation that had a large impact on target gene diversity if present in the seed. Twenty-one miRNAs were ubiquitously expressed while 31 miRNAs showed predominant expression in one or few tissues, indicating housekeeping or tissue specific functions, respectively. The miR-10 family, known to target Hox genes, were highly expressed in the developmental stages. The proportion of miR-430 family members, participating in maternal RNA clearance, was high at the earliest developmental stage.

Molecular epidemiological study on Infectious Pancreatic Necrosis Virus isolates from aquafarms in Scotland over three decades.

In order to obtain an insight into genomic changes and associated evolution and adaptation of Infectious Pancreatic Necrosis Virus (IPNV), the complete coding genomes of 57 IPNV isolates collected from Scottish aquafarms from 1982 to 2014 were sequenced and analysed. Phylogenetic analysis of the sequenced IPNV strains showed separate clustering of genogroups I, II, III and V. IPNV isolates with genetic reassortment of segment A/B of genogroup III/II were determined. About 59 % of the IPNV isolates belonged to the persistent type and 32 % to the low-virulent type, and only one highly pathogenic strain (1.79 %) was identified. Codon adaptation index calculations indicated that the IPNV major capsid protein VP2 has adapted to its salmonid host. Under-representation of CpG dinucleotides in the IPNV genome to minimize detection by the innate immunity receptors, and observed positive selection in the virulence determination sites of VP2 embedded in the variable region of the main antigenic region, suggest an immune escape mechanism driving virulence evolution. The prevalence of mostly persistent genotypes, together with the assumption of adaptation and immune escape, indicates that IPNV is evolving with the host.

Parallel evolution and adaptation to environmental factors in a marine flatfish: Implications for fisheries and aquaculture management of the turbot (Scophthalmus maximus).

Unraveling adaptive genetic variation represents, in addition to the estimate of population demographic parameters, a cornerstone for the management of aquatic natural living resources, which, in turn, represent the raw material for breeding programs. The turbot (Scophthalmus maximus) is a marine flatfish of high commercial value living on the European continental shelf. While wild populations are declining, aquaculture is flourishing in southern Europe. We evaluated the genetic structure of turbot throughout its natural distribution range (672 individuals; 20 populations) by analyzing allele frequency data from 755 single nucleotide polymorphism discovered and genotyped by double-digest RAD sequencing. The species was structured into four main regions: Baltic Sea, Atlantic Ocean, Adriatic Sea, and Black Sea, with subtle differentiation apparent at the distribution margins of the Atlantic region. Genetic diversity and effective population size estimates were highest in the Atlantic populations, the area of greatest occurrence, while turbot from other regions showed lower levels, reflecting geographical isolation and reduced abundance. Divergent selection was detected within and between the Atlantic Ocean and Baltic Sea regions, and also when comparing these two regions with the Black Sea. Evidence of parallel evolution was detected between the two low salinity regions, the Baltic and Black seas. Correlation between genetic and environmental variation indicated that temperature and salinity were probably the main environmental drivers of selection. Mining around the four genomic regions consistently inferred to be under selection identified candidate genes related to osmoregulation, growth, and resistance to diseases. The new insights are useful for the management of turbot fisheries and aquaculture by providing the baseline for evaluating the consequences of turbot releases from restocking and farming.

Whole genome duplication and transposable element proliferation drive genome expansion in Corydoradinae catfishes.

Genome size varies significantly across eukaryotic taxa and the largest changes are typically driven by macro-mutations such as whole genome duplications (WGDs) and proliferation of repetitive elements. These two processes may affect the evolutionary potential of lineages by increasing genetic variation and changing gene expression. Here, we elucidate the evolutionary history and mechanisms underpinning genome size variation in a species-rich group of Neotropical catfishes (Corydoradinae) with extreme variation in genome size-0.6 to 4.4 pg per haploid cell. First, genome size was quantified in 65 species and mapped onto a novel fossil-calibrated phylogeny. Two evolutionary shifts in genome size were identified across the tree-the first between 43 and 49 Ma (95% highest posterior density (HPD) 36.2-68.1 Ma) and the second at approximately 19 Ma (95% HPD 15.3-30.14 Ma). Second, restriction-site-associated DNA (RAD) sequencing was used to identify potential WGD events and quantify transposable element (TE) abundance in different lineages. Evidence of two lineage-scale WGDs was identified across the phylogeny, the first event occurring between 54 and 66 Ma (95% HPD 42.56-99.5 Ma) and the second at 20-30 Ma (95% HPD 15.3-45 Ma) based on haplotype numbers per contig and between 35 and 44 Ma (95% HPD 30.29-64.51 Ma) and 20-30 Ma (95% HPD 15.3-45 Ma) based on SNP read ratios. TE abundance increased considerably in parallel with genome size, with a single TE-family (TC1-IS630-Pogo) showing several increases across the Corydoradinae, with the most recent at 20-30 Ma (95% HPD 15.3-45 Ma) and an older event at 35-44 Ma (95% HPD 30.29-64.51 Ma). We identified signals congruent with two WGD duplication events, as well as an increase in TE abundance across different lineages, making the Corydoradinae an excellent model system to study the effects of WGD and TEs on genome and organismal evolution.

Single-nucleotide polymorphism discovery and panel characterization in the African forest elephant.

The continuing decline in forest elephant (Loxodonta cyclotis) numbers due to poaching and habitat reduction is driving the search for new tools to inform management and conservation. For dense rainforest species, basic ecological data on populations and threats can be challenging and expensive to collect, impeding conservation action in the field. As such, genetic monitoring is being increasingly implemented to complement or replace more burdensome field techniques. Single-nucleotide polymorphisms (SNPs) are particularly cost-effective and informative markers that can be used for a range of practical applications, including population census, assessment of human impact on social and genetic structure, and investigation of the illegal wildlife trade. SNP resources for elephants are scarce, but next-generation sequencing provides the opportunity for rapid, inexpensive generation of SNP markers in nonmodel species. Here, we sourced forest elephant DNA from 23 samples collected from 10 locations within Gabon, Central Africa, and applied double-digest restriction-site-associated DNA (ddRAD) sequencing to discover 31,851 tags containing SNPs that were reduced to a set of 1,365 high-quality candidate SNP markers. A subset of 115 candidate SNPs was then selected for assay design and validation using 56 additional samples. Genotyping resulted in a high conversion rate (93%) and a low per allele error rate (0.07%). This study provides the first panel of 107 validated SNP markers for forest elephants. This resource presents great potential for new genetic tools to produce reliable data and underpin a step-change in conservation policies for this elusive species.

An integrated framework to identify wildlife populations under threat from climate change.

Climate change is a major threat to global biodiversity that will produce a range of new selection pressures. Understanding species responses to climate change requires an interdisciplinary perspective, combining ecological, molecular and environmental approaches. We propose an applied integrated framework to identify populations under threat from climate change based on their extent of exposure, inherent sensitivity due to adaptive and neutral genetic variation and range shift potential. We consider intraspecific vulnerability and population-level responses, an important but often neglected conservation research priority. We demonstrate how this framework can be applied to vertebrates with limited dispersal abilities using empirical data for the bat Plecotus austriacus. We use ecological niche modelling and environmental dissimilarity analysis to locate areas at high risk of exposure to future changes. Combining outlier tests with genotype-environment association analysis, we identify potential climate-adaptive SNPs in our genomic data set and differences in the frequency of adaptive and neutral variation between populations. We assess landscape connectivity and show that changing environmental suitability may limit the future movement of individuals, thus affecting both the ability of populations to shift their distribution to climatically suitable areas and the probability of evolutionary rescue through the spread of adaptive genetic variation among populations. Therefore, a better understanding of movement ecology and landscape connectivity is needed for predicting population persistence under climate change. Our study highlights the importance of incorporating genomic data to determine sensitivity, adaptive potential and range shift potential, instead of relying solely on exposure to guide species vulnerability assessments and conservation planning.

Impact of Salmonid alphavirus infection in diploid and triploid Atlantic salmon (Salmo salar L.) fry.

With increasing interest in the use of triploid salmon in commercial aquaculture, gaining an understanding of how economically important pathogens affect triploid stocks is important. To compare the susceptibility of diploid and triploid Atlantic salmon (Salmo salar L.) to viral pathogens, fry were experimentally infected with Salmonid alphavirus sub-type 1 (SAV1), the aetiological agent of pancreas disease (PD) affecting Atlantic salmon aquaculture in Europe. Three groups of fry were exposed to the virus via different routes of infection: intraperitoneal injection (IP), bath immersion, or cohabitation (co-hab) and untreated fry were used as a control group. Mortalities commenced in the co-hab challenged diploid and triploid fish from 11 days post infection (dpi), and the experiment was terminated at 17 dpi. Both diploid and triploid IP challenged groups had similar levels of cumulative mortality at the end of the experimental period (41.1% and 38.9% respectively), and these were significantly higher (p < 0.01) than for the other challenge routes. A TaqMan-based quantitative PCR was used to assess SAV load in the heart, a main target organ of the virus, and also liver, which does not normally display any pathological changes during clinical infections, but exhibited severe degenerative lesions in the present study. The median viral RNA copy number was higher in diploid fish compared to triploid fish in both the heart and the liver of all three challenged groups. However, a significant statistical difference (p < 0.05) was only apparent in the liver of the co-hab groups. Diploid fry also displayed significantly higher levels of pancreatic and myocardial degeneration than triploids. This study showed that both diploid and triploid fry are susceptible to experimental SAV1 infection. The lower virus load seen in the triploids compared to the diploids may possibly be related to differences in cell metabolism between the two groups, however, further investigation is necessary to confirm this and also to assess the outcome of PD outbreaks in other developmental stages of the fish when maintained in commercial production systems.

Gene-centromere mapping in meiotic gynogenetic European seabass.

BACKGROUND: Fully isogenic lines in fish can be developed using "mitotic" gynogenesis (suppression of first zygotic mitosis following inactivation of the sperm genome). However, genome-wide verification of the steps in this process has seldom been applied. We used ddRADseq to generate SNP markers in a meiotic gynogenetic family of European seabass (Dicentrarchus labrax): (i) to verify the lack of paternal contribution in a meiotic gynogenetic family; (ii) to generate a gene-centromere map from this family; (iii) to identify telomeric markers that could distinguish mitotic gynogenetics from meiotic gynogenetics, which sometimes arise spontaneously in mitotic gynogenetic families. RESULTS: From a single meiotic gynogenetic family consisting of 79 progeny, 42 million sequencing reads (Illumina, trimmed to 148 bases) resolved 6866 unique RAD-tags. The 340 male-informative SNP markers that were identified confirmed the lack of paternal contribution. A gene-centromere map was constructed based on 804 female-informative SNPs in 24 linkage groups (2n = 48) with a total length of 1251.02 cM (initial LG assignment was based on the seabass genome assembly, dicLab v1). Chromosome arm structure could be clearly discerned from the pattern of heterozygosity in each linkage group in 18 out of 24 LGs: the other six showed anomalies that appeared to be related to issues in the genome assembly. CONCLUSION: Genome-wide screening enabled substantive verification of the production of the gynogenetic family used in this study. The large number of telomeric and subtelomeric markers with high heterozygosity values in the meiotic gynogenetic family indicate that such markers could be used to clearly distinguish between meiotic and mitotic gynogenetics.