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In population management, the most efficient method to control the increase of inbreeding and the associated loss of genetic variability is the Optimal Contributions method. This method optimizes the contributions of breeding candidates by minimizing the weighted global coancestry. Traditionally, coancestry coefficients have been estimated from pedigree data but the current availability of genome-wide information allows us to estimate them with higher precision. In recent years, developments of genomic tools in aquaculture species have been very significant. For turbot, a species with an increasing aquaculture value, the whole genome has been recently assembled and genetic and physical maps have been refined. Although several measures of genomic coancestry have been proposed, their relative efficiency for maintaining genetic variability is unknown. The objectives of this study were to compare different measures of genomic coancestry for turbot, and to evaluate their efficiency for retaining genetic variability when using the Optimal Contributions method. We used genomic data obtained through 2b-RAD technology for a domesticated population to achieve the objectives. The different genome-wide coancestry matrices compared were based on: i) the proportion of shared alleles; ii) deviations of the observed number of alleles shared by two individuals from the expected number; iii) the realized relationship matrix obtained by VanRaden's method 1; iv) the realized relationship matrix obtained by VanRaden's method 2; v) the realized relationship matrix obtained by Yang's method; and vi) identical by descent segments. The amount of genetic variability retained when using each coancestry matrix was measured as the expected heterozygosity in the next generation. Results revealed that coancestry coefficients showing high correlations between them gave similar results from the optimization. The genetic variability retained was about 5% higher when using the matrices based on the p

Titanium dioxide (TiO2) and silver (Ag) NPs are among the most used engineered inorganic nanoparticles (NPs); however, their potential effects to marine demersal fish species, are not fully understood. Therefore, this study aimed to assess the proteomic alterations induced by sub-lethal concentrations citrate-coated 25 nm ("P25") TiO2 or polyvinylpyrrolidone (PVP) coated 15 nm Ag NPs to turbot, Scophthalmus maximus. Juvenile fish were exposed to the NPs through daily feeding for 14 days. The tested concentrations were 0, 0.75 or 1.5 mg of each NPs per kg of fish per day. The determination of NPs, Titanium and Ag levels (sp-ICP-MS/ICP-MS) and histological alterations (Transmission Electron Microscopy) supported proteomic analysis performed in the liver and kidney. Proteomic sample preparation procedure (SP3) was followed by LC-MS/MS. Label-free MS quantification methods were employed to assess differences in protein expression. Functional analysis was performed using STRING web-tool. KEGG Gene Ontology suggested terms were discussed and potential biomarkers of exposure were proposed. Overall, data shows that liver accumulated more elements than kidney, presented more histological alterations (lipid droplets counts and size) and proteomic alterations. The Differentially Expressed Proteins (DEPs) were higher in Ag NPs trial. The functional analysis revealed that both NPs caused enrichment of proteins related to generic processes (metabolic pathways). Ag NPs also affected protein synthesis and nucleic acid transcription, among other processes. Proteins related to thyroid hormone transport (Serpina7) and calcium ion binding (FAT2) were suggested as biomarkers of TiO2 NPs in liver. For Ag NPs, in kidney (and at a lower degree in liver) proteins related with metabolic activity, metabolism of exogenous substances and oxidative stress (e.g.: NADH dehydrogenase and Cytochrome P450) were suggested as potential biomarkers. Data suggests adverse effects in turbot after medium/lo, Funding: European Regional Development Fund (ERDF) through Interreg Atlantic Area Program, project NANO -CULTURE [EAPA 590/2018]; Portuguese Foundation for Science and Technology (Fundacao para a Ciencia e Tecnologia; FCT) [UIDB/04423/2020, UIDP/04423/2020]

Background The high fecundity of fish species allows intense selection to be practised and therefore leads to fast genetic gains. Based on this, numerous selective breeding programmes have been started in Europe in the last decades, but in general, little is known about how the base populations of breeders have been built. Such knowledge is important because base populations can be created from very few individuals, which can lead to small effective population sizes and associated reductions in genetic variability. In this study, we used genomic information that was recently made available for turbot (Scophthalmus maximus), gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and common carp (Cyprinus carpio) to obtain accurate estimates of the effective size for commercial populations. Methods Restriction-site associated DNA sequencing data were used to estimate current and historical effective population sizes. We used a novel method that considers the linkage disequilibrium spectrum for the whole range of genetic distances between all pairs of single nucleotide polymorphisms (SNPs), and thus accounts for potential fluctuations in population size over time. Results Our results show that the current effective population size for these populations is small (equal to or less than 50 fish), potentially putting the sustainability of the breeding programmes at risk. We have also detected important drops in effective population size about five to nine generations ago, most likely as a result of domestication and the start of selective breeding programmes for these species in Europe. Conclusions Our findings highlight the need to broaden the genetic composition of the base populations from which selection programmes start, and suggest that measures designed to increase effective population size within all farmed populations analysed here should be implemented in order to manage genetic variability and ensure the sustainability of the breeding program

Background: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. Results: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. Conclusions: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families

Background: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. Results: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. Conclusions: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families

Selective breeding for improving host responses to infectious pathogens is a promising option for disease control. In fact, disease resilience, the ability of a host to survive or cope with infectious challenge, has become a highly desirable breeding goal. However, resilience is a complex trait composed of two different host defence mechanisms, namely resistance (the ability of a host to avoid becoming infected or diseased) and endurance (the ability of an infected host to survive the infection). While both could be targeted for genetic improvement, it is currently unknown how they contribute to survival, as reliable estimates of genetic parameters for both traits obtained simultaneously are scarce. A difficulty lies in obtaining endurance phenotypes for genetic analyses. In this study, we present the results from an innovative challenge test carried out in turbot whose design allowed disentangling the genetic basis of resistance and endurance to Philasterides dicentrarchi, a parasite causing scuticociliatosis that leads to substantial economic losses in the aquaculture industry. A noticeable characteristic of the parasite is that it causes visual signs that can be used for disentangling resistance and endurance. Our results showed the existence of genetic variation for both traits (heritability = 0.26 and 0.12 for resistance and endurance, respectively) and for the composite trait resilience (heritability = 0.15). The genetic correlation between resistance and resilience was very high (0.90) indicating that both are at a large extent the same trait, but no significant genetic correlation was found between resistance and endurance. A total of 18,125 SNPs obtained from 2b-RAD sequencing enabled genome-wide association analyses for detecting QTLs controlling the three traits. A candidate QTL region on linkage group 19 that explains 33% of the additive genetic variance was identified for resilience. The region contains relevant genes related to immune response and defe

A major challenge in evolutionary biology is to find an explanation for the variation in sex-determining (SD) systems across taxa and to understand the mechanisms driving sex chromosome differentiation. We studied the turbot, holding a ZW/ZZ SD system and no sex chromosome heteromorphism, by combining classical genetics and genomics approaches to disentangle the genetic architecture of this trait. RAD-Seq was used to genotype 18,214 SNPs on 1,135 fish from 36 families and a genome wide association study (GWAS) identified a ~ 6 Mb region on LG5 associated with sex (P < 0.05). The most significant associated markers were located close to sox2, dnajc19 and fxr1 genes. A segregation analysis enabled narrowing down the associated region and evidenced recombination suppression in a region overlapping the candidate genes. A Nanopore/Illumina assembly of the SD region using ZZ and WW individuals identified a single SNP fully associated with Z and W chromosomes. RNA-seq from 5-90 day-old fish detected the expression along the gonad differentiation period of a short non-coding splicing variant (ncRNA) included in a vertebrate-conserved long non-coding RNA overlapping sox2. qPCR showed that sox2 was the only differentially expressed gene between males and females at 50-55 days post fertilization, just prior the beginning of gonad differentiation. More refined information on the involvement of secondary genetic and environmental factors and their interactions on SD was gathered after the analysis of a broad sample of families. Our results confirm the complex nature of SD in turbot and support sox2 as its main driver.

Variation among families of turbot (Scophthalmus maximus) in growth, feed efficiency, and body weight variation was investigated. A total of 672 turbot (Scophthalmus maximus) originating from eight families (84 full-sibs per family) were used in this experiment. Body weight (BW) was recorded individually four times between approximately 250 and 370 days of age. Feed intake was measured for each tank during the three corresponding time periods. Feed efficiency was estimated for each tank based on the calculations of residual feed intake (RFI) and feed conversion ratio (FCR). The within-tank coefficient of variation in body weight (CV-BW) and residual body weight variation (RBWV) were calculated to evaluate group dominance dynamics. Components of variation attributable to families were estimated from linear and quadratic random regression orthogonal polynomials. The random quadratic family component explained 14% (RFI), 22% (FCR), 76% (BW), 50% (CV-BW), and 45% (RBWV) of the total variance. The family components were significant for BW, CV-BW and RBWV (p<0.001), and was very close to significance for FCR (p=0.052). The correlation between the intercept (grand mean) of RFI and FCR was highly significant (r=0.94). Intercepts of RFI and FCR were positively correlated with CV-BW and RBWV (r=0.09 to 0.12), however, the correlations were not significant. The results indicate differences between families in FCR, which may be used in selection programs aimed at improving feed efficiency.

Highly dense linkage maps enable positioning thousands of landmarks useful for anchoring the whole genome and for analysing genome properties. Turbot is the most important cultured flatfish worldwide and breeding programs in the fifth generation of selection are targeted to improve growth rate, obtain disease resistant broodstock and understand sex determination to control sex ratio. Using a Restriction-site Associated DNA approach, we genotyped 18,214 single nucleotide polymorphism in 1,268 turbot individuals from 31 full-sibling families. Individual linkage maps were combined to obtain a male, female and species consensus maps. The turbot consensus map contained 11,845 markers distributed across 22 linkage groups representing a total normalised length of 3,753.9 cM. The turbot genome was anchored to this map, and scaffolds representing 96% of the assembly were ordered and oriented to obtain the expected 22 megascaffolds according to its karyotype. Recombination rate was lower in males, especially around centromeres, and pairwise comparison of 44 individual maps suggested chromosome polymorphism at specific genomic regions. Genome comparison across flatfish provided new evidence on karyotype reorganisations occurring across the evolution of this fish group

Highly dense linkage maps enable positioning thousands of landmarks useful for anchoring the whole genome and for analysing genome properties. Turbot is the most important cultured flatfish worldwide and breeding programs in the fifth generation of selection are targeted to improve growth rate, obtain disease resistant broodstock and understand sex determination to control sex ratio. Using a Restriction-site Associated DNA approach, we genotyped 18,214 single nucleotide polymorphism in 1,268 turbot individuals from 31 full-sibling families. Individual linkage maps were combined to obtain a male, female and species consensus maps. The turbot consensus map contained 11,845 markers distributed across 22 linkage groups representing a total normalised length of 3,753.9 cM. The turbot genome was anchored to this map, and scaffolds representing 96% of the assembly were ordered and oriented to obtain the expected 22 megascaffolds according to its karyotype. Recombination rate was lower in males, especially around centromeres, and pairwise comparison of 44 individual maps suggested chromosome polymorphism at specific genomic regions. Genome comparison across flatfish provided new evidence on karyotype reorganisations occurring across the evolution of this fish group.

Variation among families of turbot (Scophthalmus maximus) in growth, feed efficiency, and body weight variation was investigated. A total of 672 turbot (Scophthalmus maximus) originating from eight families (84 full-sibs per family) were used in this experiment. Body weight (BW) was recorded individually four times between approximately 250 and 370 days of age. Feed intake was measured for each tank during the three corresponding time periods. Feed efficiency was estimated for each tank based on the calculations of residual feed intake (RFI) and feed conversion ratio (FCR). The within-tank coefficient of variation in body weight (CV-BW) and residual body weight variation (RBWV) were calculated to evaluate group dominance dynamics. Components of variation attributable to families were estimated from linear and quadratic random regression orthogonal polynomials. The random quadratic family component explained 14% (RFI), 22% (FCR), 76% (BW), 50% (CV-BW), and 45% (RBWV) of the total variance. The family components were significant for BW, CV-BW and RBWV (p<0.001), and was very close to significance for FCR (p=0.052). The correlation between the intercept (grand mean) of RFI and FCR was highly significant (r=0.94). Intercepts of RFI and FCR were positively correlated with CV-BW and RBWV (r=0.09 to 0.12), however, the correlations were not significant. The results indicate differences between families in FCR, which may be used in selection programs aimed at improving feed efficiency.

Growth is among the most important traits for animal breeding. Understanding the mechanisms underlying growth differences between individuals can contribute to improving growth rates through more efficient breeding schemes. Here, we report a transcriptomic study in muscle and brain of fast- and slow-growing turbot (Scophthalmus maximus), a relevant flatfish in European and Asian aquaculture. Gene expression and allelic association between the two groups were explored. Up-regulation of the anaerobic glycolytic pathway in the muscle of fast-growing fish was observed, indicating a higher metabolic rate of white muscle. Brain expression differences were smaller and not associated with major growth-related genes, but with regulation of feeding-related sensory pathways. Further, SNP variants showing frequency differences between fast- and slow-growing fish pointed to genomic regions likely involved in growth regulation, and three of them were individually validated through SNP typing. Although different mechanisms appear to explain growth differences among families, general mechanisms seem also to be involved, and thus, results provide a set of useful candidate genes and markers to be evaluated for more efficient growth breeding programs and to perform comparative genomic studies of growth in fish and vertebrates

Growth traits represent a main goal in aquaculture breeding programs and may be related to adaptive variation in wild fisheries. Integrating quantitative trait loci (QTL) mapping and next generation sequencing can greatly help to identify variation in candidate genes, which can result in marker-assisted selection and better genetic structure information. Turbot is a commercially important flatfish in Europe and China, with available genomic information on QTLs and genome mapping. Muscle and liver RNA-seq from 18 individuals was carried out to obtain gene sequences and markers functionally related to growth, resulting in a total of 20,447 genes and 85,344 single nucleotide polymorphisms (SNPs). Many growth-related genes and SNPs were identified and placed in the turbot genome and genetic map to explore their co-localization with growth-QTL markers. Forty-five SNPs on growth-related genes were selected based on QTL co-localization and relevant function for growth traits. Forty-three SNPs were technically feasible and validated in a wild Atlantic population, where 91% were polymorphic. The integration of functional and structural genomic resources in turbot provides a practical approach for QTL mining in this species. Validated SNPs represent a useful set of growth-related gene markers for future association, functional and population studies in this flatfish species

Disease resistance-related traits have received increasing importance in aquaculture breeding programs worldwide. Currently, genomic information offers new possibilities in breeding to address the improvement of this kind of traits. The turbot is one of the most promising European aquaculture species, and Philasterides dicentrarchi is a scuticociliate parasite causing fatal disease in farmed turbot. An appealing approach to fight against disease is to achieve a more robust broodstock, which could prevent or diminish the devastating effects of scuticociliatosis on farmed individuals. In the present study, a genome scan for quantitative trait loci (QTL) affecting resistance and survival time to P. dicentrarchi in four turbot families was carried out. The objectives were to identify QTL using different statistical approaches [linear regression (LR) and maximum likelihood (ML)] and to locate significantly associated markers for their application in genetic breeding strategies. Several genomic regions controlling resistance and survival time to P. dicentrarchi were detected. When analyzing each family separately, significant QTL for resistance were identified by the LR method in two linkage groups (LG1 and LG9) and for survival time in LG1, while the ML methodology identified QTL for resistance in LG9 and LG23 and for survival time in LG6 and LG23. The analysis of the total data set identified an additional significant QTL for resistance and survival time in LG3 with the LR method. Significant association between disease resistance-related traits and genotypes was detected for several markers, a single one explaining up to 22% of the phenotypic variance. Obtained results will be essential to identify candidate genes for resistance and to apply them in marker-assisted selection programs to improve turbot production. © 2012 The Authors.

Background The turbot (Scophthalmus maximus) is a highly appreciated European aquaculture species. Growth related traits constitute the main goal of the ongoing genetic breeding programs of this species. The recent construction of a consensus linkage map in this species has allowed the selection of a panel of 100 homogeneously distributed markers covering the 26 linkage groups (LG) suitable for QTL search. In this study we addressed the detection of QTL with effect on body weight, length and Fulton's condition factor. Results Eight families from two genetic breeding programs comprising 814 individuals were used to search for growth related QTL using the panel of microsatellites available for QTL screening. Two different approaches, maximum likelihood and regression interval mapping, were used in order to search for QTL. Up to eleven significant QTL were detected with both methods in at least one family: four for weight on LGs 5, 14, 15 and 16; five for length on LGs 5, 6, 12, 14 and 15; and two for Fulton's condition factor on LGs 3 and 16. In these LGs an association analysis was performed to ascertain the microsatellite marker with the highest apparent effect on the trait, in order to test the possibility of using them for marker assisted selection. Conclusions The use of regression interval mapping and maximum likelihood methods for QTL detection provided consistent results in many cases, although the high variation observed for traits mean among families made it difficult to evaluate QTL effects. Finer mapping of detected QTL, looking for tightly linked markers to the causative mutation, and comparative genomics are suggested to deepen in the analysis of QTL in turbot so they can be applied in marker assisted selection programs.

Background Interactions between fish and pathogens, that may be harmless under natural conditions, often result in serious diseases in aquaculture systems. This is especially important due to the fact that the strains used in aquaculture are derived from wild strains that may not have had enough time to adapt to new disease pressures. The turbot is one of the most promising European aquaculture species. Furunculosis, caused by the bacterium Aeromonas salmonicida, produces important losses to turbot industry. An appealing solution is to achieve more robust broodstock, which can prevent or diminish the devastating effects of epizooties. Genomics strategies have been developed in turbot to look for candidate genes for resistance to furunculosis and a genetic map with appropriate density to screen for genomic associations has been also constructed. In the present study, a genome scan for QTL affecting resistance and survival to A. salmonicida in four turbot families was carried out. The objectives were to identify consistent QTL using different statistical approaches (linear regression and maximum likelihood) and to locate the tightest associated markers for their application in genetic breeding strategies. Results Significant QTL for resistance were identified by the linear regression method in three linkage groups (LGs 4, 6 and 9) and for survival in two LGs (6 and 9). The maximum likelihood methodology identified QTL in three LGs (5, 6 and 9) for both traits. Significant association between disease traits and genotypes was detected for several markers, some of them explaining up to 17% of the phenotypic variance. We also identified candidate genes located in the detected QTL using data from previously mapped markers. Conclusions Several regions controlling resistance to A. salmonicida in turbot have been detected. The observed concordance between different statistical methods at particular linkage groups gives consistency to our results. The detected associated marke