Genes involved in sexual development have been identified in many teleost fishes, including the turbot, a flatfish of high commercial value in aquaculture. In this species, a major sex determination (SD) Quantitative Trait Locus has been identified, and Sox2 has been proposed as the SD gene. Although RT-PCR, qPCR, and transcriptomic analyses have been performed on turbot gonads, histological studies remain limited. Here, we examined the tissue-specific expression patterns of several sex-related genes in turbot using in situ hybridization on paraffin sections (SISH) of differentiated ovaries and testes of juvenile and adult specimens. Vasa, Foxl2, Amh, Sox2 and Sox17 transcripts were detected in both male and female gonads, whereas Cyp19a1a and Sox19 expression was restricted to the ovary; these results support previous gene expression analyses and suggest a role for these genes in gonadal development and reproductive functions in this species. SISH provides complementary information to molecular analyses by identifying the specific cell types expressing the sex-related genes analyzed in gonadal tissue, thereby offering a more comprehensive understanding of gonadal differentiation in turbot. Comparison with results reported for the gonads of other teleost species revealed similar tissue-specific gene expression patterns during sexual development.

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 similar to 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.

ABSTRACTA 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.

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 , and 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 . qPCR showed that 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 as its main driver.

Turbot (Scophthalmus maximus) is a flatfish with increasing aquaculture value in Europe and China. This species shows extreme sexual growth dimorphism, with females growing faster and achieving sexual maturity later than males – hence the interest of industry in producing all-female populations. Sex determination (SD) of turbot shows a major genetic component, the main quantitative trait loci (QTL) being located at linkage group (LG) 5. A microsatellite marker (SmaUSC-E30) at this region shows a high sexing efficiency and is used by industry for precocious sex identification. Minor secondary QTLs and some temperature influence have also been reported. Available data support a ZZ/ZW system of recent evolutionary origin in turbot. The first signs of sex differentiation are detected at 90 days post-fertilization, when the genes amh, cyp19a1a and vasa show differential expression between sexes. From this stage, female gonads undergo quick differentiation, while future male gonads remain more similar to undifferentiated gonads. Different approaches have been evaluated to increase the proportion of females in turbot, including hormone treatments and chromosome set manipulation (triploids, gynogenetics), but the most efficient and safe strategy involves a cross between ZZ males and WW superfemales, obtained through a two-generation pedigree involving sex reversal assisted by the SmaUSC-E30 marker. Triploids represent an interesting product for turbot aquaculture, because of their functional sterility, female-biased sex ratio and null impact of escapees on wild populations. Genomic strategies will be likely key to identify the SD master gene, as essential information for efficient sex monitoring and brood stock management at turbot farms.

Repetitive DNA plays a fundamental role in the organization, size and evolution of eukaryotic genomes. The sequencing of the turbot revealed a small and compact genome, as in all flatfish studied to date. The assembly of repetitive regions is still incomplete because it is difficult to correctly identify their position, number and array. The combination of classical cytogenetic techniques along with high quality sequencing is essential to increase the knowledge of the structure and composition of these sequences and, thus, of the structure and function of the whole genome. In this work, the in silico analysis of H1 histone, 5S rDNA, telomeric and Rex repetitive sequences, was compared to their chromosomal mapping by fluorescent in situ hybridization (FISH), providing a more comprehensive picture of these elements in the turbot genome. FISH assays confirmed the location of H1 in LG8; 5S rDNA in LG4 and LG6; telomeric sequences at the end of all chromosomes whereas Rex elements were dispersed along most chromosomes. The discrepancies found between both approaches could be related to the sequencing methodology applied in this species and also to the resolution limitations of the FISH technique. Turbot cytogenomic analyses have proven to add new chromosomal landmarks in the karyotype of this species, representing a powerful tool to investigate targeted genomic sequences or regions in the genetic and physical maps of this species.

The turbot, Scophthalmus maximus, belongs to the flatfishes (order Pleuronectiformes), which display substantial asymmetry of the olfactory organs and forebrain. Sox genes code for SRY-related HMG domain-bearing transcription factors involved in various developmental processes. Group B1 Sox genes as Sox2 and Sox19 appear to play major roles in neural development. Here, we characterized by in situ hybridization the developmental expression of Sox2 and Sox19 genes in metamorphic and postmetamorphic specimens and young adults of both sexes. Expression of S. maximus Sox2 (Sm-Sox2) and Sm-Sox19 mRNAs was detected in ependymal cells of different regions of the telencephalon, preoptic region, hypothalamus, and thalamus at all stages investigated. Sm-Sox2 expression but not Sm-Sox19 occurred in neurons located in particular regions such as the dorsal nucleus of the ventral telencephalon, the medial zone of the dorsal telencephalon, preoptic area and hypothalamus. Although Sm-Sox2 and Sm-Sox19 are expressed differentially in gonads, no sex differences in their expression were observed between male and female forebrains. We also investigated the topographical relation between Sox expression and cell proliferation using series double immunostained for a radial glial marker (BLBP) and cell proliferation marker (PCNA). Sm-Sox2 and Sm-Sox19 were strongly expressed in ependymal cells located in neurogenic niches revealed by the BLBP and PCNA immunostaining. Comparison with other teleosts indicates similar expression of Sox2 and Sox19 in the telencephalon, supporting conserved roles for both genes in teleost brains.

The turbot is a flatfish (Pleuronectiformes) with increasing commercial value, which has prompted active genomic research aimed at more efficient selection. Here we present the sequence and annotation of the turbot genome, which represents a milestone for both boosting breeding programmes and ascertaining the origin and diversification of flatfish. We compare the turbot genome with model fish genomes to investigate teleost chromosome evolution. We observe a conserved macrosyntenic pattern within Percomorpha and identify large syntenic blocks within the turbot genome related to the teleost genome duplication. We identify gene family expansions and positive selection of genes associated with vision and metabolism of membrane lipids, which suggests adaptation to demersal lifestyle and to cold temperatures, respectively. Our data indicate a quick evolution and diversification of flatfish to adapt to benthic life and provide clues for understanding their controversial origin. Moreover, we investigate the genomic architecture of growth, sex determination and disease resistance, key traits for understanding local adaptation and boosting turbot production, by mapping candidate genes and previously reported quantitative trait loci. The genomic architecture of these productive traits has allowed the identification of candidate genes and enriched pathways that may represent useful information for future marker-assisted selection in turbot.

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.

Fish sex determination (SD) systems are varied, suggesting evolutionary changes including either multiple evolution origins of genetic SD from nongenetic systems (such as environmental SD) and/or turnover events replacing one genetic system by another. When genetic SD is found, cytological differentiation between the two members of the sex chromosome pair is often minor or undetectable. The turbot (Scophthalmus maximus), a valuable commercial flatfish, has a ZZ/ZW system and a major SD region on linkage group 5 (LG5), but there are also other minor genetic and environmental influences. We here report refined mapping of the turbot SD region, supported by comparative mapping with model fish species, to identify the turbot master SD gene. Six genes were located to the SD region, two of them associated with gonad development (sox2 and dnajc19). All showed a high association with sex within families (P = 0), but not at the population level, so they are probably partially sex-linked genes, but not SD gene itself. Analysis of crossovers in LG5 using two families confirmed a ZZ/ZW system in turbot and suggested a revised map position for the master gene. Genetic diversity and differentiation for 25 LG5 genetic markers showed no differences between males and females sampled from a wild population, suggesting a recent origin of the SD region in turbot. We also analyzed associations with markers of the most relevant sex-related linkage groups in brill (S. rhombus), a closely related species to turbot; the data suggest that an ancient XX/XY system in brill changed to a ZZ/ZW mechanism in turbot.

The 3rd International Symposium Genomics in Aquaculture (GIA), 4-6 September 2013, Bodo, Norway

Production of all-female populations in turbot can increase farmer’s benefits since sexual dimorphism in growth in this species is among the highest within marine fish, turbot females reaching commercial size 3–6 months earlier than males. Puberty in males occurs earlier than in females, which additionally slows their growth. Thus, elucidating the mechanisms of sex determination and gonad differentiation is a relevant goal for turbot production. A ZZ/ZW sex determination mechanism has been suggested for this species, and four sex-related quantitative trait loci (QTL) were detected, the major one located in linkage group (LG) 5 and the three minor ones in LG6, LG8, and LG21. In the present work, we carried out a linkage analysis for several sex-related markers: (1) three anonymous sex-associated RAPD and (2) several candidate genes related to sex determination and gonad differentiation in other species (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2, Cyp19a, Cyp19b). We focused our attention on their co-localization with the major and minor sex-related QTL trying to approach to the master sex-determining gene of this species. Previously described growth-related QTL were also considered since the association observed between growth and sex determination in fish. Amh, Dmrta2, and one RAPD were located in LG5, while Sox9 and Sox17 (LG21), Cyp19b (LG6), and a second RAPD (LG8) co-mapped with suggestive sex-related QTL, thus supporting further analyses on these genes to elucidate the genetic basis of this relevant trait for turbot farming.

Turbot is one of the most important farmed fish in Europe. This species exhibits a considerable sexual dimorphism in growth and sexual maturity that makes the all-female production recommended for turbot farming. Our knowledge about the genetic basis of sex determination and the molecular regulation of gonad differentiation in this species is still limited. Our goal was to identify and compare gene expression and functions between testes and ovaries in adults in order to ascertain the relationship between the genes that could be involved in the gonad differentiation or related to the sex determination system. The identification of differentially expressed sex related genes is an initial step towards understanding the molecular mechanisms of gonad differentiation. For this, we carried out a transcriptome analysis based on cDNA-AFLP technique which allowed us to obtain an initial frame on sex-specific gene expression that will facilitate further analysis especially along the critical gonad differentiating period. With the aim of widening the study on sex-biased gene expression we reproduced the same experiments in two somatic tissues: liver and brain. We have selected the liver because it is the most analyzed one regarding sexual dimorphic gene expression and due to its importance in steroid hormones metabolism and the brain because the functional relationship between brain and gonad is documented. We found slight but important differences between sexes which deserve further investigation.

AQUA 2018, #WeRAquaculture we are the producers and investors, the scientists and technical advisors, the legislators and educators, the students, civil society organizations and consumers of farmed aquatic products, 25-29 August 2018, Montpellier, France