Comparative expression analysis in mature gonads, liver and brain of turbot (Scophthalmus maximus) by cDNA-AFLPS

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 investigationThis research work was financially supported by the Xunta of Galicia (07MMA004200PR) to A. Viñas. X. Taboada was supported by a fellowship from the European Social Fund and Consellería de Educación e Ordenación Universitaria- Xunta de Galicia (Spain)S

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

This is a post-peer-review, pre-copyedit version of an article published in Chromosoma. The final authenticated version is available online at: https://doi.org/10.1007/s00412-014-0452-2Bacterial 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 assemblyThis study was supported by Spain’s Ministerio de Ciencia e Innovación (AGL2009-13273), Consolider Ingenio Aquagenomics (CSD200700002) and Xunta de Galicia (09MMA011261PR; 10MMA200027PR). Samples for cytogenetic analysis were kindly supplied by Cluster de Acuicultura de GaliciaS

Multiple evidences suggest sox2 as the main driver of a young and complex sex determining ZW/ZZ system in turbot (Scophthalmus maximus)

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.Postprin

Fine Mapping and Evolution of the Major Sex Determining Region in Turbot (Scophthalmus maximus)

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.This research work was supported by the Consellería de Educación e Ordenación Universitaria and the Dirección Xeral de I+D Xunta de Galicia (project 10MMA200027PR) and by the Spanish Government (Consolider Ingenio Aquagenomics: CSD2007-00002 project) and Spanish Ministerio de Ciencia e Innovación (AGL2009-13273) projectsS

Whole genome sequencing of turbot (Scophthalmus maximus; Pleuronectiformes):a fish adapted to demersal life

12 páginas, 5 figuras.-- Antonio Figueras ... et al.-- This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly citedThe 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 turbotThis work was funded by the Spanish Government: projects Consolider Ingenio: Aquagenomics (CSD2007-00002) and Metagenoma de la Península Ibérica (CSD2007-00005), Ministerio de Economía y Competitividad and European Regional Development Funds (AGL2012-35904), and Ministerio de Economía y Competitividad (AGL2014-51773 and AGL2014-57065-R); and Local Government Xunta de Galicia (GRC2014/010). P.P. and D.R. gratefully acknowledge the Spanish Ministerio de Educación for their FPU fellowships (AP2010-2408, AP2012-0254). Funding to pay the Open Access publication charges for this article was provided by the Ministerio de Economía y Competitividad (AGL2014-51773) and Xunta de Galicia (GRC2014/010)Peer reviewe

Comparative expression analysis in mature gonads, liver and brain of turbot (Scophthalmus maximus) by cDNA-AFLPS

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. © 2011 Elsevier B.V.This research work was financially supported by the Xunta of Galicia (07MMA004200PR) to A. Viñas. X. Taboada was supported by a fellowship from the European Social Fund and Consellería de Educación e Ordenación Universitaria- Xunta de Galicia (Spain).Peer Reviewe

Reproduction and Sex Control in Turbot

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 farmsPeer Reviewe

Fine mapping of the major sex-determining locus in the Turbot (Scophthalmus maximus l.)

The 3rd International Symposium Genomics in Aquaculture (GIA), 4-6 September 2013, Bodø, NorwayPeer reviewe

Genetic architecture of sex determination in turbot, Scophthalmus maximus

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, FranceTurbot (Scophthalmus maximus) is a flatfish with increasing aquaculture value in Europe and China. This species shows extreme sexual growth dimorphism, females growing faster and becoming sexually mature 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, but minor QTLs and temperature influence have also been reported. Available data suggest a ZZ/ZW system of recent evolutionary origin, since recombination is not restricted and no genetic divergence at this region is observed males and females. In this study, we carried out a GWAS analysis of SD in this species using 18,165 SNPs in a large set of 36 families. Although, previous findings were confirmed (major SD region at LG5), ~30% of families showed a different pattern suggesting association with other LGs or environmental influence. Standard SD LG5 families were used to narrow down to 531 kb the region where the master gene is putatively located. This region was deeply analyzed by re-sequencing ZZ and WW individuals to look for a diagnostic difference between sexes related to SD. Furthermore, candidate genes were scrutinized for structural differences and their expression profiles studied along the critical SD period. All data support that SD in this species meets to a complex trait with the major locus being recently recruitedPeer Reviewe

Fine Mapping and Evolution of the Major Sex Determining Region in Turbot (Scophthalmus maximus)

10 pages, 4 figures, 1 table, supporting information http://g3journal.org/content/4/10/1871/suppl/DC1Fish 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. © 2014 Taboada et al.This research work was supported by the Consellería de Educación e Ordenación Universitaria and the Dirección Xeral de I+D Xunta de Galicia (project 10MMA200027PR) and by the Spanish Government (Consolider Ingenio Aquagenomics: CSD2007-00002 project) and Spanish Ministerio de Ciencia e Innovación (AGL2009-13273) projectsPeer Reviewe