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8 pages, 5 figures, supplementary material https://doi.org/10.1016/j.ygeno.2021.07.007 ; DNA methylation is one of the main epigenetic mechanisms that regulate gene expression in a manner that depends on the genomic context and varies considerably across taxa. This DNA modification was first found in nuclear genomes of eukaryote several decades ago and it has also been described in mitochondrial DNA. It has recently been shown that mitochondrial DNA is extensively methylated in mammals and other vertebrates. Our current knowledge of mitochondrial DNA methylation in fish is very limited, especially in non-model teleosts. In this study, using whole-genome bisulfite sequencing, we determined methylation patterns within non-CpG (CH) and CpG (CG) contexts in the mitochondrial genome of Nile tilapia, a non-model teleost of high economic importance. Our results demonstrate the presence of mitochondrial DNA methylation in this species predominantly within a non-CpG context, similarly to mammals. We found a strand-specific distribution of methylation, in which highly methylated cytosines were located on the minus strand. The D-loop region had the highest mean methylation level among all mitochondrial loci. Our data provide new insights into the potential role of epigenetic mechanisms in regulating metabolic flexibility of mitochondria in fish, with implications in various biological processes, such as growth and development ; This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no 683210, 2016) and by the Research Council of Norway under the Toppforsk programme (grant agreement no 250548/F20, 2016) ; With the institutional support of the 'Severo Ochoa Centre of Excellence' accreditation (CEX2019-000928-S) ; Peer reviewed

12 pages, 6 figures, 2 tables, supplementary material https://doi.org/10.1016/j.ygeno.2021.06.037.-- Data availability: The DNA hydroxymethylation dataset of this study is available in the SRA (NCBI) repository, under the accession number PRJNA665628. The RNA sequencing dataset of this study is available in the GEO (NCBI) repository, under the accession number GSE158910 ; In vertebrates, the somatotropic axis comprising the pituitary gland, liver and muscle plays a major role in myogenesis. Its output in terms of muscle growth is highly affected by nutritional and environmental cues, and thus likely epigenetically regulated. Hydroxymethylation is emerging as a DNA modification that modulates gene expression but a holistic characterization of the hydroxymethylome of the somatotropic axis has not been investigated to date. Using reduced representation 5-hydroxymethylcytosine profiling we demonstrate tissue-specific localization of 5-hydroxymethylcytosines at single nucleotide resolution. Their abundance within gene bodies and promoters of several growth-related genes supports their pertinent role in gene regulation. We propose that cytosine hydroxymethylation may contribute to the phenotypic plasticity of growth through epigenetic regulation of the somatotropic axis ; This work was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreements nos. 683210 and 812986) and by the Research Council of Norway under the Toppforsk programme (grant agreement no 250548/F20) ; With the institutional support of the 'Severo Ochoa Centre of Excellence' accreditation (CEX2019-000928-S) ; Peer reviewed

Abstract Background Genomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry. Results Expressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database ("Turbot 2 database") was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences ("Turbot 3 database"), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50–90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified. Conclusions The combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs ; The current work was granted by the Spanish Government thanks to a Consolider Project (Project Aquagenomics, ref. CDS2007-0002) and to projects AGL2006-13158-C03 and AGL2009-13282-C01 and C02. LR was supported by an Aquagenomics postdoctoral contract and BGP was supported by an Isidro Parga Pondal research fellowship from the Xunta de Galicia (Spain). Authors want to thank M.J. Redondo (IATS), María Vázquez (CETGA) and Ana Paula Losada (USC) for their assistance with the infection trial. The authors also thank the Unit of Information Resources for Research (URICI-CSIC) for the co-financing of this publication in Open Access ; We also acknowledge institutional support from the Unit of Information Resources for Research at the "Consejo Superior de Investigaciones Científicas" (CSIC) for the article-processing charges contribution ; Peer Reviewed

16 pages, 6 figures, supplementary material https://www.frontiersin.org/articles/10.3389/fcell.2022.828124/full#supplementary-material.-- Data Availability Statement: According to national legislation/guidelines, specifically the Administrative Regulations of the People's Republic of China on Human Genetic Resources (http://www.gov.cn/zhengce/content/2019-06/10/content_5398829.htm, http://english.www.gov.cn/policies/latest_releases/2019/06/10/content_281476708945462.htm), no additional raw data is available at this time. Data of this project can be accessed after an approval application to the China National Genebank (CNGB, https://db.cngb.org/cnsa/). Please refer to https://db.cngb.org/, or email: CNGBdb@cngb.org for detailed application guidance. The accession code CNP0002319 should be included in the application ; Oogenesis is a highly orchestrated process that depends on regulation by autocrine/paracrine hormones and growth factors. However, many details of the molecular mechanisms that regulate fish oogenesis remain elusive. Here, we performed a single-cell RNA sequencing (scRNA-seq) analysis of the molecular signatures of distinct ovarian cell categories in adult Chinese tongue sole (Cynoglossus semilaevis). We characterized the successive stepwise development of three germ cell subtypes. Notably, we identified the cellular composition of fish follicle walls, including four granulosa cell types and one theca cell type, and we proposed important transcription factors (TFs) showing high activity in the regulation of cell identity. Moreover, we found that the extensive niche–germline bidirectional communications regulate fish oogenesis, whereas ovulation in fish is accompanied by the coordination of simultaneous and tightly sequential processes across different granulosa cells. Additionally, a systems biology analysis of the homologous genes shared by Chinese tongue sole and macaques revealed remarkably conserved biological processes in germ cells and granulosa cells across vertebrates. Our results provide key insights into the cell-type-specific mechanisms underlying fish oogenesis at a single-cell resolution, which offers important clues for exploring fish breeding mechanisms and the evolution of vertebrate reproductive systems ; This work was supported by the National Nature Science Foundation of China (31722058, 31802275 and 31472269); the National Key R&D Program of China (2018YFD0900301); the AoShan Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology (2017ASTCP-ES06); the Taishan Scholar Project Fund of Shandong of China to C.S.; the National Ten-Thousands Talents Special Support Program to C.S.; the Central Public-interest Scientific Institution Basal Research Fund, CAFS (No.2020TD19); and the China Agriculture Research System (CARS-47-G03) ; With the institutional support of the 'Severo Ochoa Centre of Excellence' accreditation (CEX2019-000928-S) ; Peer reviewed

15 pages, 6 figures, supplementary material https://doi.org/10.1007/s10126-019-09886-x ; The European sea bass is one of the most important cultured fish in Europe and has a marked sexual growth dimorphism in favor of females. It is a gonochoristic species with polygenic sex determination, where a combination between still undifferentiated genetic factors and environmental temperature determines sex ratios. The molecular mechanisms responsible for gonadal sex differentiation are still unknown. Here, we sampled fish during the gonadal developmental period (110 to 350 days post fertilization, dpf), and performed a comprehensive transcriptomic study by using a species-specific microarray. This analysis uncovered sex-specific gonadal transcriptomic profiles at each stage of development, identifying larger number of differentially expressed genes in ovaries when compared to testis. The expression patterns of 54 reproduction-related genes were analyzed. We found that hsd17β10 is a reliable marker of early ovarian differentiation. Further, three genes, pdgfb, snx1, and nfy, not previously related to fish sex differentiation, were tightly associated with testis development in the sea bass. Regarding signaling pathways, lysine degradation, bladder cancer, and NOD-like receptor signaling were enriched for ovarian development while eight pathways including basal transcription factors and steroid biosynthesis were enriched for testis development. Analysis of the transcription factor abundance showed an earlier increase in females than in males. Our results show that, although many players in the sex differentiation pathways are conserved among species, there are peculiarities in gene expression worth exploring. The genes identified in this study illustrate the diversity of players involved in fish sex differentiation and can become potential biomarkers for the management of sex ratios in the European sea bass and perhaps other cultured species ; This work was supported by Spanish Government Consolider-Ingenio 2010 grant "Aquagenomics" (ref. CDS2007-0002) to SMK, FM, SZ and FP. BC was supported by an Aquagenomics contract; LR was supported by Aquagenomics postdoctoral contract; ND was supported by Aquagenomics predoctoral contract. NSB was supported by AGL2016-78710-R ; Peer Reviewed

14 pages, 7 figures, 2 tables, supplementary material https://doi.org/10.1016/j.ygeno.2021.04.007.-- Availability of data and materials: All dataset generated during this study are included (in this article and as supplementary information files) or are available in public repositories. Turbot Genome Sequencing data are at NCBI databases (Bioproject: PRJNA631898) (https://www.ncbi.nlm.nih.gov/genome/?term=turbot) and BioProject es PRJNA649485 (https://www.ncbi.nlm.nih.gov/bioproject/649485; Accessión numbers: SRX8843737, SRX8843739, SRX8843738) ; 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 ; This work was supported by the Spanish Ministry of Economy and Competitiveness, Grant: AGL2014-57065-R, by the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 81792 (AQUA-FAANG) and by Consellería de Educación, Universidade e Formación Profesional. Xunta de Galicia, Grant number: ED431C 2018/28 ; With funding from the Spanish government through the 'Severo Ochoa Centre of Excellence' accreditation (CEX2019-000928-S) ; Peer reviewed

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 ; This work was supported by the Spanish Ministry of Economy and Competitiveness, Grant: AGL2014-57065-R, by the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 81792 (AQUA-FAANG) and by Consellería de Educación, Universidade e Formación Profesional. Xunta de Galicia, Grant number:ED431C 2018/28 ; SI