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      Transcriptomics Analysis of Crassostrea hongkongensis for the Discovery of Reproduction-Related Genes

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          Abstract

          Background

          The reproductive mechanisms of mollusk species have been interesting targets in biological research because of the diverse reproductive strategies observed in this phylum. These species have also been studied for the development of fishery technologies in molluscan aquaculture. Although the molecular mechanisms underlying the reproductive process have been well studied in animal models, the relevant information from mollusks remains limited, particularly in species of great commercial interest. Crassostrea hongkongensis is the dominant oyster species that is distributed along the coast of the South China Sea and little genomic information on this species is available. Currently, high-throughput sequencing techniques have been widely used for investigating the basis of physiological processes and facilitating the establishment of adequate genetic selection programs.

          Results

          The C. hongkongensis transcriptome included a total of 1,595,855 reads, which were generated by 454 sequencing and were assembled into 41,472 contigs using de novo methods. Contigs were clustered into 33,920 isotigs and further grouped into 22,829 isogroups. Approximately 77.6% of the isogroups were successfully annotated by the Nr database. More than 1,910 genes were identified as being related to reproduction. Some key genes involved in germline development, sex determination and differentiation were identified for the first time in C. hongkongensis ( nanos, piwi, ATRX, FoxL2, β-catenin, etc.). Gene expression analysis indicated that vasa, nanos, piwi, ATRX, FoxL2, β-catenin and SRD5A1 were highly or specifically expressed in C. hongkongensis gonads. Additionally, 94,056 single nucleotide polymorphisms (SNPs) and 1,699 simple sequence repeats (SSRs) were compiled.

          Conclusions

          Our study significantly increased C. hongkongensis genomic information based on transcriptomics analysis. The group of reproduction-related genes identified in the present study constitutes a new tool for research on bivalve reproduction processes. The large group of molecular markers discovered in this study will be useful for population screening and marker assisted selection programs in C. hongkongensis aquaculture.

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          Most cited references66

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          Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation.

          In mammals, the transcription factor SRY, encoded by the Y chromosome, is normally responsible for triggering the indifferent gonads to develop as testes rather than ovaries. However, testis differentiation can occur in its absence. Here we demonstrate in the mouse that a single factor, the forkhead transcriptional regulator FOXL2, is required to prevent transdifferentiation of an adult ovary to a testis. Inducible deletion of Foxl2 in adult ovarian follicles leads to immediate upregulation of testis-specific genes including the critical SRY target gene Sox9. Concordantly, reprogramming of granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages occurs with testosterone levels comparable to those of normal XY male littermates. Our results show that maintenance of the ovarian phenotype is an active process throughout life. They might also have important medical implications for the understanding and treatment of some disorders of sexual development in children and premature menopause in women.
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            Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx

            Background New methods are needed for genomic-scale analysis of emerging model organisms that exemplify important biological questions but lack fully sequenced genomes. For example, there is an urgent need to understand the potential for corals to adapt to climate change, but few molecular resources are available for studying these processes in reef-building corals. To facilitate genomics studies in corals and other non-model systems, we describe methods for transcriptome sequencing using 454, as well as strategies for assembling a useful catalog of genes from the output. We have applied these methods to sequence the transcriptome of planulae larvae from the coral Acropora millepora. Results More than 600,000 reads produced in a single 454 sequencing run were assembled into ~40,000 contigs with five-fold average sequencing coverage. Based on sequence similarity with known proteins, these analyses identified ~11,000 different genes expressed in a range of conditions including thermal stress and settlement induction. Assembled sequences were annotated with gene names, conserved domains, and Gene Ontology terms. Targeted searches using these annotations identified the majority of genes associated with essential metabolic pathways and conserved signaling pathways, as well as novel candidate genes for stress-related processes. Comparisons with the genome of the anemone Nematostella vectensis revealed ~8,500 pairs of orthologs and ~100 candidate coral-specific genes. More than 30,000 SNPs were detected in the coral sequences, and a subset of these validated by re-sequencing. Conclusion The methods described here for deep sequencing of the transcriptome should be widely applicable to generate catalogs of genes and genetic markers in emerging model organisms. Our data provide the most comprehensive sequence resource currently available for reef-building corals, and include an extensive collection of potential genetic markers for association and population connectivity studies. The characterization of the larval transcriptome for this widely-studied coral will enable research into the biological processes underlying stress responses in corals and evolutionary adaptation to global climate change.
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              DMRT1 prevents female reprogramming in the postnatal mammalian testis

              Sex in mammals is determined in the foetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells 1 . This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the foetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving Wnt/β-catenin signaling (Supplemental Fig. 1) 2 . In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogramme granulosa cells into Sertoli cells 2 . Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor 3 in mouse Sertoli cells, even in adults, activates Foxl2 and reprogrammes Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced, and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the foetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates 4,5 and Dmrt1-related sexual regulators are conserved throughout metazoans 3 . Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the etiology of human syndromes linked to DMRT1, including disorders of sexual differentiation 6 and testicular cancer 7 .
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 August 2015
                2015
                : 10
                : 8
                : e0134280
                Affiliations
                [1 ]Key Laboratory of Marine Bio-resource Sustainable Utilization, Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
                [2 ]South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou, China
                [3 ]Qingyuan Polytechnic, Qingyuan, China
                The Ohio State University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: YT YZ. Performed the experiments: YT YZ JC. Analyzed the data: YT YZ JH. Contributed reagents/materials/analysis tools: SX YHZ JL. Wrote the paper: YT YZ ZY JH. Led and supervised the study: YZ ZY. Tissue and larval sampling: YZ YT SX YZ JL. RNA extraction: YZ JL YT. 454 sequencing: the University of Illinois, USA. Assembled and annotated the transcriptome: JH YT. RT-qPCR and mRNA in situ hybridization: YT JC. SSR and SNP identification: JH YT.

                Article
                PONE-D-15-08893
                10.1371/journal.pone.0134280
                4530894
                26258576
                7d0c5789-207e-49b2-9c62-d49edcd264c4
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 3 March 2015
                : 7 July 2015
                Page count
                Figures: 8, Tables: 5, Pages: 24
                Funding
                Support was provided by High Tech Research and Development Program of China (863 program) [ http://www.863.gov.cn/] no.2012AA10A405-3 to YZ; National Natural Science Foundation of China [ http://www.nsfc.gov.cn/] no.41306145 to YT; National Natural Science Foundation of China [ http://www.nsfc.gov.cn/] no. 31101927 to JC; Pearl River S&T Nova Program of Guangzhou [ http://www.gzsi.gov.cn/] no. 2013J2200095 to YZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                Sequencing data are deposited in the NIH Short Read Archive database with Run accession number SRR949615.

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