The sex-specific transcriptome of the hermaphrodite sparid sharpsnout seabream (Diplodus puntazzo)

Background: Teleosts are characterized by a remarkable breadth of sexual mechanisms including various forms of hermaphroditism. Sparidae is a fish family exhibiting gonochorism or hermaphroditism even in closely related species. The sparid Diplodus puntazzo (sharpsnout seabream), exhibits rudimentary hermaphroditism characterized by intersexual immature gonads but single-sex mature ones. Apart from the intriguing reproductive biology, it is economically important with a continuously growing aquaculture in the Mediterranean Sea, but limited available genetic resources. Our aim was to characterize the expressed transcriptome of gonads and brains through RNA-Sequencing and explore the properties of genes that exhibit sex-biased expression profiles. Results: Through RNA-Sequencing we obtained an assembled transcriptome of 82,331 loci. The expression analysis uncovered remarkable differences between male and female gonads, while male and female brains were almost identical. Focused search for known targets of sex determination and differentiation in vertebrates built the sex-specific expression profile of sharpsnout seabream. Finally, a thorough genetic marker discovery pipeline led to the retrieval of 85,189 SNPs and 29,076 microsatellites enriching the available genetic markers for this species. Conclusions: We obtained a nearly complete source of transcriptomic sequence as well as marker information for sharpsnout seabream, laying the ground for understanding the complex process of sex differentiation of this economically valuable species. The genes involved include known candidates from other vertebrate species, suggesting a conservation of the toolkit between gonochorists and hermaphrodites

The Gene Toolkit Implicated in Functional Sex in Sparidae Hermaphrodites: Inferences From Comparative Transcriptomics

Sex-biased gene expression is the mode through which sex dimorphism arises from a nearly identical genome, especially in organisms without genetic sex determination. Teleost fishes show great variations in the way the sex phenotype forms. Among them, Sparidae, that might be considered as a model family displays a remarkable diversity of reproductive modes. In this study, we sequenced and analyzed the sex-biased transcriptome in gonads and brain (the tissues with the most profound role in sexual development and reproduction) of two sparids with different reproductive modes: the gonochoristic common dentex, Dentex dentex, and the protandrous hermaphrodite gilthead seabream, Sparus aurata. Through comparative analysis with other protogynous and rudimentary protandrous sparid transcriptomes already available, we put forward common male and female-specific genes and pathways that are probably implicated in sex-maintenance in this fish family. Our results contribute to the understanding of the complex processes behind the establishment of the functional sex, especially in hermaphrodite species and set the groundwork for future experiments by providing a gene toolkit that can improve efforts to control phenotypic sex in finfish in the ever-increasingly important field of aquaculture

Broadening the miRNA Catalogue in Livestock Species

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The “MetaCopepod” project: Designing an integrated DNA metabarcoding and image analysis approach to study and monitor the diversity of zooplanktonic copepods and cladocerans in the Mediterranean Sea

The timely and accurate analysis of marine zooplankton diversity is a challenge in ecological and monitoring studies. Morphology-based identification of taxa, which requires taxonomy experts, is time consuming and cannot provide accurate resolution at species level in several cases (e.g. immature stages, cryptic species, broken specimens). The “MetaCopepod” project is aimed at overcoming these limitations by developing a high-throughput and cost effective methodology that integrates DNA metabarcoding and image analysis. Utilizing the accuracy of DNA metabarcoding in species recognition and the quantitative results of image analysis, zooplankton diversity (mainly of copepods and cladocerans) is assessed both qualitatively (species' composition) and quantitatively (abundance, biomass and size-distribution). To achieve this goal, bulk zooplankton samples are first scanned and analyzed with ZooImage and then massively sequenced for a selected fragment of the mitochondrial 16S rRNA gene. Through a bioinformatic pipeline, sequences are compared to a reference genetic database, constructed within the project, and identified at species- level. The methodology was calibrated by using both mock and taxonomically identified samples and demonstrated on samples collected monthly from monitoring stations across the Mediterranean Sea. It is currently optimized for higher integration and accuracy and is expected to become a powerful tool for monitoring zooplankton in the long term and for early warning of bioinvasions and other ecosystem change

The genomic basis of color pattern polymorphism in the Harlequin ladybird

© 2018 The Authors Many animal species comprise discrete phenotypic forms. A common example in natural populations of insects is the occurrence of different color patterns, which has motivated a rich body of ecological and genetic research [1–6]. The occurrence of dark, i.e., melanic, forms displaying discrete color patterns is found across multiple taxa, but the underlying genomic basis remains poorly characterized. In numerous ladybird species (Coccinellidae), the spatial arrangement of black and red patches on adult elytra varies wildly within species, forming strikingly different complex color patterns [7, 8]. In the harlequin ladybird, Harmonia axyridis, more than 200 distinct color forms have been described, which classic genetic studies suggest result from allelic variation at a single, unknown, locus [9, 10]. Here, we combined whole-genome sequencing, population-based genome-wide association studies, gene expression, and functional analyses to establish that the transcription factor Pannier controls melanic pattern polymorphism in H. axyridis. We show that pannier is necessary for the formation of melanic elements on the elytra. Allelic variation in pannier leads to protein expression in distinct domains on the elytra and thus determines the distinct color patterns in H. axyridis. Recombination between pannier alleles may be reduced by a highly divergent sequence of ∼170 kb in the cis-regulatory regions of pannier, with a 50 kb inversion between color forms. This most likely helps maintain the distinct alleles found in natural populations. Thus, we propose that highly variable discrete color forms can arise in natural populations through cis-regulatory allelic variation of a single gene. More than 200 distinct color forms have been described in natural populations of the harlequin ladybird, Harmonia axyridis. Gautier et al. show that this variation is controlled by the transcription factor Pannier. Pannier is necessary to produce black pigment, and its expression pattern prefigures the coloration pattern in each color form

Inducible transgenic mice reveal resting dendritic cells as potent inducers of CD8+ T cell tolerance

Dendritic cells (DC) are inducers of immune responses par excellence. They also seem responsible for the induction of peripheral T cell tolerance. To investigate these opposite functions of DC, we generated a Cre/LoxP-based system that allows inducible antigen presentation by DC in vivo. This enables us to study the immunogical consequences of antigen presentation by resting versus mature DC without adoptively transferring DC and with physiological numbers of endogenous, naive responder T cells. We found that presentation of LCMV-derived CTL epitopes by resting DC resulted in antigen-specific tolerance, which could not be broken by subsequent infection with LCMV. On the other hand, antigen presentation by activated DC primed endogenous CTL to expand and to develop protective effector function

Direct amplification of length polymorphisms (DALP), or how to get and characterize new genetic markers in many species.

International audienceDirect amplification of length polymorphisms (DALP) uses an arbitrarily primed PCR (AP-PCR) to produce genomic fingerprints and to enable sequencing of DNA polymorphisms in virtually any species. Oligonucleotide pairs were designed to each produce a specific multi-banded pattern and all the fragments thus generated can be directly sequenced with the same two universal M13 sequencing primers. This strategy combines the advantages of a high resolution fingerprint technique and the possibility of characterizing the polymorphisms. The use of family members as templates in the multi-locus detection step allows a direct test of allele transmission, as well as early mapping of the markers or selection of loci associated with some traits or diseases. We used this method to detect micro-deletions/insertions and microsatellite DNA loci useful in population genetics studies, but it could be applied in many other fields of biology, such as genome mapping for definition of polymorphic sequence tagged sites, directly localized on a genetic map

NOBLAST and JAMBLAST: New Options for BLAST and a Java Application Manager for BLAST results

International audienceNOBLAST (New Options for BLAST) is an open source program that provides a new user-friendly tabular output format for various NCBI BLAST programs (Blastn, Blastp, Blastx, Tblastn, Tblastx, Mega BLAST and Psi BLAST) without any use of a parser and provides E-value correction in case of use of segmented BLAST database. JAMBLAST using the NOBLAST output allows the user to manage, view and filter the BLAST hits using a number of selection criteria

Species-wide homogeneity of nuclear ribosomal ITS2 sequences in the spider mite Tetranychus urticae contrasts with extensive mitochondrial COI polymorphism.

International audienceWe compared patterns of intraspecific polymorphism of two markers with contrasted modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the phytophagous mite Tetranychus urticae Koch. The second internal transcribed spacer (ITS2) of rDNA and a fragment in the mtDNA gene coding for Cytochrome Oxidase I (COI), were PCR-amplified and sequenced in samples of various geographical origins distributed worldwide. The 15 COI haplotypes found fell into two major phylogenetic lineages differing by an average of 5% nucleotide divergence. Samples from the Mediterranean basin were represented in both lineages, and showed no phylogeographical structure. The other samples, from temperate regions of the northern hemisphere, were clustered in one of the lineages and displayed little variation, indicating a recent colonization of this region. In contrast, no variation at all was found at the ITS2 in this species. We sequenced both COI and ITS2 in four other species of the genus Tetranychus and found that, despite the absence of intraspecific polymorphism, ITS appears to evolve 2.5 times faster than COI. We argue that rDNA homogeneity over the species range of T. urticae results from the high colonization potential of this species, preventing long-term differentiation. Preliminary data on two other mite species (Amphitetranychus viennensis Zacher and Mononychellus progresivus Doreste) with stricter ecological requirements and more restricted colonization potential revealed substantial and concordant geographical differentiation for both ITS2 and COI

Evaluating the performance of available tools for building de novo transcriptome hybrid assemblies by combining reads of different length

One of the greatest avenues opened by next genera&on sequencing (NGS) technologies is the possibility to sequence on large scale species with no prior genomic informa&on, the so called non-model species, at reasonable cost. This has revolu&onized the way biologists approach ques&ons, allowing the implementa&on of experiments previously considered impossible. However, unlike model species, non-model species lack a reference genome or transcriptome assembly, which encouraged the community to develop various strategies to deal with the challenge of construc&ng a reference assembly de novo. Short reads oQen lead to the assembly of incomplete con&gs with low error rate, while longer reads improve greatly the coverage of the sequenced transcripts but are error-prone. A generally recognized need is to increase the length of used reads, but current technologies cannot offer long reads without increasing the error rate at the same &me. Although the combina&on of short and long reads promises to bring together the advantages of both read types, i.e. long reads and low error rate, it is ques&onable whether this is applicable with the available tools. Here, we aim at comparing de novo assemblies incorpora&ng reads produced by different sequencing plaRorms, and in par&cular short reads from Illumina HiSeq2000 (100 bp PE), a bit longer reads produced by Illumina MiSeq (300bp PE), and long reads produced by the PacBio technology (1500 bp). We use as test case the transcriptome sequencing of the whitefly Bemisia tabbaci 1 and assess the efficiency of available soQware developed to combine reads with different lengths. Finally, we provide basic guidelines for improving the assemblies produced for non-model species and propose future direc&ons to deal with this challenge faced by most researchers of organismal biology