Development of 55 novel polymorphic microsatellite loci for the critically endangered L. (Actinopterygii: Perciformes: Percidae) and cross-species amplification in five other percids

International audienceBy combining biotin-enrichment protocol and next generation pyrosequencing, through 454 GS-FLX Titanium technology, 55 polymorphic microsatellites loci with perfect motif were isolated from the Rhone streber (), a critically endangered European fish species. Eight multiplex PCR kits were optimised in order to genotype a total of 58 polymorphic loci, including three previously published loci. The level of genetic diversity was assessed for 68 , 30 , 33 and four individuals. Amplification success was also assessed on and using single individuals. These markers will be useful to investigate the population structure of the highly fragmented Rhone streber. They represent a powerful tool for conservation issues and evolutionary approaches of this endemic species. Moreover, part of our markers demonstrated applicability to other percid species, allowing for potential applications to fisheries and aquaculture management

Cross-species amplification of 41 microsatellites in European cyprinids: A tool for evolutionary, population genetics and hybridization studies

<p>Abstract</p> <p>Background</p> <p>Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date.</p> <p>Findings</p> <p>Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from <it>Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma </it>and <it>Leuciscus leuciscus</it>) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species. The level of genetic diversity was assessed in <it>Alburnus alburnus, Alburnoides bipunctatus, C. genei, C. n. nasus, C. soetta, C. t. toxostoma, L. idus, L. leuciscus, Pachychilon pictum, Rutilus rutilus, Squalius cephalus </it>and <it>Telestes souffia</it>. The applicability of the markers was also tested on <it>Abramis brama, Blicca bjoerkna </it>and <it>Scardinius erythrophtalmus </it>specimens. Overall, between 24 and 37 of these markers revealed polymorphic for the investigated species and 23 markers amplified for all the 15 European cyprinid species.</p> <p>Conclusions</p> <p>The developed set of markers demonstrated its performance in discriminating European cyprinid species. Furthermore, it allowed detecting and characterizing hybrid individuals. These microsatellites will therefore be useful to perform comparative evolutionary and population genetics studies dealing with European cyprinids, what is of particular interest in conservation issues and constitutes a tool of choice to conduct hybridization studies.</p

From Late Miocene to Holocene: Processes of Differentiation within the Telestes Genus (Actinopterygii: Cyprinidae)

Investigating processes and timing of differentiation of organisms is critical in the understanding of the evolutionary mechanisms involved in microevolution, speciation, and macroevolution that generated the extant biodiversity. From this perspective, the Telestes genus is of special interest: the Telestes species have a wide distribution range across Europe (from the Danubian district to Mediterranean districts) and have not been prone to translocation. Molecular data (mtDNA: 1,232 bp including the entire Cyt b gene; nuclear genome: 11 microsatellites) were gathered from 34 populations of the Telestes genus, almost encompassing the entire geographic range. Using several phylogenetic and molecular dating methods interpreted in conjunction with paleoclimatic and geomorphologic evidence, we investigated the processes and timing of differentiation of the Telestes lineages. The observed genetic structure and diversity were largely congruent between mtDNA and microsatellites. The Messinian Salinity Crisis (Late Miocene) seems to have played a major role in the speciation processes of the genus. Focusing on T. souffia, a species occurring in the Danube and Rhone drainages, we were able to point out several specific events from the Pleistocene to the Holocene that have likely driven the differentiation and the historical demography of this taxon. This study provides support for an evolutionary history of dispersal and vicariance with unprecedented resolution for any freshwater fish in this region

Representativeness of microsatellite distributions in genomes, as revealed by 454 GS-FLX Titanium pyrosequencing

<p>Abstract</p> <p>Background</p> <p>Microsatellites are markers of choice in population genetics and genomics, as they provide useful insight into patterns and processes as diverse as genome evolutionary dynamics and demographic processes. The acquisition of microsatellites through multiplex-enriched libraries and 454 GS-FLX Titanium pyrosequencing is a promising new tool for the isolation of new markers in unknown genomes. This approach can also be used to evaluate the extent to which microsatellite-enriched libraries are representative of the genome from which they were isolated. In this study, we deciphered potential discrepancies in microsatellite content recovery for two reference genomes (<it>Apis mellifera </it>and <it>Danio rerio</it>), selected on the basis of their extreme heterogeneity in terms of the proportions and distributions of microsatellites on chromosomes.</p> <p>Results</p> <p>The <it>A. mellifera </it>genome, in particular, was found to be highly heterogeneous, due to extremely high rates of recombination, with hotspots, but the only bias consistently introduced into pyrosequenced multiplex-enriched libraries concerned sequence length, with the overrepresentation of sequences 160 to 320 bp in length. Other deviations from expected proportions or distributions of motifs on chromosomes were observed, but the significance and intensity of these deviations was mostly limited. Furthermore, no consistent adverse competition between multiplexed probes was observed during the motif enrichment phase.</p> <p>Conclusions</p> <p>This approach therefore appears to be a promising strategy for improving the development of microsatellites, as it introduces no major bias in terms of the proportions and distribution of microsatellites.</p

L'apron du Rhône (Zingel asper), un poisson emblématique: Répartition, prospection et dynamique de la population du Verdon

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Etude du régime alimentaire de l'apron du Rhône dans le Verdon

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VTAM: A robust pipeline for validating metabarcoding data using optimized parameters based on internal controls

Metabarcoding has become a powerful approach to study biodiversity from environmental samples but it is still prone to some pitfalls. Several papers have called for good practice in study design, data production and analyses to ensure repeatability and comparability between studies. Notably, the importance of mock community samples, negative controls, and replicates is frequently highlighted (Alberdi et al. 2018, O'Rourke et al. 2020). However, their use in bioinformatics pipelines is often limited to post hoc verification of expectations by the user. Indeed, one of the biggest challenges in metabarcoding analyses is to take into account the trade-off between false positive (FP) and false negative (FN) occurrences. We thus developed the VTAM (Validation and Taxonomic Assignation of Metabarcoding data) pipeline, which is the first tool to use explicitly the negative control and mock samples to find optimal parameters to minimize false positive and negative occurrences. In addition, VTAM addresses all known technical error types including tag-jumps, repeatability among replicates, and also it is able to integrate more than one overlapping markers to further minimize false negative occurrences.In order to evaluate VTAM, we compared it with two other pipelines: a pipeline based on DADA2 (Callahan et al. 2016) and LULU (Frøslev et al. 2017), and a pipeline based on OBITools3 (Boyer et al. 2016) and metabaR (Zinger et al. 2020). Two datasets from fish and bat diet studies were analysed with the three different pipelines. Based on mock and negative samples, we demonstrate that VTAM showed the best precision for mock samples in both datasets, while specificity in negative controls were comparable among the three pipelines (Fig. 1).VTAM therefore constitutes a complete pipeline to filter and validate metabarcoding data, from raw FASTQ data to Amplicon Sequence Variant tables with taxonomic assignments. Our pipeline aggregates a series of features rarely grouped in a single pipeline and performs a non-arbitrary parameter optimization based on internal control samples to generate conservative but informative metabarcoding datasets. We believe VTAM provides a very valuable tool for the validation of metabarcoding data, which is essential for conducting robust analyses of biodiversity

Two multiplexed sets of 21 and 18 microsatellites for Phoxinus phoxinus (L.) and Gobio gobio (L.) developed by cross-species amplification

International audienceA total of 68 cyprinid microsatellites were screened for cross-species amplification in order to develop three PCR multiplex kits for the Eurasian minnow (Phoxinus phoxinus L.) and the gudgeon (Gobio gobio L.). The multiplex kits cluster 21 and 18 microsatellites for P. phoxinus and G. gobio, respectively. All loci were evaluated using ~80 individuals sampled from three distinct populations. By multiplexing the markers, we aim to decrease genotyping costs and increase time efficiency. These two microsatellite sets represent a valuable tool for ecological, evolutionary, conservation and management issues