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Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (

Background Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients. Methods MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2’deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34+ cells with lentiviral vectors encoding the pri-miR-10a precursor. Results MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors. Conclusions MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion. Electronic supplementary material The online version of this article (10.1186/s12885-018-4993-2) contains supplementary material, which is available to authorized users.

De novo OTU picking from large metabarcoding read datasets is at the same time a current and a complex task, and several methods coexist to perform it. We present here the outcome of a collective project developed within Working Group on « Data Analysis and Storage » in DNAqua.net. Our aim has been to organize a thorough comparison of OTU composition according to some selected methods called by the wrappers, in a diversity of situations. This has been done by disposing of a set of different datasets, and a set of different methods, applying each method on each dataset, and comparing the results. We have deliberately chosen to work with cleaned datasets only, and not to include cleaning in the process. We have worked with a set of about 60 different datasets, some environmental, some as mock communities, produced by six teams, in different countries (D, F, I, T, UK), each with specific markers for different organisms. All datasets have been cleaned beforehand by the team proposing it. We have installed four different tools for building OTUs by unsupervised clustering : Swarm (Mahé et al. 2015), Vsearch (Rognes et al. 2016) with the same receipe for all datasets, usearch (Edgar 2010) with a unique command, the same for all datasets, and yapotu, which computes pairwise Smith-Waterman distances between all reads of a given dataset, and then clusters them with graph based techniques. Yapotu approach is expected to be the most accurate one, as there are no heuristics in the calculations. We have harmonized common input/output format for the four methods, to make comparisons. Here is a summary of the indicators selected for comparing results. We have first computed basic indicators per sample and method, like the number of OTU, the number of singletons, the number of OTUs with ten reads or more (after dereplication), and the fraction of reads that have been allocated to an OTU. The four methods displayed a great variety of counts, with highest number of OTUs and singletons for Swarm, then slighltly equivalent figures (but a smaller number of singletons) for yapotu, and significantly smaller counts for Vsearch and Usearch. However, the counts for the number of OTUs with 10 reads or more are much more convergent between the four methods. We have then compared rank-size curves, which have been computed for all pairs (sample by method). Here again, yapotu and swarm results are very similar, whereas Vsearch and Usearch sometimes are close to the former pattern, sometimes very different (I attach a figure?) We then have computed 10 different diversity indices, like OTU richness, Shannon, Chao, eveness. Here again, results provided by Swarm and Yapotu are very similar, with very strong correlations between indices over all samples by method, whereas the correlations with Vsearch and Usearch is very poor. Finally, we have computed all contingency tables (in a sparse format) between all pairs of methods (hence, 6 pairs) for all samples, which accurately describe whether OTUs composition are similar or dissimilar between methods. We have observed that swarm OTUs are systematically nested within yapotu OTUs, and most often, there is a one to one correspondence between a Swarm and a Yapotu OTU. As a conclusion, we show that - Swarm and yapotu yield very similar results including for fine details, the only diffrence being a larger number of singletons provided by Swarm ; - This shows that Swarm OTUs are very close to OTUs built by single linkage Clustering on Smith-Waterman pairwise distances, and consolidates these approaches. - Very often, both Vsearch and Usearch diverge from those convergent results, but not always, and it is not easy to understand when and why. Some further investigations are needed therefore. All datasets will be publicly available for further benchmarking of a wider set of methods and datasets.

One of the approaches to cure HIV is the use of therapeutic vaccination. We have launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles in aviremic patients under antiretroviral therapy (ART). A HIV-1 polypeptidic therapeutic vaccine based on viral sequence data obtained from circulating blood was proposed; here, our aim was to compare the proviral DNA in blood and gut-associated lymphoid tissue (GALT) at two different levels : nucleotide sequences and potential CTL epitopes. The reverse transcriptase was sequenced in both compartments using next generation sequencing (NGS) in samples from nine individuals, two of which had also single genome sequencing (SGS) performed; phylogenetic trees were established and compared; CTL epitopes were also identified according to their potential affinity for the HLA alleles. The proviral sequences of both compartments intra-patient exhibited a very low genetic divergence while it was possible to differentiate the sequences inter-patients; SGS analysis of two couples of samples confirmed that there was not a compartmentalization of the sequences intra-patient.When we simulated the CTL epitopes which can be presented by the corresponding HLA alleles in both compartments, no significant difference was observed. We conclude that the proviral DNA sequences in blood and GALT are similar and that the epitope analysis in blood can be considered as relevant to that observed in the GALT, a hard-to-reach major compartment, and can therefore be used for therapeutic vaccine approaches.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast,de novo ad hocprimer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20 to 40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence, quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

Starmerella bacillaris is an ascomycetous yeast ubiquitously present in grapes and fermenting grape musts. In this report, we present the draft genome sequence of the S. bacillaris type strain CBS 9494, isolated from sweet botrytized wines, which will contribute to the study of this genetically heterogeneous wine yeast species.

Volume 7, no. 3, e00872-18, 2018, [https://doi.org/10.1128/MRA.00872-18][1]. Page 1: The article title should read as given above. Page 1, line 1: “ Starmerella bacillaris (syn., Candida zemplinina )” should read “ Candida zemplinina (syn., Starmerella bacillaris ).” Pages 1 and 2: “ S.

Starmerella bacillaris is an ascomycetous yeast ubiquitously present in grapes and fermenting grape musts. In this report, we present the draft genome sequence of the S. bacillaris type strain CBS 9494, isolated from sweet botrytized wines, which will contribute to the study of this genetically heterogeneous wine yeast species.

International audience; Cacao (Theobroma cacao L.) is an important economic crop, yet studies of its domestication history and early uses are limited. Traditionally, cacao is thought to have been first domesticated in Mesoamerica. However, genomic research shows that T. cacao’s greatest diversity is in the upper Amazon region of northwest South America, pointing to this region as its centre of origin. Here, we report cacao use identified by three independent lines of archaeological evidence—cacao starch grains, absorbed theobromine residues and ancient DNA—dating from approximately 5,300 years ago recovered from the Santa Ana-La Florida (SALF) site in southeast Ecuador. To our knowledge, these findings constitute the earliest evidence of T. cacao use in the Americas and the first unequivocal archaeological example of its pre-Columbian use in South America. They also reveal the upper Amazon region as the oldest centre of cacao domestication yet identified.

MicroRNAs are key factors in the regulation of gene expression and their deregulation has been directly linked to various pathologies such as cancer. The use of small molecules to tackle the overexpression of oncogenic miRNAs has proved its efficacy and holds the promise for therapeutic applications. Here we describe the screening of a 640-compound library and the identification of polyamine derivatives interfering with in vitro Dicer-mediated processing of the oncogenic miR-372 precursor (pre-miR-372). The most active inhibitor is a spermine-amidine conjugate that binds to the pre-miR-372 with a KD of 0.15 µM, and inhibits its in vitro processing with a IC50 of 1.06 µM. The inhibition of miR-372 biogenesis was confirmed in gastric cancer cells overexpressing miR-372 and a specific inhibition of proliferation through de-repression of the tumor suppressor LATS2 protein, a miR-372 target, was observed. This compound modifies the expression of a small set of miRNAs and its selective biological activity has been confirmed in patient-derived ex vivo cultures of gastric carcinoma. Polyamine derivatives are promising starting materials for future studies about the inhibition of oncogenic miRNAs and, to the best of our knowledge, this is the first report about the application of functionalized polyamines as miRNAs interfering agents.

Understanding the mechanisms behind the typicity of regional wines inevitably brings attention to microorganisms associated with their production. Oenococcus oeni is the main bacterial species involved in wine and cider making. It develops after the yeast-driven alcoholic fermentation and performs the malolactic fermentation, which improves the taste and aromatic complexity of most wines. Here, we have evaluated the diversity and specificity of O. oeni strains in six regions. A total of 235 wines and ciders were collected during spontaneous malolactic fermentations and used to isolate 3,212 bacterial colonies. They were typed by multilocus variable analysis, which disclosed a total of 514 O. oeni strains. Their phylogenetic relationships were evaluated by a second typing method based on single nucleotide polymorphism (SNP) analysis. Taken together, the results indicate that each region holds a high diversity of strains that constitute a unique population. However, strains present in each region belong to diverse phylogenetic groups, and the same groups can be detected in different regions, indicating that strains are not genetically adapted to regions. In contrast, greater strain identity was seen for cider, white wine, or red wine of Burgundy, suggesting that genetic adaptation to these products occurred. IMPORTANCE This study reports the isolation, genotyping, and geographic distribution analysis of the largest collection of O. oeni strains performed to date. It reveals that there is very high diversity of strains in each region, the majority of them being detected in a single region. The study also reports the development of an SNP genotyping method that is useful for analyzing the distribution of O. oeni phylogroups. The results show that strains are not genetically adapted to regions but to specific types of wines. They reveal new phylogroups of strains, particularly two phylogroups associated with white wines and red wines of Burgundy. Taken together, the results shed light on the diversity and specificity of wild strains of O. oeni , which is crucial for understanding their real contribution to the unique properties of wines.

Introduction Les plaquettes jouent un role essentiel dans l’hemostase et la thrombose. Malgre la mise en œuvre de la leucoreduction, les effets indesirables receveurs (EIR) n’ont pas disparu. Le but de ce travail est de caracteriser l’ensemble des modifications du transcriptome plaquettaire dans les melanges de concentres plaquettaires (MCP) impliques dans un EIR. Methodes Une analyse transcriptomique des plaquettes a ete realisee dans 6 MCP associes a l’apparition d’un EIR versus 6 temoins apparies. Le sequencage a ete realise avec la technologie Ion-Proton®, l’alignement des reads avec le logiciel STAR sur le genome de reference hg19, la quantification des genes a l’aide de l’algorithme EM du logiciel Partek® Flow. Les packages R (DESeq2, Edge R, Limma voom) ont ete utilises pour les analyses statistiques. Les proteines codant pour les genes differentiellement exprimees (DE) qui impactent sur les processus biologiques (BP) et sur les voies de signalisation ont ete etudiees via une analyse d’interaction gene-gene (String db) et une analyse fonctionnelle (PANTHER). Resultats Soixante dix neuf genes significativement DE (p > 0,05) ont ete identifies. Un reseau constitue de 15 genes fortement connectes a ete mis en evidence, avec SPTBN1 comme gene hub. L’analyse fonctionnelle revele, entre autre, un enrichissement de la voie de transduction du signal impliquant RHOC et ARHGAP30 (retrouves en interaction dans String db). Conclusion Des modifications profondes du transcriptome des MCP ont ete observees. Ces observations renforcent l’idee que l’apparition d’un EIR puisse etre associee a la modification de la structure plaquettaire et plus precisement par remodelage de son cytosquelette.

National audience

Considered as a sister species of Saccharomyces cerevisiae, S. uvarum is, to a lesser extent, an interesting species for fundamental and applied research studies. Despite its potential interest as a new gene pool for fermenting agents, the intraspecific molecular genetic diversity of this species is still poorly investigated. In this study, we report the use of nine microsatellite markers to describe S. uvarum genetic diversity and population structure among 108 isolates from various geographical and substrate origins (wine, cider and natural sources). Our combined microsatellite markers set allowed differentiating 89 genotypes. In contrast to S. cerevisiae genetic diversity, wild and human origin isolates were intertwined. A total of 75% of strains were proven to be homozygotes and estimated heterozygosity suggests a selfing rate above 0.95 for the different population tested here. From this point of view, the S. uvarum life cycle appears to be more closely related to S. paradoxus or S. cerevisiae of natural resources than S. cerevisiae wine isolates. Population structure could not be correlated to distinct geographic or technological origins, suggesting lower differentiation that may result from a large exchange between human and natural populations mediated by insects or human activities.

Three wine estates (designated A, B, and C) were sampled in Sauternes, a typical appellation of the Bordeaux wine area producing sweet white wine. From those wine estates, 551 yeast strains were collected between 2012 and 2014, added to 102 older strains from 1992 to 2011 from wine estate C. All the strains were analyzed through 15 microsatellite markers, resulting in 503 unique Saccharomyces cerevisiae genotypes, revealing high genetic diversity and a low presence of commercial yeast starters. Population analysis performed using F st genetic distance or ancestry profiles revealed that the two closest wine estates, B and C, which have juxtaposed vineyard plots and common seasonal staff, share more related isolates with each other than with wine estate A, indicating exchange between estates. The characterization of isolates collected 23 years ago at wine estate C in relation to recent isolates obtained at wine estate B revealed the long-term persistence of isolates. Last, during the 2014 harvest period, a temporal succession of ancestral subpopulations related to the different batches associated with the selective picking of noble rotted grapes was highlighted. IMPORTANCE High genetic diversity of S. cerevisiae isolates from spontaneous fermentation on wine estates in the Sauternes appellation of Bordeaux was revealed. Only 7% of all Sauternes strains were considered genetically related to specific commercial strains. The long-term persistence (over 20 years) of S. cerevisiae profiles on a given wine estate is highlighted.

A large quantity of high quality RNA is often required in the analysis of gene expression. However, RNA extraction from samples taken from woody plants is generally complex, and represents the main limitation to study gene expression, particularly in refractory species like conifers. Standard RNA extraction protocols are available but they are highly time consuming, and not adapted to large scale extraction. Here we present a high-throughput RNA extraction protocol. This protocol was adapted to a micro-scale by modifying the classical cetyltrimethylammonium (CTAB) protocol developed for pine: (i) quantity of material used (100-200 mg of sample), (ii) disruption of samples in microtube using a mechanical tissue disrupter, and (iii) the use of SSTE buffer. One hundred samples of woody plant tissues/organs can be easily treated in two working days. An average of 15 \ig of high quality RNA per sample was obtained. The RNA extracted is suitable for applications such as real time reverse transcription polymerase chain reaction, cDNA library construction or synthesis of complex targets for microarray analysis.

International audience; The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with Saccharomyces cerevisiae has been extensively investigated these last few years, and several interesting features including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed 10 microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localization. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature were associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments.

International audience; The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

International audience; In order to determine the mechanical resistance of several forest tree species to rockfall, an inventory of the type of damage sustained in an active rockfall corridor was carried out in the French Alps. The diameter, spatial position and type of damage incurred were measured in 423 trees. Only 5% of trees had sustained damage above a height of 1.3 m and in damaged trees, 66% of broken or uprooted trees were conifers. Larger trees were more likely to be wounded or dead than smaller trees, although the size of the wounds was relatively smaller in larger trees. The species with the least proportion of damage through stem breakage, uprooting or wounding was European beech (Fagus sylvatica L.). Winching tests were carried out on two conifer species, Norway spruce (Picea abies L.) and Silver .r (Abies alba Mill.), as well as European beech, in order to verify the hypothesis that beech was highly resistant to rockfall and that conifers were more susceptible to uprooting or stem breakage. Nineteen trees were winched downhill and the force necessary to cause failure was measured. The energy (Efail) required to break or uproot a tree was then calculated. Most .r trees failed in the stem and spruce usually failed through uprooting. Beech was either uprooted or broke in the stem and was twice as resistant to failure as .r, and three times more resistant than spruce. Efail was strongly related to stem diameter in European beech only, and was signi.cantly higher in this species compared to Norway spruce. Results suggest that beech would be a better species to plant with regards to protection against rockfall. Nevertheless, all types of di.erent abiotic stresses on any particular alpine site should be considered by the forest manager, as planting only broadleaf species may compromise the protecting capacity of the forest e.g. in the case of snow avalanches. In order to determine the mechanical resistance of several forest tree species to rockfall, an inventory of the type of damage sustained in an active rockfall corridor was carried out in the French Alps. The diameter, spatial position and type of damage incurred were measured in 423 trees. Only 5% of trees had sustained damage above a height of 1.3 m and in damaged trees, 66% of broken or uprooted trees were conifers. Larger trees were more likely to be wounded or dead than smaller trees, although the size of the wounds was relatively smaller in larger trees. The species with the least proportion of damage through stem breakage, uprooting or wounding was European beech (Fagus sylvatica L.). Winching tests were carried out on two conifer species, Norway spruce (Picea abies L.) and Silver .r (Abies alba Mill.), as well as European beech, in order to verify the hypothesis that beech was highly resistant to rockfall and that conifers were more susceptible to uprooting or stem breakage. Nineteen trees were winched downhill and the force necessary to cause failure was measured. The energy (Efail) required to break or uproot a tree was then calculated. Most .r trees failed in the stem and spruce usually failed through uprooting. Beech was either uprooted or broke in the stem and was twice as resistant to failure as .r, and three times more resistant than spruce. Efail was strongly related to stem diameter in European beech only, and was signi.cantly higher in this species compared to Norway spruce. Results suggest that beech would be a better species to plant with regards to protection against rockfall. Nevertheless, all types of di.erent abiotic stresses on any particular alpine site should be considered by the forest manager, as planting only broadleaf species may compromise the protecting capacity of the forest e.g. in the case of snow avalanches.

Vetiver grass (Vetiveria zizanioides), also known as Chrysopogon zizanioides, is a graminaceous plant native to tropical and subtropical India. The southern cultivar is sterile; it flowers but sets no seeds. It is a densely tufted, perennial grass that is considered sterile outside its natural habitat. It grows 0.5–1.5 m high, stiff stems in large clumps from a much branched root stock. The roots of vetiver grass are fibrous and reported to reach depths up to 3 m thus being able to stabilize the soil and its use for this purpose is promoted by the World Bank. Uprooting tests were carried out on vetiver grass in Spain in order to ascertain the resistance the root system can provide when torrential runoffs and sediments are trying to uproot the plant. Uprooting resistance of each plant was correlated to the shoot and root morphological characteristics. In order to investigate any differences between root morphology of vetiver grass in its native habitat reported in the literature, and the one planted in a sub-humid environment in Spain, excavation techniques were used to show root distribution in the soil. Results show that vetiver grass possesses the root strength to withstand torrential runoff. Planted in rows along the contours, it may act as a barrier to the movement of both water and soil. However, the establishment of the vetiver lags behind the reported rates in its native tropical environment due to adverse climatic conditions in the Mediterranean. This arrested development is the main limitation to the use of vetiver in these environments although its root strength is more than sufficient.

International audience; Lignin is a major chemical compound of wood and one of the most abundant organic biopolymers on earth. It accumulates in the secondary cell wall of xylem cells and is a major target for tree breeders because of its foreseen role in the emerging bioeconomy. In this study, we paved the way toward an accelerated domestication of a widely grown tree species, Eucalyptus urophylla, by molecular breeding. To this end, we first described the pattern of nucleotide variation occurring at seven structural and regulatory genes of the lignin biosynthesis pathway and found high levels of average nucleotide and haplotype diversity per gene (pi = 0.0065 and Hd = 0.853). Then, taking advantage of a pre-existing factorial mating design, a candidate-gene-based quantitative trait locus (QTL) detection strategy was used to compare the variation of lignin quality (syringyl by guaiacyl ratio (S/G)) with the nucleotidic variability in these seven genes in 304 genotypes belonging to 33 connected full-sib families. Two genes, encoding cinnamoyl-CoA reductase (CCR) and a Rho-like GTPase (ROP1), were shown to be linked to the variation of S/G through different single and multi-locus single-nucleotide polymorphism (SNP)- and haplotype-based association methods. Providing that relevant candidate genes are selected and their patterns of nucleotide diversity is accurately described, we showed that quantitative trait nucleotides (QTNs) can be detected taking advantage of pre-existing field experiments and trait measurements gathered in the framework of a forest tree breeding program.

International audience; The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (~1900 years ago), and to the Neolithic era for bioprocesses (~4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.

International audience; Although many yeasts are useful for food production and beverage, some species may cause spoilage with important economic loss. This is the case of Dekkera/Brettanomyces bruxellensis, a contaminant species that is mainly associated with fermented beverages (wine, beer, cider and traditional drinks). To better control Brettanomyces spoilage, rapid and reliable genotyping methods are necessary to determine the origins of the spoilage, to assess the effectiveness of preventive treatments and to develop new control strategies. Despite several previously published typing methods, ranging from classical molecular methods (RAPD, AFLP, REA-PFGE, mtDNA restriction analysis) to more engineered technologies (infrared spectroscopy), there is still a lack of a rapid, reliable and universal genotyping approach. In this work, we developed eight polymorphic microsatellites markers for the Brettanomyces/Dekkera bruxellensis species. Microsatellite typing was applied to the genetic analysis of wine and beer isolates from Europe, Australia and South Africa. Our results suggest that B. bruxellensis is a highly disseminated species, with some strains isolated from different continents being closely related at the genetic level. We also focused on strains isolated from two Bordeaux wineries on different substrates (grapes, red wines) and for different vintages (over half a century). We showed that all B. bruxellensis strains within a cellar are strongly related at the genetic level, suggesting that one clonal population may cause spoilage over decades. The microsatellite tool now paves the way for future population genetics research of the B. bruxellensis species.

Background The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies. Results In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable. Conclusion This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

International audience; Microsatellites have been popular molecular markers ever since their advent in the late eighties. Despite growing competition from new genotyping and sequencing techniques, the use of these versatile and cost-effective markers continues to increase, boosted by successive technical advances. First, methods for multiplexing PCR have considerably improved over the last years, thereby decreasing genotyping costs and increasing throughput. Second, next-generation sequencing technologies allow the identification of large numbers of microsatellite loci at reduced cost in non-model species. As a consequence, more stringent selection of loci is possible, thereby further enhancing multiplex quality and efficiency. However, current practices are lagging behind. By surveying recently published population genetic studies relying on simple sequence repeats, we show that more than half of the studies lack appropriate quality controls and do not make use of multiplex PCR. To make the most of the latest technical developments, we outline the need for a well-established strategy including standardized high-throughput bench protocols and specific bioinformatic tools, from primer design to allele calling.

International audience; The bacterial communities associated with the cockle (Cerastoderma edule) were investigated at the individual level through a 10-month monitoring programme. Temporal changes and those changes associated with a common parasite of the cockle, Bucephalus minimus, were investigated by monthly sampling of individuals, selected based on their shell length (cohort monitoring). Cockle bacterial community abundance (CBCA) and diversity (CBCD) were estimated by epifluorescence microscopy counts and automated ribosomal intergenic spacer analysis, respectively. CBCA showed a temporal pattern peaking at 30 × 106 cells per gram of cockle flesh and intervalval liquid in October and a significant 1.8-fold increase linked with B. minimus occurrence. CBCD was characterized by 112 ± 26 intergenic transcribed spacer (ITS) per individual and showed a relative homology between individuals (52 ± 6%, Jaccard similarity) in spite of more than 30% of rare ITS. Consistent with an undisturbed evolution of the condition index of the studied cohort individuals as an estimate of their physiological state, neither temporal nor parasite-induced change in CBCA has been related to marked changes in CBCD.

Background Plants are subjected to continuous stimuli from the environment and have evolved an ability to respond through various growth and development processes. Phototropism and gravitropism responses enable the plant to reorient with regard to light and gravity. Results We quantified the speed of maritime pine seedlings to reorient with regard to light and gravity over 22 days. Seedlings were inclined at 15, 30 and 45 degrees with vertical plants as controls. A lateral light source illuminated the plants and stem movement over time was recorded. Depending on the initial angle of stem lean, the apical response to the lateral light source differed. In control and 15° inclined plants, the apex turned directly towards the light source after only 2 h. In plants inclined at 30° and 45°, the apex first reoriented in the vertical plane after 2 h, then turned towards the light source after 24 h. Two-dimensional gel electrophoresis coupled with mass spectrometry was then used to describe the molecular response of stem bending involved in photo- and gravi-tropism after 22 hr and 8 days of treatment. A total of 486 spots were quantitatively analyzed using image analysis software. Significant changes were determined in the protein accumulation of 68 protein spots. Early response gravitropic associated proteins were identified, which are known to function in energy related and primary metabolism. A group of thirty eight proteins were found to be involved in primary metabolism and energy related metabolic pathways. Degradation of Rubisco was implicated in some protein shifts. Conclusions Our study demonstrates a rapid gravitropic response in apices of maritime pine seedlings inclined >30°. Little or no response was observed at the stem bases of the same plants. The primary gravitropic response is concomitant with a modification of the proteome, consisting of an over accumulation of energy and metabolism associated proteins, which may allow the stem to reorient rapidly after bending.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Background Domestication and breeding involve the selection of particular phenotypes, limiting the genomic diversity of the population and creating a bottleneck. These effects can be precisely estimated when the location of domestication is established. Few analyses have focused on understanding the genetic consequences of domestication and breeding in fruit trees. In this study, we aimed to analyse genetic structure and changes in the diversity in sweet cherry Prunus avium L. Results Three subgroups were detected in sweet cherry, with one group of landraces genetically very close to the analysed wild cherry population. A limited number of SSR markers displayed deviations from the frequencies expected under neutrality. After the removal of these markers from the analysis, a very limited bottleneck was detected between wild cherries and sweet cherry landraces, with a much more pronounced bottleneck between sweet cherry landraces and modern sweet cherry varieties. The loss of diversity between wild cherries and sweet cherry landraces at the S-locus was more significant than that for microsatellites. Particularly high levels of differentiation were observed for some S-alleles. Conclusions Several domestication events may have happened in sweet cherry or/and intense gene flow from local wild cherry was probably maintained along the evolutionary history of the species. A marked bottleneck due to breeding was detected, with all markers, in the modern sweet cherry gene pool. The microsatellites did not detect the bottleneck due to domestication in the analysed sample. The vegetative propagation specific to some fruit trees may account for the differences in diversity observed at the S-locus. Our study provides insights into domestication events of cherry, however, requires confirmation on a larger sampling scheme for both sweet cherry landraces and wild cherry.

BackgroundThere is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size ( approximately 23.8 Gb/C).Methodology/principal findingsA 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates).Conclusions/significanceThis study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.

FR2116; International audience

Vetiver grass (Vetiveria zizanioides), also known as Chrysopogon zizanioides, is a graminaceous plant native to tropical and subtropical India. The southern cultivar is sterile; it flowers but sets no seeds. It is a densely tufted, perennial grass that is considered sterile outside its natural habitat. It grows 0.5‐1.5 m high, stiff stems in large clumps from a much branched root stock. The roots of vetiver grass are fibrous and reported to reach depths up to 3 m thus being able to stabilise the soil and its use for this purpose is promoted by the World Bank. Uprooting tests were carried out on vetiver grass in Spain in order to ascertain the resistance the root system can provide when torrential runoffs and sediments are trying to uproot the plant. Uprooting resistance of each plant was correlated to the shoot and root morphological characteristics. In order to investigate any differences between root morphology of vetiver grass in its native habitat reported in the literature, and the one planted in a sub-humid environment in Spain, excavation techniques were used to show root distribution in the soil. Results show that vetiver grass possesses the root strength to withstand torrential runoff. Planted in rows along the contours, it may act as a barrier to the movement of both water and soil. However, the establishment of the vetiver lags behind the reported rates in its native tropical environment due to adverse climatic conditions in the Mediterranean. This arrested development is the main limitation to the use of vetiver in these environments although its root strength is more than suffcient.

To determine which are the most important characters governing mechanical resistance to rockfall and wind loading, static winching tests were carried out on three tree species: Silver fir (Abies alba Mill.),European beech (Fagus sylvatica L.) and Norway spruce (Picea abies L.) in a mixed forest stand. Trees were winched to an angle of 0.25° at the stem base, both up- and downhill in order to compare how the same individual reacts when tested in two different directions. Trees were then winched to failure. Strain gauges were attached to the stem and one up- and downhill lateral root in order to determine the distribution of strain within the tree during overturning. Root morphology was then measured for all trees which uprooted during failure. No significant differences were found in the force necessary to winch trees up- and downhill in any species, either to an angle of 0.25° or to failure. Strain was significantly higher in lateral roots of Silver fir than in roots of Norway spruce and European beech when winched downhill. Downhill roots of Norway spruce were largely held in tension when trees were pulled downhill, whereas in Silver fir and European beech, they were held in compression. When trees were pulled uphill, no significant differences were found between species, and strain decreased along the lateral root of downhill roots only. European beech possessed a significantly greater number of roots than either Norway spruce or Silver fir. Norway spruce possessed a higher proportion of total root length near the soil surface, whereas European beech had the greatest proportion in the intermediate depth class and Silver fir had the highest maximal root depth. Norway spruce had a significantly lower proportion of oblique roots than the other two species, resulting in a plate-like root system which was less resistant to overturning than Silver fir or European beech.

Expression patterns of hundreds of transcripts in apical buds were monitored during bud flushing in sessile oak (Quercus petraea), in order to identify genes differentially expressed between the quiescent and active stage of bud development. Different transcriptomic techniques combining the construction of suppression subtractive hybridization (SSH) libraries and the monitoring of gene expression using macroarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were performed to dissect bud burst, with a special emphasis on the onset of the process. We generated 801 expressed sequence tags (ESTs) derived from six developmental stages of bud burst. Macroarray experiment revealed a total of 233 unique transcripts exhibiting differential expression during the process, and a putative function was assigned to 65% of them. Cell rescue/defense-, metabolism-, protein synthesis-, cell cycle- and transcription-related transcripts were among the most regulated genes. Macroarray and real-time RT-PCR showed that several genes exhibited contrasted expressions between quiescent and swelling buds, such as a putative homologue of the transcription factor DAG2 (Dof Affecting Germination 2), previously reported to be involved in the control of seed germination in Arabidopsis thaliana. These differentially expressed genes constitute relevant candidates for signaling pathway of bud burst in trees.

Growth, development and productivity of long-lived organisms such as forest trees are continuously challenged by abiotic stresses, and may also be greatly affected by predicted climatic change. As a first step toward creating stress-resistant maritime pine (Pinus pinaster Ait.) varieties by marker-assisted breeding, we describe the identification and characterization of water-stress-responsive genes in hydroponically grown seedlings that were well watered (-0.08 MPa) or subjected to water deprivation (-0.45 MPa) by the addition of polyethylene glycol. The cDNA amplified fragment-length polymorphism (cDNA-AFLP) technique was used to identify genes regulated by water deprivation. Approximately 4000 transcript-derived fragments (TDFs) were screened, of which 28 increased and 20 decreased in seedlings subjected to water deprivation. Of these 48 TDFs, 62.6% corresponded to proteins of known function, which indicate the main mechanisms involved in the osmotic stress response (photosynthesis, carbohydrate metabolism, cell wall synthesis and plant defense). We found that 16.6% of the 48 TDFs were similar to Arabidopsis thaliana (L.) Heynh gene products, 10.4% were similar to Pinus taeda L. expressed sequence tags (ESTs) and 10.4% did not match any sequences in the public databases. The relative abundance of these transcripts was quantitatively analyzed by reverse northern of both needle and root tissues, confirming the effectiveness of the cDNA-AFLP technique in detecting differentially expressed genes. The identification and characterization of water-stress-responsive genes provide new insights into the nature of the machinery involved in the response to water deprivation in a forest tree.

International audience

International audience; The trans-β-farnesene synthase has been purified from basal and middle parts of growing needles of maritime pine (Pinus pinaster Ait.). This enzyme catalyses the conversion of farnesyl diphosphate to trans-β-farnesene, an acyclic sesquiterpene hydrocarbon. Its acidic property facilited its isolation by ionexchange chromatography. Its purification was continued on hydroxylapatite column and gel permeation and was greatly improved by dye binding chromatography. SDS-gel electrophoresis revealed a single band in the range of 45 kD for this enzyme which has been 1,030-fold purified. Trans-β-farnesene synthase requires for divalent cations with Mg2+ preferred to Mn2+ and for thiol reagents. Effects of inhibitors were examined and the enzyme activity was strongly affected by p-hydroxymercuribenzoic acid. The Km for farnesyl diphosphate was in the range of 5 μM and the optimum pH of activity was between 7-7.3.

International audience; The effects of long-term water stress on water and terpene contents of the foliage of Cupressus sempervirens were studied. A great deal of water was lost over 2 months before a remarkable stabilization. A strong decrease of all the classes of terpenes accompanied this dehydration. Mono- and sesquiterpene hydrocarbons and free terpenols were almost entirely metabolized, whereas esters and terpene glycosides rose slightly and remained at a constant level when the water content had stabilized. Although a significant part of the mono- and sesquiterpene hydrocarbons was emitted in the early stage of stress application, the major part was used by the plant in response to the drought conditions.

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To test for different stem reorientation strategies in leaning seedlings of maritime pine (Pinus pinaster Ait.) and loblolly pine (Pinus taeda L.), we inclined dormant plants at 0°, 45° and 90° for 35 days in optimal growthconditions. Stem angle to the vertical was measured at regular intervals. After 22 and 35 days, bending tests were carried out to determine stem elasticity and stiffness. The quantity of compression wood (CW) formed was also measured. Results showed that after 24 h, the apices of leaning maritime pine stems had completely reoriented to the vertical, whereas no such primary tropic response was observed in loblolly pine. After 22 days, the significantly stiffer loblolly pines had begun straightening up from the stem base, but not maritime pine. After 35 days, the overall secondary reorientation process was more efficient in loblolly pine, with significantly more CW produced. In both species, CW had formed immediately on tilting stems, i.e. no early wood cells had formed. As maritime pine originates from a dry to semi-arid region, having less CW will mean that water uptake will be greater for the same xylem area compared to loblolly pine which possesses more CW. In establishing maritime pine seedlings, rapid apical orientation with regard to light and gravity will compensate for a slower straightening up mechanism in the more lignified parts of the stem. Therefore, juvenile needle exposure to patches of light will be maximised at all times.

International audience

Root tensile strength is an important factor to consider when choosing suitable species for reinforcing soil on unstable slopes. Tensile strength has been found to increase with decreasing root diameter, however, it is not known how this phenomenon occurs. We carried out tensile tests on roots 0.2¿12.0 mm in diameter of three conifer and two broadleaf species, in order to determine the relationship between tensile strength and diameter. Two species, Pinus pinaster Ait. and Castanea sativa Mill., were then chosen for a quantitative analysis of root cellulose content. Cellulose is responsible for tensile strength in wood due to its microfibrillar structure. Results showed that in all species, a significant power relationship existed between tensile strength and root diameter, with a sharp increase of tensile strength in roots with a diameter 1.0 mm, Fagus sylvatica L. was the most resistant to failure, followed by Picea abies L. and C. sativa., P. pinaster and Pinus nigra Arnold roots were the least resistant in tension for the same diameter class. Extremely high values of strength (132-201 MPa) were found in P. abies, C. sativa and P. pinaster, for the smallest roots (0.4 mm in diameter). The power relationship between tensile strength and root diameter cannot only be explained by a scaling effect typical of that found in fracture mechanics. Therefore, this relationship could be due to changes in cellulose content as the percentage of cellulose was also observed to increase with decreasing root diameter and increasing tensile strength in both P. pinaster and C. sativa.