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19 Pág., Microgeographical adaptation occurs when the effects of directional selection persist despite gene flow. Traits and genetic loci under selection can then show adaptive divergence, against the backdrop of little differentiation at other traits or loci. How common such events are and how strong the selection is that underlies them remain open questions. Here, we discovered and analysed microgeographical patterns of genomic divergence in four European and Mediterranean conifers with widely differing life-history traits and ecological requirements (Abies alba MIll., Cedrus atlantica [Endl.] Manetti, Pinus halepensis Mill. and Pinus pinaster Aiton) by screening pairs from geographically close forest stands sampled along steep ecological gradients. We inferred patterns of genomic divergence by applying a combination of divergence outlier detection methods, demographic modelling, Approximate Bayesian Computation inferences and genomic annotation to genomic data. Surprisingly for such small geographical scales, we showed that selection is strong in all species but generally affects different loci in each. A clear signature of selection was systematically detected on a fraction of the genome, of the order of 0.1%-1% of the loci depending on the species. The novel modelling method we designed for estimating selection coefficients showed that the microgeographical selection coefficient scaled by population size (Ns) was 2-30. Our results convincingly suggest that selection maintains within-population diversity at microgeographical scales in spatially heterogeneous environments. Such genetic diversity is likely to be a major reservoir of adaptive potential, helping populations to adapt under fluctuating environmental conditions., Agence Nationale de la Recherche, Grant/ Award Number: FLAG project (ANR-12- ADAP-0007-01); European Commission, Grant/Award Number: TOPWOOD project (MSCA-RISE-2014-645654- TOPWOOD) and grant agreement No. 676876 (GenTree); Ministère de l'Écologie, du Développement Durable et de l'Énergie, Grant/Award Number: GRAAL project (10-MCGOT-GICC-8-CVS-139)

High genetic variation and extensive gene flow may help forest trees with adapting to ongoing climate change, yet the genetic bases underlying their adaptive potential remain largely unknown. We investigated range-wide patterns of potentially adaptive genetic variation in 64 populations of European beech (Fagus sylvatica L.) using 270 SNPs from 139 candidate genes involved either in phenology or in stress responses. We inferred neutral genetic structure and processes (drift and gene flow) and performed differentiation outlier analyses and gene-environment association (GEA) analyses to detect signatures of divergent selection.Beech range-wide genetic structure was consistent with the species’ previously identified postglacial expansion scenario and recolonization routes. Populations showed high diversity and low differentiation along the major expansion routes. A total of 52 loci were found to be putatively under selection and 15 of them turned up in multiple GEA analyses. Temperature and precipitation related variables were equally represented in significant genotype-climate associations. Signatures of divergent selection were detected in the same proportion for stress response and phenology-related genes. The range-wide adaptive genetic structure of beech appears highly integrated, suggesting a balanced contribution of phenology and stress-related genes to local adaptation, and of temperature and precipitation regimes to genetic clines. Our results imply a best-case scenario for the maintenance of high genetic diversity during range shifts in beech (and putatively other forest trees) with a combination of gene flow maintaining within-population neutral diversity and selection maintaining between-population adaptive differentiation.

Drought and waterlogging impede tree growth and may even lead to tree death. Oaks, an emblematic group of tree species, have evolved a range of adaptations to cope with these constraints. The two most widely distributed European species, pedunculate (PO; Quercus robur L.) and sessile oak (SO; Quercus petraea Matt. Lieb), have overlapping ranges, but their respective distribution are highly constrained by local soil conditions. These contrasting ecological preferences between two closely related and frequently hybridizing species constitute a powerful model to explore the functional bases of the adaptive responses in oak. We exposed oak seedlings to waterlogging and drought, conditions typically encountered by the two species in their respective habitats, and studied changes in gene expression in roots using RNA-seq. We identified genes that change in expression between treatments differentially depending on species. These “species × environment”-responsive genes revealed adaptive molecular strategies involving adventitious and lateral root formation, aerenchyma formation in PO, and osmoregulation and ABA regulation in SO. With this experimental design, we also identified genes with different expression between species independently of water conditions imposed. Surprisingly, this category included genes with functions consistent with a role in intrinsic reproductive barriers. Finally, we compared our findings with those for a genome scan of species divergence and found that the expressional candidate genes included numerous highly differentiated genetic markers between the two species. By combining transcriptomic analysis, gene annotation, pathway analyses, as well as genome scan for genetic differentiation among species, we were able to highlight loci likely involved in adaptation of the two species to their respective ecological niches.

Research conductedWith the ongoing global warming, there are serious concerns about the persistence of locally adapted populations. Indeed, with the raising of temperature, the phenological cycle of tree species may be strongly affected since higher winter temperatures may have a negative impact on endodormancy release if chilling requirements are not fulfilled during winter and late frost in spring may expose trees if buds flush too early. Thus, Environmental gradients (showing continuous variations of environmental conditions) constitute a design of choice to analyze the effect of winter dormancy in locally adapted population.MethodsIn the present study, we used an elevation gradient in the Pyrenees to explore the gene expression network involved in dormancy regulation in natural populations of sessile oak locally adapted to temperature. Terminal buds were harvested during dormancy induction and release at different elevations. Then, gene expression was quantified using RNAseq and we used a likelihood ratio test to identify genes displaying significant dormancy, elevation or dormancy-by-elevation interaction effects.Key resultsOur results highlight molecular processes in locally adapted populations along this elevation cline, and made it possible to identify key dormancy-by-elevation responsive genes revealing that locally adapted populations have evolved distinct molecular strategies to adapt their bud phenology in response to environmental variation (i.e. temperature).

The pace of tree microevolution during Anthropocene warming is largely unknown. We used a retrospective approach to monitor genomic changes in oak trees since the Little Ice Age (LIA). Allelic frequency changes were assessed from whole-genome pooled sequences for four age-structured cohorts of sessile oak (Quercus petraea) dating back to 1680, in each of three different oak forests in France. The genetic covariances of allelic frequency changes increased between successive time periods, highlighting genome-wide effects of linked selection. We found imprints of convergent linked selection in the three forests during the late LIA, and a shift of selection during more recent time periods. The changes in allelic covariances within and between forests mirrored the documented changes in the occurrence of extreme events (droughts and frosts) over the last three hundred years. The genomic regions with the highest covariances were enriched in genes involved in plant responses to pathogens and abiotic stresses (temperature and drought). These responses are consistent with the reported sequence of frost (or drought) and disease damage ultimately leading to the oak dieback after extreme events. Our results therefore provide evidence of selection operating on long-lived species during recent climatic changes.

The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here, we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in

The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 Mb and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb of the genome in which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome- wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate.

International audience; Most existing forests are subjected to natural and human-mediated selection pressures, which have increased due to climate change and the increasing needs of human societies for wood, fibre and fuel resources. It remains largely unknown how these pressures trigger evolutionary changes. We address this issue here for temperate European oaks (Quercus petraeaandQ. robur), which grow in mixed stands, under even-aged management regimes. We screened numerous functional traits for univariate selection gradients and for expected and observed genetic changes over two successive generations. In both species, growth, leaf morphology and physiology, and defence-related traits displayed significant selection gradients and predicted shifts, whereas phenology, water metabolism, structure and resilience-related traits did not. However, the direction of the selection response and the potential for adaptive evolution differed between the two species.Quercus petraeahad a much larger phenotypic and genetic variance of fitness thanQ. robur. This difference raises concerns about the adaptive response ofQ. roburto contemporary selection pressures. Our investigations suggest thatQ. roburwill probably decline steadily, particularly in mixed stands withQ. petraea, consistent with the contrasting demographic dynamics of the two species.

International audience; Isolating genetic markers is often costly and time-consuming for non-model fungal species. However, these markers are of primary importance to identify the origin of invasive species and to infer their reproductive mode and dispersal ability. We slightly modified a recent molecular method to quickly isolate and validate single-nucleotide polymorphism (SNP) markers, from a first Erysiphe alphitoides draft genome, one of the main causal agent of oak powdery mildew in Europe. Although the draft assembly was strongly fragmented (555,289 contigs), we successfully isolated 1700 SNPs from 75 single-copy genes conserved in most fungal genomes. Ninety percent of them allowed to clearly distinguish the two main Erysiphe species reported on European oaks: E. alphitoides and E. quercicola. Thirty-six SNPs, located in distinct genes, were then validated using a strategy of MassArray genotyping on 95 E. alphitoides isolates sampled in Europe. This genotyping showed that only monospore isolates had the expected haploid signature, whereas direct genotyping from field leaves showed signature of mixed infection. Considering haploid isolates, these markers led to the first results of population genetic diversity, and suggested that E. quercicola may have a more asexual reproduction than its sister species, E. alphitoides.

SummaryThe tree of life is highly reticulate, with the history of population divergence buried amongst phylogenies deriving from introgression and lineage sorting. In this study, we test the hypothesis that there are regions of the oak (Quercus, Fagaceae) genome that are broadly informative about phylogeny and investigate global patterns of oak diversity.We utilize fossil data and restriction-site associated DNA sequencing (RAD-seq) for 632 individuals representing ca. 250 oak species to infer a time-calibrated phylogeny of the world’s oaks. We use reversible-jump MCMC to reconstruct shifts in lineage diversification rates, accounting for among-clade sampling biases. We then map the > 20,000 RAD-seq loci back to a recently published oak genome and investigate genomic distribution of introgression and phylogenetic support across the phylogeny.Oak lineages have diversified among geographic regions, followed by ecological divergence within regions, in the Americas and Eurasia. Roughly 60% of oak diversity traces back to four clades that experienced increases in net diversification due to climatic transitions or ecological opportunity.The support we find for the phylogeny contrasts with high genomic heterogeneity in phylogenetic signal and introgression. Oaks are phylogenomic mosaics, and their diversity may in fact depend on the gene flow that shapes the oak genome.

International audience; Fungal communities associated with plants and soil influence plant fitness and ecosystem functioning. They are frequently studied by metabarcoding approaches targeting the ribosomal internal transcribed spacer (ITS), but there is no consensus concerning the most appropriate bioinformatic approach for the analysis of these data. We sequenced an artificial fungal community composed of 189 strains covering a wide range of Ascomycota and Basidiomycota, to compare the performance of 360 software and parameter combinations. The most sensitive approaches, based on the USEARCH and VSEARCH clustering algorithms, detected almost all fungal strains but greatly overestimated the total number of strains. By contrast, approaches using DADA2 to detect amplicon sequence variants were the most effective for recovering the richness and composition of the fungal community. Our results suggest that analyzing single forward (R1) sequences with DADA2 and no filter other than the removal of low-quality and chimeric sequences is a good option for fungal community characterization. (C) 2019 Elsevier Ltd and British Mycological Society. All rights reserved.

GnpIS is an information system designed to integrate and link genomic, genetic and environmental data into a single environment dedicated to plant (crops and forest trees) and fungi data. GnpIS is regularly improved with new functionalities answering specific needs raised by scientists and released several times a year. We propose to illustrate the integrated genome annotation system we set up with a focus on the interoperability between genomic and genetic data (e.g. Markers, QTL) present in GnpIS-core, through the use case Quercus robur (the pedunculate oak), a large, complex and highly heterozygous genome.This genome annotation system relies on GMOD interfaces such as WebApollo/JBrowse and Intermine to make these data available under a user-friendly environment. All annotations and analysis results (Transposable Elements (TEs), genes, ncRNA ...) and functional annotation (protein-coding genes) were obtained using powerful and robust pipelines: (i) REPET used to detect, classify and annotate TEs representing 50% of the genome; (ii) Eugene which integrates ab initio and similarity gene finding softwares to predict gene models; (iii) ncRNA were annotated using different tools to annotate lncRNA, miRNA, rRNA, tRNA (iv) A functional annotation pipeline mainly based on Interproscan and comparative genomics was performed on the 25,808 highly confident predicted proteins. This system allows experts to analyze their protein families of interest and curate/validate gene structure.All together these resources provide a framework to study the two key evolutionary processes that explain the remarkable diversity found within the Quercus genus: local adaptation and speciation.

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.

Background Meiotic recombination is a fundamental evolutionary process. It not only generates diversity, but influences the efficacy of natural selection and genome evolution. There can be significant heterogeneity in recombination rates within and between species, however this variation is not well understood outside of a few model taxa, particularly in forest trees. Eucalypts are forest trees of global economic importance, and dominate many Australian ecosystems. We studied recombination rate in Eucalyptus globulus using genetic linkage maps constructed in 10 unrelated individuals, and markers anchored to the Eucalyptus reference genome. This experimental design provided the replication to study whether recombination rate varied between individuals and chromosomes, and allowed us to study the genomic attributes and population genetic parameters correlated with this variation. Results Recombination rate varied significantly between individuals (range = 2.71 to 3.51 centimorgans/megabase [cM/Mb]), but was not significantly influenced by sex or cross type (F1 vs. F2). Significant differences in recombination rate between chromosomes were also evident (range = 1.98 to 3.81 cM/Mb), beyond those which were due to variation in chromosome size. Variation in chromosomal recombination rate was significantly correlated with gene density (r = 0.94), GC content (r = 0.90), and the number of tandem duplicated genes (r = −0.72) per chromosome. Notably, chromosome level recombination rate was also negatively correlated with the average genetic diversity across six species from an independent set of samples (r = −0.75). Conclusions The correlations with genomic attributes are consistent with findings in other taxa, however, the direction of the correlation between diversity and recombination rate is opposite to that commonly observed. We argue this is likely to reflect the interaction of selection and specific genome architecture of Eucalyptus. Interestingly, the differences amongst chromosomes in recombination rates appear stable across Eucalyptus species. Together with the strong correlations between recombination rate and features of the Eucalyptus reference genome, we maintain these findings provide further evidence for a broad conservation of genome architecture across the globally significant lineages of Eucalyptus.

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National audience; The large, complex and highly heterozygous genome of pedunculate oak (Quercus robur) was sequenced using a whole-genome shotgun approach [1] . Roche 454 GS-FLX sequence reads were assembled into contigs and combined with Illumina reads from paired-end, mate-pair libraries and true synthetic long reads to build a total of 8,827 scaffolds (1.46 Gb total size; N50=821 kb). Both haplotypes were merged into an haploid version and 12 pseudomolecules were established using a high-density linkage map [2] combined with a syntenome approach using the peach genome sequence.The structural (Transposable Elements (TEs), genes, ncRNA) and functional annotation of automatically predicted genes relies on powerful and robust pipelines: (i) REPET package [3] [4] was first used to de novo detect, classify and annotate TEs representing about 50% of the genome; (ii) Eugene was trained and launched to integrate ab initio and similarity gene finding software to finally predict 43,240 genes including 29,665 highly confident gene models; (iii) ncRNA were predicted using feelcn (lncRNA), similarities against databases and small RNAseq data analysis (miRNA), RNAmmer (rRNA), tRNAscan-SE (tRNA) and Infernal package (other non-coding RNA) (iv) A functional annotation pipeline mainly based on Interproscan to search for patterns/motifs and Blast based comparative genomics was launched onto the 43,240 predicted proteins. The assignation of a provisional definition for predicted protein according to the results of the most reliable tools and their occurrence in Oak annotation was produced (D. Goodstein method, personal communication). We will present here these pipelines and the results of this annotation.We also set up an integrated genome annotation system (dedicated to oak) based on GMOD web interfaces such as WebApollo/JBrowse and Intermine to make these data available under a user-friendly environment. This system allowed experts to analyze their respective protein families of interest and curate/validate gene structure. We will also present the interoperability between these genomic data and genetic data produced in Quercus (SNPs, linkage maps, QTLs) available in GnpIS [5] an information System for plants. All together these resources provide a framework to study the two key evolutionary processes that explain the remarkable diversity found within the Quercus genus: local adaptation and speciation.

International audience; This article documents the public availability of transcriptomic resources for (i) the Hazelnut tree (Corylus avellana L.) and (ii) the oriental rat flea and primary plague vector, Xenopsylla cheopis.

Testing how populations are locally adapted and predicting their response to their future environment is of key importance in view of climate change. Landscape genomics is a powerful approach to investigate genes and environmental factors involved in local adaptation. In a pooled amplicon sequencing approach of 94 genes in 71 populations, we tested if >3'500 single nucleotide polymorphisms (SNPs) in the three most common oak species in Switzerland (Quercus petraea, Q. pubescens, Q. robur) show an association with abiotic factors related to local topography, historical climate, and soil characteristics. In the analysis including all species, the most frequently associated environmental factors were those best describing the habitats of the species. In the species-specific analyses, the most important environmental factors and associated SNPs greatly differed among species. However, we identified one SNP and seven genes that were associated to the same environmental factor across all species. We finally used regressions of allele frequencies of the most strongly associated SNPs along environmental gradients to predict the risk of non-adaptedness (RONA), which represents the average change in allele frequency at climate-associated loci theoretically required to match future climatic conditions. RONA is considerable for some populations and species (up to 48% in single populations) and strongly differs among species. Given the long generation time of oaks, some of the required allele frequency changes might not be realistic to achieve based on standing genetic variation. Hence, future adaptedness requires gene flow or planting of individuals carrying beneficial alleles from habitats currently matching future climatic conditions. This article is protected by copyright. All rights reserved.

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Maritime pine provides essential ecosystem services in the south-western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three-generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene-based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies. © 2016 John Wiley & Sons Ltd.

International audience; Background Increasing our understanding of the genetic architecture of complex traits, through analyses of genotype-phenotype associations and of the genes/polymorphisms accounting for trait variation, is crucial, to improve the integration of molecular markers into forest tree breeding. In this study, two full-sib families and one breeding population of maritime pine were used to identify quantitative trait loci (QTLs) for height growth and stem straightness, through linkage analysis (LA) and linkage disequilibrium (LD) mapping approaches. Results The populations used for LA consisted of two unrelated three-generation full-sib families (n = 197 and n = 477). These populations were assessed for height growth or stem straightness and genotyped for 248 and 217 markers, respectively. The population used for LD mapping consisted of 661 founders of the first and second generations of the breeding program. This population was phenotyped for the same traits and genotyped for 2,498 single-nucleotide polymorphism (SNP) markers corresponding to 1,652 gene loci. The gene-based reference genetic map of maritime pine was used to localize and compare the QTLs detected by the two approaches, for both traits. LA identified three QTLs for stem straightness and two QTLs for height growth. The LD study yielded seven significant associations (P ≤ 0.001): four for stem straightness and three for height growth. No colocalisation was found between QTLs identified by LA and SNPs detected by LD mapping for the same trait. Conclusions This study provides the first comparison of LA and LD mapping approaches in maritime pine, highlighting the complementary nature of these two approaches for deciphering the genetic architecture of two mandatory traits of the breeding program.

International audience; Genomic resources have recently been developed for a number of species of Fagaceae, with the purpose of identifying the genetic factors underlying the adaptation of these long-lived, biologically predominant, commercially and ecologically important species to their environment. The sequencing of genomes of the size of the oak genome (740 Mb/C) is now becoming both possible and affordable due to breakthroughs in sequencing technology. However, an understanding of the composition and structure of the oak genome is required before launching a sequencing initiative. We constructed random (Rd) and hypomethylated (Hp) genomic libraries for pedunculate oak (Quercus robur) and carried out a sample sequencing of 2.33 and 2.36 Mb of shotgun DNA from the Rd and Hp libraries, respectively, to provide a first insight into the repetitive element and gene content of the oak genome. We found striking similarities between Rd sequences and previously analyzed BAC end sequences of pedunculate oak, with a similar percentage of known repeat elements (5.56%), an almost identical simple sequence repeat density (i.e., 29 SSRs per 100 kb), an identical profile of SSR motifs (in descending order of frequency—dinucleotide, pentanucleotide, trinucleotide, tetranucleotide, and hexanucleotide motifs). Conversely, the Hp fraction was, as expected, enriched in nuclear genes (2.44-fold enrichment). This enrichment was associated with a lower frequency of retrotransposons than for Rd sequences. We also identified twice as many SSR motifs in the Rd library as in the Hp library. This work provides useful information before opening a new chapter in oak genome sequencing.

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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.

Background Many northern-hemisphere forests are dominated by oaks. These species extend over diverse environmental conditions and are thus interesting models for studies of plant adaptation and speciation. The genomic toolbox is an important asset for exploring the functional variation associated with natural selection. Results The assembly of previously available and newly developed long and short sequence reads for two sympatric oak species, Quercus robur and Quercus petraea, generated a comprehensive catalog of transcripts for oak. The functional annotation of 91 k contigs demonstrated the presence of a large proportion of plant genes in this unigene set. Comparisons with SwissProt accessions and five plant gene models revealed orthologous relationships, making it possible to decipher the evolution of the oak genome. In particular, it was possible to align 9.5 thousand oak coding sequences with the equivalent sequences on peach chromosomes. Finally, RNA-seq data shed new light on the gene networks underlying vegetative bud dormancy release, a key stage in development allowing plants to adapt their phenology to the environment. Conclusion In addition to providing a vast array of expressed genes, this study generated essential information about oak genome evolution and the regulation of genes associated with vegetative bud phenology, an important adaptive traits in trees. This resource contributes to the annotation of the oak genome sequence and will provide support for forward genetics approaches aiming to link genotypes with adaptive phenotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1331-9) contains supplementary material, which is available to authorized users.

International audience; An Illumina Infinium SNP genotyping array was constructed for European white oaks. Six individuals of Quercus petraea and Q. robur were considered for SNP discovery using both previously obtained Sanger sequences across 676 gene regions (1371 in vitro SNPs) and Roche 454 technology sequences from 5112 contigs (6542 putative in silico SNPs). The 7913 SNPs were genotyped across the six parental individuals, full-sib progenies (one within each species and two interspecific crosses between Q. petraea and Q. robur) and three natural populations from south-western France that included two additional interfertile white oak species (Q. pubescens and Q. pyrenaica). The genotyping success rate in mapping populations was 80.4% overall and 72.4% for polymorphic SNPs. In natural populations, these figures were lower (54.8% and 51.9%, respectively). Illumina genotype clusters with compression (shift of clusters on the normalized x-axis) were detected in ~25% of the successfully genotyped SNPs and may be due to the presence of paralogues. Compressed clusters were significantly more frequent for SNPs showing a priori incorrect Illumina genotypes, suggesting that they should be considered with caution or discarded. Altogether, these results show a high experimental error rate for the Infinium array (between 15% and 20% of SNPs potentially unreliable and 10% when excluding all compressed clusters), and recommendations are proposed when applying this type of high-throughput technique. Finally, results on diversity levels and shared polymorphisms across targeted white oaks and more distant species of the Quercus genus are discussed, and perspectives for future comparative studies are proposed.

To help define the pathologies associated with yeast cells as they age, we analyzed the transcriptome of young and old cells isolated by elutriation, which allows isolation of biochemical quantities of old cells much further advanced in their life span than old cells prepared by the biotin-streptavidin method. Both 18-generation-old wild-type yeast and 8-generation-old cells from a prematurely aging mutant (dna2-1), with a defect in DNA replication, were evaluated. Genes involved in gluconeogenesis, the glyoxylate cycle, lipid metabolism, and glycogen production are induced in old cells, signifying a shift toward energy storage. We observed a much more extensive generalized stress response known as the environmental stress response (ESR), than observed previously in biotin-streptavidin-isolated cells, perhaps because the elutriated cells were further advanced in their life span. In addition, there was induction of DNA repair genes that fall in the so-called DNA damage “signature” set. In the dna2-1 mutant, energy production genes were also induced. The response in the dna2-1 strain is similar to the telomerase delete response, genes whose expression changes during cellular senescence in telomerase-deficient cells. We propose that these results suggest, albeit indirectly, that old cells are responding to genome instability.

A total of 1113 oak trees from 222 populations originating from eight countries (Austria, Bosnia-Herzegovina, Croatia, Hungary, Romania, Slovakia, Slovenia, Yugoslavia) were sampled in natural populations or in provenance tests. The sampled trees belong to four different species (Quercus robur, Quercus petraea, Quercus pubescens, Quercus frainetto) and to several putative subspecies. Variation at four chloroplast DNA (cpDNA) fragments was studied using restriction enzymes, resulting in the detection of 12 haplotypes. One haplotype was present in 36% of the trees, and six were found in 6–17% of the trees. The haplotypes are shared extensively between species and subspecies. They belong to three different lineages (A, C and E) and are phylogeographically structured in the region investigated. Haplotypes of lineage E dominate to the east of the Carpathian mountains in Romania, whereas the Carpathian Basin seems to have been colonised along several different colonisation routes, from the Balkan peninsula but also from Italy. The data support the possible role of climatic instability during the late glacial period in shaping this complex geographic structure. The presence of several secondary refugia could be inferred in the region, which have played a major role in the second step of recolonisation, at the onset of the Holocene period. # 2002 Elsevier Science B.V. All rights reserved.

International audience; Systematic sequencing is the method of choice for generating genomic resources for molecular marker development and candidate gene identification in non-model species. We generated 47,357 Sanger ESTs and 2.2M Roche-454 reads from five cDNA libraries for European beech (Fagus sylvatica L.). This tree species of high ecological and economic value in Europe is among the most representative trees of deciduous broadleaf forests. The sequences generated were assembled into 21,057 contigs with MIRA software. Functional annotations were obtained for 85% of these contigs, from the proteomes of four plant species, Swissprot accessions and the Gene Ontology database. We were able to identify 28,079 in silico SNPs for future marker development. Moreover, RNAseq and qPCR approaches identified genes and gene networks regulated differentially between two critical phenological stages preceding vegetative bud burst (the quiescent and swelling buds stages). According to climatic model based projection, some European beech populations may be endangered, particularly at the southern and eastern edges of the European distribution range, which are strongly affected by current climate change. This first genomic resource for the genus Fagus should facilitate the identification of key genes for beech adaptation and management strategies for preserving beech adaptability.

The accessibility of high-throughput genotyping technologies has contributed greatly to the development of genomic resources in non-model organisms. High-density genotyping arrays have only recently been developed for some economically important species such as conifers. The potential for using genomic technologies in association mapping and breeding depends largely on the genome wide patterns of diversity and linkage disequilibrium in current breeding populations. This study aims to deepen our knowledge regarding these issues in maritime pine, the first species used for reforestation in south western Europe. Using a new map merging algorithm, we first established a 1,712 cM composite linkage map (comprising 1,838 SNP markers in 12 linkage groups) by bringing together three already available genetic maps. Using rigorous statistical testing based on kernel density estimation and resampling we identified cold and hot spots of recombination. In parallel, 186 unrelated trees of a mass-selected population were genotyped using a 12k-SNP array. A total of 2,600 informative SNPs allowed to describe historical recombination, genetic diversity and genetic structure of this recently domesticated breeding pool that forms the basis of much of the current and future breeding of this species. We observe very low levels of population genetic structure and find no evidence that artificial selection has caused a reduction in genetic diversity. By combining these two pieces of information, we provided the map position of 1,671 SNPs corresponding to 1,192 different loci. This made it possible to analyze the spatial pattern of genetic diversity (H e ) and long distance linkage disequilibrium (LD) along the chromosomes. We found no particular pattern in the empirical variogram of H e across the 12 linkage groups and, as expected for an outcrossing species with large effective population size, we observed an almost complete lack of long distance LD. These results are a stepping stone for the development of strategies for studies in population genomics, association mapping and genomic prediction in this economical and ecologically important forest tree species.

Article fédérateur entre toutes les équipes Européennes travaillant sur le pin maritime. Assemblage du transcriptome des données venant de France (INRA Orléans + INRA Pierroton) + Espagne + Portugal = projet Sustainpine; International audience; Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine 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.

Waterlogging causes stressful conditions for perennial species. The temporary overabundance of water in waterlogged soil can induce hypoxia in the rhizosphere, leading to root death, tree decline and even dieback. Two closely related members of the European white oak complex, pedunculate (Quercus robur L.) and sessile (Quercus petraea Matt. Liebl.) oaks, have different ecological characteristics, especially regarding their adaptation to soil waterlogging. The tolerance of waterlogging observed in pedunculate oak is driven principally by its ability to produce adaptive structures, hypertrophied lenticels and adventitious roots, and to switch rapidly its metabolism to the fermentative pathway. This study had two objectives: (i) to identify genes important for adaptation to waterlogging and (ii) to gain insight into the molecular mechanisms involved in hypertrophied lenticel formation in pedunculate oak. We subjected seedlings of the two species to hypoxia by maintaining the water level 2 cm above the collar. The immersed part of the stem (i.e., containing hypertrophied lenticels in pedunculate oak) was sampled after 9 days of waterlogging stress and its gene expression was investigated by RNA-seq. Genes displaying differential expression between the two species were identified with the DESeq R package and a false discovery rate of 0.001. We found that 3705 contigs were differentially regulated between the two species. Twenty-two differentially expressed genes were validated by real-time quantitative polymerase chain reaction. The suberin biosynthesis pathway was found to be upregulated in pedunculate oak, consistent with molecular mechanisms analogous to those operating in the radial oxygen loss barrier in waterlogging-tolerant species.

International audience

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National audience

Single Nucleotide Polymorphisms (SNPs) are the most abundant form of genetic variation in the genome. In this poster, we reviewed what has been achieved in terms of highly-multiplexed SNP genotyping assay construction in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. Seven custom SNP-assays (384 and 1536-plex), oriented towards broad applications, have been designed. We illustrated here the usefulness of this genotyping technology to address specific questions related to i/ genetic diversity and population structure analysis, ii/ linkage and QTL mapping and iii/ association mapping. With respect to genetic diversity and differentiation, a custom VeraCode assay for 384 SNPs mostly based on a larger array designed for linkage mapping (see below) allowed to obtain less blurred, albeit similar, breeding zone boundaries than a set of 12 nuSSRs screened on the same individuals. Levels of diversity were also more accurately estimated, showing clear differences among gene pools. Interestingly, a relatively small subset of SNPs would be enough to develop an application tool for origin certification, which could have a notable impact on current operational practices. In terms of linkage mapping, a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (in vitro SNPs) and from Sanger-derived Expressed Sequenced Tags (in silico SNPs) was established. Offspring from two mapping pedigrees were genotyped. A consensus map comprising 357 SNPs from 292 different loci was constructed and the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. Moreover, QTL detection for different traits is underway. In terms of association mapping, a custom GoldenGate assay with 384 SNPs was built and used to genotype 160 unrelated plus-trees from a half-sib experimental design for which breeding values (for height growth, circumference, stem straightness at 8 years, lignin content and extractives at 31 years) were available. Taking into account multiple testing, one single SNP in a gene encoding a putative fasciclin-like arabinogalactan protein was found to be associated with growth traits. We conclude that the VeraCode/GoldenGate assays can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This first generation of SNP-arrays has been recently upgraded to an Infinium-array (containing 10.5k SNPs) thanks to deep sequencing based on new generation sequencing technologies. The Infinium-array also includes SNPs from comparative orthologous sequences with other major conifer species, providing a wider collection of anchor points for comparative genomics among these major groups of forest trees.