In forest tree populations, the timing of budbreak (TBB) depends on several factors, both environmental and genetic. The genomic architecture underlying this trait is still not fully characterized. So far, common garden experiments have highlighted a few genomic regions with little heritability, while the whole spectrum of TBB variation observed in the wild still remains unexplored. We performed an in situ genome-wide association study (GWAS) to investigate the genetic and environmental bases of phenotypic variation in budbreak in two wild range-edge populations of the temperate continental tree, European beech (Fagus sylvatica L.) surveyed in two consecutive years. We found distinct significant loci between populations and among years, suggesting that local adaptation has occurred and that genotype-by-environment interactions contribute to the observed variation. The phenotypic variance explained by the significant loci is between 0.2% and 54% for field data and 0% to 59% for modelled data, which suggests that while some traits are polygenic, some others are influenced by a few loci with large effects in the population, which demonstrates the relevance of conducting in situ GWAS in multiple populations. Our findings provide novel insights into the complex genetic architecture of TBB in forest trees. The genetic diversity of budbreak control provides opportunities for selection that could be used through assisted migration to help prepare beech forests to face climate change.

Abstract Key message By monitoring a field experiment over nine years, we investigated the impacts of the two main pathogen species Erysiphe quercicola S. Takam. and U. Braun and Erysiphe alphitoides (Griffon and Maubl.) U. Braun and S. Takam causing powdery mildew on a young cohort of pedunculate oak (Quercus robur L.), both from a demographic and genetic point of view using SNP markers. We show that survival rate is affected by mean disease severity. But while the growth-related tolerance to infection of the oak individual seems to be more determinant than resistance against infection, no equalizing effect of the disease could be detected. Context Studies on the effects of pathogens on the survival and population dynamics of forest trees are scarce. Yet a better understanding of these interactions could prove strategic in the challenging context of climate change. Aims Our general objective was to characterize the demographic and genetic impact of the two main pathogen species Erysiphe quercicola S. Takam. and U. Braun and Erysiphe alphitoides (Griffon and Maubl.) U. Braun and S. Takam causing powdery mildew in the early stages of a Quercus robur L. population. Methods An ad hoc field design with two disease exposures, natural and protected, was surveyed over nine years. This enabled a detailed phenotypic monitoring of 1733 emerging individuals from 15 progenies, and the genotyping of 68% of them. Results The pathogen induced high levels of seedling mortality several years after sowing, associated with reduced growth and capacity to overwinter. Fast-growing families showed the highest survival rate under both natural and protected disease exposure. Contrary to a possible trade-off hypothesis between growth and defense, family height potential was not negatively related to disease resistance across the studied oak mother trees. While supporting a deleterious effect of very low individual heterozygosity on the probability of survival, average genomic diversity was not significantly affected by mortality associated with powdery mildew. Our study also points to a few candidate genes for several fitness-related traits. Conclusion Overall, our results suggest that in oak natural populations, infection levels (related to resistance sensu stricto) may be less determinant than growth-related tolerance to infection for the fate of seedlings. However, an equalizing effect of powdery mildew on relative oak genotype performances cannot be excluded at later stages.

The demographic and genetic impacts of powdery mildew on the early stages of an oak population were studied in an ad hoc field design with two disease exposures. This enabled a detailed phenotypic monitoring of 1,733 emerging individuals from 15 progenies over nine years, and the genotyping of 68% of them. The pathogen induced high levels of seedling mortality several years after sowing, associated with reduced growth and capacity to overwinter. The probability of juvenile survival could be predicted from mean disease severity in early years and acorn weight. Fast-growing families showed the highest survival rate under both natural and protected disease exposure. Correlatively, no equalizing effect of increased powdery mildew pressure on the relative contribution of mother trees to the next generation could be detected. Contrary to a possible trade-off hypothesis between growth and defense, family height potential was not negatively related to disease resistance across the studied oak mother trees. Overall, our results suggest that in Quercus robur natural populations, infection levels (related to resistance sensu stricto ) may be less determinant than growth-related tolerance to infection for the fate of seedlings. However, an equalizing effect of powdery mildew on relative oak genotype performances cannot be excluded at later stages since such an effect was already visible on height. Average genomic diversity was not significantly affected by mortality associated with powdery mildew. However, our study brings support to a deleterious effect of very low individual heterozygosity on the probability of survival across the different families. Finally, our study points to a few candidate genes for several fitness-related traits.

Oaks are dominant forest tree species widely distributed across the Northern Hemisphere, where they constitute natural resources of economic, ecological, social and historical value. Hybridisation and adaptive introgression have long been thought to be major drivers of their ecological success. Therefore, the maintenance of species barriers remains a key question, given the extent of interspecific gene flow. In this study, we made use of the tremendous genetic variation among four European white oak species (31 million single nucleotide polymorphisms (SNPs)) to infer the evolutionary history of these species, study patterns of genetic differentiation and identify reproductive barriers. We first analysed the ecological and historical relationships among these species and inferred a long-term strict isolation followed by a recent and extensive postglacial contact using approximate Bayesian computation. Assuming this demographic scenario, we then performed backward simulations to generate the expected distributions of differentiation under neutrality to scan their genomes for reproductive barriers. We finally identified important intrinsic and ecological functions driving the reproductive isolation. We discussed the importance of identifying the genetic basis for the ecological preferences between these oak species and its implications for the renewal of European forests under global warming.

Forest tree species are increasingly subject to severe mortalities from exotic pests, pathogens, and invasive organisms, accelerated by climate change. Such forest health issues are threatening multiple species and ecosystem sustainability globally. One of the most extreme examples of forest ecosystem disruption is the extirpation of the American chestnut ( Castanea dentata ) caused by the introduction of chestnut blight and root rot pathogens from Asia. Asian species of chestnut are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome for Chinese chestnut ( C. mollissima ) “Vanuxem,” one of the donors of disease resistance for American chestnut restoration. From the de novo assembly of the complete genome (725.2 Mb in 14,110 contigs), over half of the sequences have been anchored to the 12 genetic linkage groups. The anchoring is validated by genetic maps and in situ hybridization to chromosomes. We demonstrate the value of the genome as a platform for research and species restoration, including signatures of selection differentiating American chestnut from Chinese chestnut to identify important candidate genes for disease resistance, comparisons of genome organization with other woody species, and a genome-wide examination of progress in backcross breeding for blight resistance. This reference assembly should prove of great value in the understanding, improvement, and restoration of chestnut species.

SummaryThe tree of life is highly reticulate, with the history of population divergence emerging from populations of gene phylogenies that reflect histories of introgression, lineage sorting and divergence. In this study, we investigate global patterns of oak diversity and test the hypothesis that there are regions of the oak genome that are broadly informative about phylogeny.We utilize fossil data and restriction‐site associatedDNAsequencing (RAD‐seq) for 632 individuals representing nearly 250Quercusspecies to infer a time‐calibrated phylogeny of the world's oaks. We use a reversible‐jump Markov chain Monte Carlo method to reconstruct shifts in lineage diversification rates, accounting for among‐clade sampling biases. We then map the > 20 000RAD‐seq loci back to an annotated 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, probably in response to climatic transitions or ecological opportunity.The strong support 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.

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.

Exotic pathogens cause severe damage in natural populations in the absence of coevolutionary dynamics with their hosts. However, some resistance to such pathogens may occur in naive populations. The objective of this study was to investigate the genetics of this so-called 'exapted' resistance to two pathogens of Asian origin (Erysiphe alphitoides and Phytophthora cinnamomi) in European oak. Host-pathogen compatibility was assessed by recording infection success and pathogen growth in a full-sib family of Quercus robur under controlled and natural conditions. Two high-resolution genetic maps anchored on the reference genome were used to study the genetic architecture of resistance and to identify positional candidate genes. Two genomic regions, each containing six strong and stable quantitative trait loci (QTLs) accounting for 12-19% of the phenotypic variation, were mainly associated with E. alphitoides infection. Candidate genes, especially genes encoding receptor-like-kinases and galactinol synthases, were identified in these regions. The three QTLs associated with P. cinnamomi infection did not colocate with QTLs found for E. alphitoides. These findings provide evidence that exapted resistance to E. alphitoides and P. cinnamomi is present in Q. robur and suggest that the underlying molecular mechanisms involve genes encoding proteins with extracellular signaling functions.

The Camelid Genome Variation Database: An Integrative Database for Studying Camelid Genetic Evolution, Biological Characteristics and Desert Adaption Tuya Saren, Haiqing Liu and Rimutu Ji, (1)Inner Mongolia Institute of Camel Research, Alxa, Inner Mongolia, China, (2)Inner Mongolia Agricultural University, Huhhot, China Aims: The camelids including Bactrian camel (Camelus bactrianus), dromedary (Camelus dromedaries) and alpaca (Vicugna pacos), are important economically livestock for transportation and production (meat, milk and wool). During the long-term natural selection, they have acquired many unique abilities to adapt to the desert environment. In recent decades, their genetic diversity, evolutionary history and biological characteristics have been elucidated by independent studies. However, these studies of camelids were scattered and the amount of available data were still limited. Our database Camel GVD is designed to provide comprehensive resources for camelid genome studies. Methods: Camel GVD is a web-based large-scale whole genome variation database, including single nucleotide polymorphisms (SNPs), insertions and deletions (Indels). Results: It contains a collection of ~19 million variants identified by whole-genome sequencing of over 100 camels from our original research. It also contains all the public available camelid genome data. Camel GVD integrates a comprehensive collection of variation related information, including variation annotations, associated genes/proteins/transcripts, sample locations, population introductions, and allele frequencies. It offers several functions, for users to search, display and retrieve the variations and their annotations. Camel GVD also provides several data browsing functionalities under the “Browse” pull-down menu, including variant alignment, coverage and genome feature files. Users can select one sample or several samples to browse the information. It also provides a special function to display gene information of several camel features based on the existing studies to provide convenience for users. In addition, the vcf files can be downloaded for advanced data mining and analysis. Thus, Camel GVD is expected to become a bioinformatics platform for studying the evolution, genetic breeding, biological characteristics and functional genes of camelids. Conclusion: Our database Camel GVD is the first large-scale, and so far the most comprehensive collection of genomic variant data from over 100 individual camelids. Compared with one similar database, our database provides both SNPs and Indels information, and also provides functional gene information associated with camel genetic and biological characteristics. This database is helpful to facilitate the future studies of population genetics and molecular biology in camelids.

In the post-genomics era, non-model species like most Fagaceae still lack operational diversity resources for population genomics studies. We completed the analyses of Sanger sequences produced from over 800 gene fragments covering ~530 kb across the genic partition of European oaks in a range-wide sampling of 25 individuals (11 Quercus petraea, 13 Q. robur, one Q. ilex as an outgroup). Regions targeted represented broad functional categories potentially involved in species ecological preferences, and a random set of genes. Using a high-quality dedicated pipeline, we provide a detailed characterization of over 14500 polymorphisms, including ~12500 SNPs -218 being triallelic-, over 1500 insertion-deletions, and ~200 novel di- and tri-nucleotide SSR loci. This catalog also includes various summary statistics within and among species, gene ontology information, and standard formats to assist loci choice for genotyping projects. The distribution of nucleotide diversity and differentiation across genic regions are also described for the first time in those species (mean θπ close to ~0.0049 in Q. petraea and to ~0.0045 in Q. robur across random regions, and mean FST ~0.13 across SNPs). Robust patterns were observed which emphasize a slightly but significantly higher diversity in Q. petraea, across a random gene set and in the abiotic stress functional category, and a heterogeneous landscape of both diversity and differentiation. These patterns are discussed in the context of both species documented introgression history despite strong reproductive barriers. The quality, representativity in terms of species genomic diversity, and usefulness of the resources provided are discussed for possible applications in medium scale landscape ecology projects, and as a reference resource for validation purposes in larger-scale re-sequencing projects. These are preferentially recommended in oaks in contrast to SNP array development, given the large nucleotide variation and low levels of linkage disequilibrium revealed.

Reproduction, one of the main components of plant fitness, is highly variable in response to environmental cues, but little is known about the genetic determinism underlying reproduction-related traits in forest tree species. There is therefore an urgent need to characterize the genetic architecture of those traits if we are to predict the evolutionary trajectories of forest populations facing rapidly changing environment and mitigate their impacts. Using a full-sib family of pedunculate oak (Quercus robur), we investigated the within population variability of seed production and mean seed mass during four consecutive years. Reproductive traits were highly variable between trees and between years. The high narrow sense heritability and evolvability estimated underline the important genetic effect on the variability in seed production and mean seed mass. Despite a large variability over years, reproductive traits show significant genetic correlation between years. Furthermore, for the first time in forest tree species, quantitative trait loci (QTLs) associated with seed production and mean mass of a seed have been identified. While it is commonly assumed and observed that fitness traits have low narrow sense heritabilities, our findings show that reproduction-related traits may undergo evolutionary changes under selective pressure and may be determinant for tree adaptation.

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.

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.

Summary Historical trajectories of tree species during the late Quaternary have been well reconstructed through genetic and palaeobotanical studies. However, many congeneric tree species are interfertile, and the timing and contribution of introgression to species divergence during their evolutionary history remains largely unknown. We quantified past and current gene flow events between four morphologically divergent oak species (Quercus petraea, Q. robur, Q. pyrenaica, Q. pubescens), by two independent inference methods: diffusion approximation to the joint frequency spectrum (∂a∂i) and approximate Bayesian computation (ABC). For each pair of species, alternative scenarios of speciation allowing gene flow over different timescales were evaluated. Analyses of 3524 single nucleotide polymorphisms (SNPs) randomly distributed in the genome, showed that these species evolved in complete isolation for most of their history, but recently came into secondary contact, probably facilitated by the most recent period of postglacial warming. We demonstrated that: there was sufficient genetic differentiation before secondary contact for the accumulation of barriers to gene flow; and current European white oak genomes are a mosaic of genes that have crossed species boundaries and genes impermeable to gene flow.

A new method for obtaining internal views of tree trunks was recently developed using X-ray computed tomography (CT). This technology makes it possible to observe and measure rameal traces that are left by latent buds, sequential branches, and epicormic branches in the wood. Epicormic branches are undesirable for producing high-value solid wood, especially in Quercus robur, an important hardwood forest tree species in Europe, which is prone to epicormic branches that develop from abundant latent buds. For the very first time, branching-related traits deduced from X-ray CT observation make it possible to analyze the genetic architecture of oak branching through a quantitative trait locus (QTL) analysis. Highly significant QTLs were detected for traits related to latent buds and epicormic branches. The number and effect of these QTLs suggest a moderate genetic determinism for the formation of latent buds and the development of epicormic branches. Three hotspots were found, grouping QTLs for different branching traits. An analysis of the common physiological denominators of these coincident traits suggests that their genetic controls are related to either the regulation of the axillary meristem initiation or to bud dormancy. Conversely, the position of only the separate QTL related to the number of sequential branches suggests an independent genetic control.

We developed the densest single-nucleotide polymorphism (SNP)-based linkage genetic map to date for the genus Quercus An 8k gene-based SNP array was used to genotype more than 1,000 full-sibs from two intraspecific and two interspecific full-sib families of Quercus petraea and Quercus robur A high degree of collinearity was observed between the eight parental maps of the two species. A composite map was then established with 4,261 SNP markers spanning 742 cM over the 12 linkage groups (LGs) of the oak genome. Nine genomic regions from six LGs displayed highly significant distortions of segregation. Two main hypotheses concerning the mechanisms underlying segregation distortion are discussed: genetic load vs. reproductive barriers. Our findings suggest a predominance of pre-zygotic to post-zygotic barriers.

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 whether >3500 single nucleotide polymorphisms ( SNP s) 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 SNP s greatly differed among species. However, we identified one SNP and seven genes that were associated with the same environmental factor across all species. We finally used regressions of allele frequencies of the most strongly associated SNP s along environmental gradients to predict the risk of nonadaptedness ( 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.