Christophe Plomion

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

Effective population size (N_e) is a pivotal evolutionary parameter with crucial implications in conservation practice and policy. Genetic methods to estimate N_e have been preferred over demographic methods because they rely on genetic data rather than time‐consuming ecological monitoring. Methods based on linkage disequilibrium (LD), in particular, have become popular in conservation as they require a single sampling and provide estimates that refer to recent generations. A software program based on the LD method, GONE, looks particularly promising to estimate contemporary and recent‐historical N_e (up to 200 generations in the past). Genomic datasets from non‐model species, especially plants, may present some constraints to the use of GONE, as linkage maps and reference genomes are seldom available, and SNP genotyping is usually based on reduced‐representation methods. In this study, we use empirical datasets from four plant species to explore the limitations of plant genomic datasets when estimating N_e using the algorithm implemented in GONE, in addition to exploring some typical biological limitations that may affect N_e estimation using the LD method, such as the occurrence of population structure. We show how accuracy and precision of N_e estimates potentially change with the following factors: occurrence of missing data, limited number of SNPs/individuals sampled, and lack of information about the location of SNPs on chromosomes, with the latter producing a significant bias, previously unexplored with empirical data. We finally compare the N_e estimates obtained with GONE for the last generations with the contemporary N_e estimates obtained with the programs currentNe and NeEstimator.

Purpose of Review: In this review, we synthesise current knowledge on trade-offs among traits in key fitness dimensions and identify major research gaps with the intention of laying the groundwork for a rapid advance in tree breeding for multiple objectives as a key contribution to the sustainability of planted forests in the future. Recent Findings: Trade-offs among growth, reproduction, defence, stress tolerance and product quality predicted theoretically have been reported experimentally in many breeding programmes. Among these trade-offs, the genetic linkage between resistance against biotic threats and growth (or other relevant traits) is particularly critical for the current and future management of forest genetic resources. Maintaining tree growth and wood quality in the novel environments of the future requires the assessment of genetic correlations of target traits with phenology, closely linked to survival to temperature extremes. Improving our current knowledge on the genetic trade-offs of drought tolerance as a breeding objective in forest trees obligates a more precise definition of both the specific traits and the experimental conditions. Published evidence suggests that common target traits in breeding programmes may trade-off with reproductive success and fire-adaptation, and the simultaneous improvement of growth and wood quality traits still remains as a constraint in traditional tree breeding. Summary: Changing environments combined with pests and diseases are challenging plantation forestry worldwide, which implies an urgent need to develop new improvement strategies to build the resilience of forestry for our future environments. It is essential to have a better understanding of how traits interact, especially those important for production, climate and biotic threat resilience, but much of the information is still missing. Since many key trade-offs are affected by the environment, we need new studies under novel environments to forecast levels of multi-trait integration in breeding populations.

Somatic mutations potentially play a role in plant evolution, but common expectations pertaining to plant somatic mutations remain insufficiently tested. Unlike in most animals, the plant germline is assumed to be set aside late in development, leading to the expectation that plants accumulate somatic mutations along growth. Therefore, several predictions were made on the fate of somatic mutations: mutations have generally low frequency in plant tissues; mutations at high frequency have a higher chance of intergenerational transmission; branching topology of the tree dictates mutation distribution; and exposure to UV (ultraviolet) radiation increases mutagenesis. To provide insights into mutation accumulation and transmission in plants, we produced two high-quality reference genomes and a unique dataset of 60 high-coverage whole-genome sequences of two tropical tree species, Dicorynia guianensis (Fabaceae) and Sextonia rubra (Lauraceae). We identified 15,066 de novo somatic mutations in D. guianensis and 3,208 in S. rubra , surprisingly almost all found at low frequency. We demonstrate that 1) low-frequency mutations can be transmitted to the next generation; 2) mutation phylogenies deviate from the branching topology of the tree; and 3) mutation rates and mutation spectra are not demonstrably affected by differences in UV exposure. Altogether, our results suggest far more complex links between plant growth, aging, UV exposure, and mutation rates than commonly thought.

Effective population size ( N _e ) is a pivotal evolutionary parameter with crucial implications in conservation practice and policy. Genetic methods to estimate N _e have been preferred over demographic methods because they rely on genetic data rather than time-consuming ecological monitoring. Methods based on linkage disequilibrium, in particular, have become popular in conservation as they require a single sampling and provide estimates that refer to recent generations. A recently developed software based on linkage disequilibrium, GONE, looks particularly promising to estimate contemporary and recent-historical N _e (up to 200 generations in the past). Genomic datasets from non-model species, especially plants, may present some constraints to the use of GONE, as linkage maps and reference genomes are seldom available, and SNPs genotyping is usually based on reduced-representation methods. In this study, we use empirical datasets from four plant species to explore the limitations of plant genomic datasets when estimating N _e using the algorithm implemented in GONE, in addition to exploring some typical biological limitations that may affect N _e estimation using the linkage disequilibrium method, such as the occurrence of population structure. We show how accuracy and precision of N _e estimates potentially change with the following factors: occurrence of missing data, limited number of SNPs/individuals sampled, and lack of information about the location of SNPs on chromosomes, with the latter producing a significant bias, previously unexplored with empirical data.

Somatic mutations potentially play a role in plant evolution, but common expectations pertaining to plant somatic mutations remain insufficiently tested. Unlike in most animals, the plant germline is assumed to be set aside late in development, leading to the expectation that plants accumulate somatic mutations along growth. Therefore, several predictions were made on the fate of somatic mutations: mutations have generally low frequency in plant tissues; mutations at high frequency have a higher chance of intergenerational transmission; branching topology of the tree dictates mutation distribution; and, exposure to UV radiation increases mutagenesis. To provide new insights into mutation accumulation and transmission in plants, we produced two high-quality reference genomes and a unique dataset of 60 high-coverage whole-genome sequences of two tropical tree species, Dicorynia guianensis (Fabaceae) and Sextonia rubra (Lauraceae). We identified 15,066 de novo somatic mutations in D. guianensis and 3,208 in S. rubra , surprisingly almost all found at low frequency. We demonstrate that: 1) low-frequency mutations can be transmitted to the next generation; 2) mutation phylogenies deviate from the branching topology of the tree; and 3) mutation rates and mutation spectra are not demonstrably affected by differences in UV exposure. Altogether, our results suggest far more complex links between plant growth, ageing, UV exposure, and mutation rates than commonly thought. Significance Statement: The origin and fate of new mutations have received less attention in plants than in animals. Similarly to animals, plant mutations are expected to accumulate with growth and time, and under exposure to UV light. However, contrary to animals, plant reproductive organs form late in an individual’s development, allowing the transmission to the progeny of mutations accumulated along growth. Here, we resequenced DNA from different branches differentially exposed to sunlight of two tropical tree species. We showed that new mutations are generally rare in plant tissues and do not mimic branching patterns but can nevertheless be transmitted to the progeny. Our findings provide a new perspective on heritable plant mutation and its pivotal role as the engine of evolution.

Specialized or secondary metabolites play a key role in plant resistance against abiotic stresses and defences against bioaggressors. For example, in sessile oaks Quercus petraea , phenolics contribute to reduce herbivore damage and improve drought resistance. Here, we explored the natural variation of specialized metabolites in nine European provenances of sessile oaks and aimed to detect its underlying genetic bases. We sampled mature leaves from high and low branches on 225 sessile oak trees located in a common garden and used untargeted metabolomics to characterise the variation of 217 specialized metabolites. In addition, we used whole genome low-depth sequencing to genotype individuals for 1.4M genetic markers. We found that leaf specialized metabolites displayed extensive within-provenance variation, but very little differentiation between provenances. In addition, a genome-wide association study allowed detecting significant associations for 42% of these metabolites. Hence, our results suggest that genetic variation for most leaf specialized metabolites is unlikely to be locally adaptive, however lack of differentiation among populations suggests selection acts locally to maintain diversity at loci associated with leaf specialized metabolites variation.

The effect of past environmental changes on the demography and genetic diversity of natural populations remains a contentious issue and has rarely been investigated across multiple, phylogenetically distant species. Here, we performed comparative population genomic analyses and demographic inferences for seven widely distributed and ecologically contrasted European forest tree species based on concerted sampling of 164 populations across their natural ranges. For all seven species, the effective population size, N _e , increased or remained stable over many glacial cycles and up to 15 million years in the most extreme cases. Hence, surprisingly, the drastic environmental changes associated with the Pleistocene glacial cycles have had little impact on the level of genetic diversity of dominant forest tree species, despite major shifts in their geographic ranges. Based on their trajectories of N _e over time, the seven tree species can be divided into three major groups, highlighting the importance of life history and range size in determining synchronous variation in genetic diversity, over time. Altogether, our results indicate that forest trees were able to retain their evolutionary potential over very long periods of time despite strong environmental changes. One-Sentence Summary: Population genomic analyses reveal that forest trees retained their evolutionary potential over the Quaternary.

Through repeated cycles of selection and recombination, tree breeding programs deliver genetically improved varieties for a range of target traits such as biomass production, stem form, resistance to biotic stresses, wood properties, etc. However, in the context of increased drought and heat waves, it is not yet known whether growth performance will impede drought resistance. To address this question, we compared the hydraulic properties, such as hydraulic efficiency and hydraulic safety, in four varieties over successive generations of genetically improved maritime pines (i.e., Pinus pinaster Aït.) for growth and stem form. We measured 22 functional traits related to hydraulic efficiency, hydraulic safety, xylem anatomy and wood density. We found that improved varieties presented higher hydraulic conductivity with larger tracheid lumen size and tracheid lumen fraction, and smaller wall thickness reinforcement and tracheid density, but not at the cost of reduced embolism resistance. The reported absence of trade-off between hydraulic conductivity and embolism resistance is a strong asset to improve biomass productivity, through increased hydraulic efficiency, without impacting drought resistance, and should enable new maritime pine varieties to cope with a drier climate. Our study is one of the first to reveal the hydraulic mechanisms over successive generations of genetic improvement for tree growth. It provides guidelines for sustainable forest management through breeding for other forest tree species.

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.