Genome-wide methylome stability and parental effects in the worldwide distributed Lombardy poplar.

BACKGROUND: Despite the increasing number of epigenomic studies in plants, little is known about the forces that shape the methylome in long-lived woody perennials. The Lombardy poplar offers an ideal opportunity to investigate the impact of the individual environmental history of trees on the methylome. RESULTS: We present the results of three interconnected experiments on Lombardy poplar. In the first experiment, we investigated methylome variability during a growing season and across vegetatively reproduced generations. We found that ramets collected over Europe and raised in common conditions have stable methylomes in symmetrical CG-contexts. In contrast, seasonal dynamics occurred in methylation patterns in CHH context. In the second experiment, we investigated whether methylome patterns of plants grown in a non-parental environment correlate with the parental climate. We did not observe a biological relevant pattern that significantly correlates with the parental climate. Finally, we investigated whether the parental environment has persistent carry-over effects on the vegetative offspring's phenotype. We combined new bud set observations of three consecutive growing seasons with former published bud set data. Using a linear mixed effects analysis, we found a statistically significant but weak short-term, parental carry-over effect on the timing of bud set. However, this effect was negligible compared to the direct effects of the offspring environment. CONCLUSIONS: Genome-wide cytosine methylation patterns in symmetrical CG-context are stable in Lombardy poplar and appear to be mainly the result of random processes. In this widespread poplar clone, methylation patterns in CG-context can be used as biomarkers to infer a common ancestor and thus to investigate the recent environmental history of a specific Lombardy poplar. The Lombardy poplar shows high phenotypic plasticity in a novel environment which enabled this clonal tree to adapt and survive all over the temperate regions of the world.

Sequencing of Complete Chloroplast Genomes.

In this chapter, frequently used methods for elucidating sequence and structure of chloroplast genomes are reviewed, as a current best practice guide. This concerns methods for DNA extraction, sequencing library preparation, and bioinformatics (assembly, verification, annotation, and sequence comparisons). Recommendations for standard data reporting practices are given-chloroplast genome sequencing reports can be highly formalized, and publication in the form of standard data reports is the best option for comparison and meta-analysis purposes.

Epigenetic Analysis through MSAP-NGS Coupled Technology: The Case Study of White Poplar Monoclonal Populations/Stands.

Over the last several decades, several lines of evidence have shown that epigenetic modifications modulate phenotype and mediate an organism's response to environmental stimuli. Plant DNA is normally highly methylated, although notable differences exist between species. Many biomolecular techniques based on PCR have been developed to analyse DNA methylation status, however a qualitative leap was made with the advent of next-generation sequencing (NGS). In the case of large, repetitive, or not-yet-sequenced genomes characterised by a high level of DNA methylation, the NGS analysis of bisulphite pre-treated DNA is expensive and time consuming, and moreover, in some cases data analysis is a major challenge. Methylation-sensitive amplification polymorphism (MSAP) analysis is a highly effective method to study DNA methylation. The method is based on the comparison of double DNA digestion profiles (EcoRI-HpaII and EcoRI-MspI) to reveal methylation pattern variations. These are often attributable to pedoclimatic and stress conditions which affect all organisms during their lifetime. In our study, five white poplar (Populus alba L.) specimens were collected from different monoclonal stands in the Maltese archipelago, and their DNA was processed by means of an innovative approach where MSAP analysis was followed by NGS. This allowed us to identify genes that were differentially methylated among the different specimens and link them to specific biochemical pathways. Many differentially methylated genes were found to encode transfer RNAs (tRNAs) related to photosynthesis or light reaction pathways. Our results clearly demonstrate that this combinatorial method is suitable for epigenetic studies of unsequenced genomes like P. alba (at the time of study), and to identify epigenetic variations related to stress, probably caused by different and changing pedoclimatic conditions, to which the poplar stands have been exposed.

SNP marker development in Pinus sylvestris L. in stress-responsive genes characterized from Pinus cembra L. transcriptomes.

Massively parallel sequencing of cDNA is an efficient route for generating sequence collections that represent expressed genes under different environmental control. The analysis of their sequence helps in developing molecular markers, such as SNPs, which represent a useful tool in detecting adaptive signals in populations. In this study novel PCR markers, based on stress responsive genes, were designed from the transcriptome of the haploxylon Swiss stone pine (Pinus cembra L.) and tested for SNPs in the diploxylon Scots pine (Pinus sylvestris L.). 84 primers were tested on P. sylvestris DNA samples originating from three different types of habitat. After sequencing and BLAST search of the amplified products, parts of 19 different candidate genes were analysed by considering the polymorphic sites, insertions/deletions as well as synonymous and non-synonymous SNPs. In a total of 3735 sites no indels, eight synonymous and 11 non-synonymous SNPs were found. By providing de novo molecular markers developed in P. cembra and tested for transferability in Scots pine, our results give support for the use of de novo markers targeting conserved regions across different pines. The SNPs detected may have important applications in further studies of adaptive genetic variation, providing tools to study relevant genes important in the long-term adaptation of pine species.

A Reference Genome Sequence for the European Silver Fir (Abies alba Mill.): A Community-Generated Genomic Resource.

Silver fir (Abies alba Mill.) is a keystone conifer of European montane forest ecosystems that has experienced large fluctuations in population size during during the Quaternary and, more recently, due to land-use change. To forecast the species' future distribution and survival, it is important to investigate the genetic basis of adaptation to environmental change, notably to extreme events. For this purpose, we here provide a first draft genome assembly and annotation of the silver fir genome, established through a community-based initiative. DNA obtained from haploid megagametophyte and diploid needle tissue was used to construct and sequence Illumina paired-end and mate-pair libraries, respectively, to high depth. The assembled A. alba genome sequence accounted for over 37 million scaffolds corresponding to 18.16 Gb, with a scaffold N50 of 14,051 bp. Despite the fragmented nature of the assembly, a total of 50,757 full-length genes were functionally annotated in the nuclear genome. The chloroplast genome was also assembled into a single scaffold (120,908 bp) that shows a high collinearity with both the A. koreana and A. sibirica complete chloroplast genomes. This first genome assembly of silver fir is an important genomic resource that is now publicly available in support of a new generation of research. By genome-enabling this important conifer, this resource will open the gate for new research and more precise genetic monitoring of European silver fir forests.

Prediction of hydraulic conductivity loss from relative water loss: new insights into water storage of tree stems and branches.

More frequently occurring, drought waves call for a deeper understanding of tree hydraulics and fast and easily applicable methods to measure drought stress. The aim of this study was to establish empirical relationships between the percent loss of hydraulic conductivity (PLC) and the relative water loss (RWL) in woody stem axes with different P50 , i.e. the water potential (Ψ) that causes 50% conductivity loss. Branches and saplings of temperate conifer (Picea abies, Larix decidua) and angiosperm species (Acer campestre, Fagus sylvatica, Populus x canescens, Populus tremula, Sorbus torminalis) and trunk wood of mature P. abies trees were analyzed. P50 was calculated from hydraulic measurements following bench top dehydration or air injection. RWL and PLC were fitted by linear, quadratic or cubic equations. Species- or age-specific RWLs at P50 varied between 10 and 25% and P88 , the Ψ that causes 88% conductivity loss, between 18 and 44%. P50 was predicted from the relationship between Ψ and the RWL. The predictive quality for P50 across species was almost 1:1 (r2 = 0.99). The approach presented allows thus reliable and fast prediction of PLC from RWL. Branches and saplings with high hydraulic vulnerability tended to have lower RWLs at P50 and at P88 . The results are discussed with regard to the different water storage capacities in sapwood and survival strategies under drought stress. Potential applications are screening trees for drought sensitivity and a fast interpretation of diurnal, seasonal or drought induced changes in xylem water content upon their impact on conductivity loss.

Variability in DNA Methylation and Generational Plasticity in the Lombardy Poplar, a Single Genotype Worldwide Distributed Since the Eighteenth Century.

In the absence of genetic diversity, plants rely on the capacity of phenotypic plasticity to cope with shifts in environmental conditions. Understanding the mechanisms behind phenotypic plasticity and how local phenotypic adjustments are transferred to clonal offspring, will provide insight into its ecological and evolutionary significance. Epigenetic changes have recently been proposed to play a crucial role in rapid environmental adaptation. While the contribution of epigenetic changes to phenotypic plasticity has been extensively studied in sexual reproducing model organisms, little work has been done on vegetative generations of asexual reproducing plant species. We studied the variability of DNA methylation and bud set phenology of the Lombardy poplar (Populus nigra cv. Italica Duroi), a cultivated tree representing a single genotype worldwide distributed since the eighteenth century. Bud set observations and CpG methyl polymorphisms were studied on vegetative offspring resulting from cuttings grown for one season in a common glasshouse environment. The cuttings were collected from 60 adult Lombardy poplars growing in different environments. The physiological condition of the cuttings was determined by measuring weight and nutrient condition. Methylation sensitive amplified polymorphisms were used to obtain global patterns of DNA methylation. Using logistic regression models, we investigated correlations among epigenotype, bud phenology, and the climate at the home site of the donor trees, while accounting for physiological effects. We found significant epigenetic variation as well as significant variation in bud phenology, in the absence of genetic variation. Remarkably, phenology of bud set observed at the end of the growing season in the common environment was significantly correlated with climate variables at the home site of the mother trees, specifically the average temperature of January and monthly potential evapotranspiration. Although we could not directly detect significant effects of epigenetic variation on phenology, our results suggest that, in the Lombardy poplar, epigenetic marks contribute to the variation of phenotypic response that can be transferred onto asexually reproduced offspring resulting in locally adapted ecotypes. This contributes to the growing evidence that epigenetic-based transgenerational inheritance might be relevant for adaptation and evolution in contrasting or rapidly changing environments.

Discrimination of Picea chihuahuana Martinez populations on the basis of climatic, edaphic, dendrometric, genetic and population traits.

BACKGROUND: Picea chihuahuana, which is endemic to Mexico, is currently listed as "Endangered" on the Red List. Chihuahua spruce is only found in the Sierra Madre Occidental (SMO), Mexico. About 42,600 individuals are distributed in forty populations. These populations are fragmented and can be classified into three geographically distinct clusters in the SMO. The total area covered by P. chihuahuana populations is less than 300 ha. A recent study suggested assisted migration as an alternative to the ex situ conservation of P. chihuahuana, taking into consideration the genetic structure and diversity of the populations and the predictions regarding the future climate of the habitat. However, detailed background information is required to enable development of plans for protecting and conserving species and for successful assisted migration. Thus, it is important to identify differences between populations in relation to environmental conditions. The genetic diversity of populations, which affect vigor, evolution and adaptability of the species, must also be considered. In this study, we examined 14 populations of P. chihuahuana, with the overall aim of discriminating the populations and form clusters of this species. METHODS: Each population was represented by one 50 × 50 m plot established in the center of its respective location. Climate, soil, dasometric, density variables and genetic and species diversities were assessed in these plots for further analyses. The putatively neutral and adaptive AFLP markers were used to calculate genetic diversity. Affinity Propagation (AP) clustering technique and k-means clustering algorithm were used to classify the populations in the optimal number of clusters. Later stepwise binomial logistic regression was applied to test for significant differences in variables of the southern and northern P. chihuahuana populations. Spearman's correlation test was used to analyze the relationships among all variables studied. RESULTS: The binomial logistic regression analysis revealed that seven climate variables, the geographical longitude and sand proportion in the soil separated the southern from northern populations. The northern populations grow in more arid and continental conditions and on soils with lower sand proportion. The mean genetic diversity using all AFLP studied of P. chihuahuana was significantly correlated with the mean temperature in the warmest month, where warmer temperatures are associated to larger genetic diversity. Genetic diversity of P. chihuahuana calculated with putatively adaptive AFLP was not statistically significantly correlated with any environmental factor. DISCUSSION: Future reforestation programs should take into account that at least two different groups (the northern and southern cluster) of P. chihuahuana exist, as local adaptation takes place because of different environmental conditions.

Selection against recombinant hybrids maintains reproductive isolation in hybridizing Populus species despite F1 fertility and recurrent gene flow.

Natural hybrid zones have proven to be precious tools for understanding the origin and maintenance of reproductive isolation (RI) and therefore species. Most available genomic studies of hybrid zones using whole- or partial-genome resequencing approaches have focused on comparisons of the parental source populations involved in genome admixture, rather than exploring fine-scale patterns of chromosomal ancestry across the full admixture gradient present between hybridizing species. We have studied three well-known European 'replicate' hybrid zones of Populus alba and P. tremula, two widespread, ecologically divergent forest trees, using up to 432 505 single-nucleotide polymorphisms (SNPs) from restriction site-associated DNA (RAD) sequencing. Estimates of fine-scale chromosomal ancestry, genomic divergence and differentiation across all 19 poplar chromosomes revealed strikingly contrasting results, including an unexpected preponderance of F1 hybrids in the centre of genomic clines on the one hand, and genomically localized, spatially variable shared variants consistent with ancient introgression between the parental species on the other. Genetic ancestry had a significant effect on survivorship of hybrid seedlings in a common garden trial, pointing to selection against early-generation recombinants. Our results indicate a role for selection against recombinant genotypes in maintaining RI in the face of apparent F1 fertility, consistent with the intragenomic 'coadaptation' model of barriers to introgression upon secondary contact. Whole-genome resequencing of hybridizing populations will clarify the roles of specific genetic pathways in RI between these model forest trees and may reveal which loci are affected most strongly by its cyclic breakdown.

Epigenetic Diversity of Clonal White Poplar (Populus alba L.) Populations: Could Methylation Support the Success of Vegetative Reproduction Strategy?

The widespread poplar populations of Sardinia are vegetatively propagated and live in different natural environments forming large monoclonal stands. The main goals of the present study were: i) to investigate/measure the epigenetic diversity of the poplar populations by determining their DNA methylation status; ii) to assess if and how methylation status influences population clustering; iii) to shed light on the changes that occur in the epigenome of ramets of the same poplar clone. To these purposes, 83 white poplar trees were sampled at different locations on the island of Sardinia. Methylation sensitive amplified polymorphism analysis was carried out on the genomic DNA extracted from leaves at the same juvenile stage. The study showed that the genetic biodiversity of poplars is quite limited but it is counterbalanced by epigenetic inter-population molecular variability. The comparison between MspI and HpaII DNA fragmentation profiles revealed that environmental conditions strongly influence hemi-methylation of the inner cytosine. The variable epigenetic status of Sardinian white poplars revealed a decreased number of population clusters. Landscape genetics analyses clearly demonstrated that ramets of the same clone were differentially methylated in relation to their geographic position. Therefore, our data support the notion that studies on plant biodiversity should no longer be restricted to genetic aspects, especially in the case of vegetatively propagated plant species.

Genome-wide patterns of differentiation and spatially varying selection between postglacial recolonization lineages of Populus alba (Salicaceae), a widespread forest tree.

Studying the divergence continuum in plants is relevant to fundamental and applied biology because of the potential to reveal functionally important genetic variation. In this context, whole-genome sequencing (WGS) provides the necessary rigour for uncovering footprints of selection. We resequenced populations of two divergent phylogeographic lineages of Populus alba (n = 48), thoroughly characterized by microsatellites (n = 317), and scanned their genomes for regions of unusually high allelic differentiation and reduced diversity using > 1.7 million single nucleotide polymorphisms (SNPs) from WGS. Results were confirmed by Sanger sequencing. On average, 9134 high-differentiation (≥ 4 standard deviations) outlier SNPs were uncovered between populations, 848 of which were shared by ≥ three replicate comparisons. Annotation revealed that 545 of these were located in 437 predicted genes. Twelve percent of differentiation outlier genome regions exhibited significantly reduced genetic diversity. Gene ontology (GO) searches were successful for 327 high-differentiation genes, and these were enriched for 63 GO terms. Our results provide a snapshot of the roles of 'hard selective sweeps' vs divergent selection of standing genetic variation in distinct postglacial recolonization lineages of P. alba. Thus, this study adds to our understanding of the mechanisms responsible for the origin of functionally relevant variation in temperate trees.

Analysis of variation in chloroplast DNA sequences.

This chapter introduces and reviews methods for analyzing variation in chloroplast DNA, mainly by polymerase chain reaction (PCR) and subsequent revelation of polymorphisms. Sources for chloroplast primers are discussed, as well as methods such as Sanger sequencing, PCR followed by restriction fragment length polymorphism (RFLP), gel electrophoresis, fragment analysis on automated DNA sequencers, denaturing high-performance liquid chromatography (dHPLC), and next-generation sequencing (NGS). A special section deals with peculiarities of chloroplast DNA variation, such as tandem repeats and mini- and microsatellites.

Recombinant hybrids retain heterozygosity at many loci: new insights into the genomics of reproductive isolation in Populus.

The maintenance of species barriers in the face of gene flow is often thought to result from strong selection against intermediate genotypes, thereby preserving genetic differentiation. Most speciation genomic studies thus aim to identify exceptionally divergent loci between populations, but divergence will be affected by many processes other than reproductive isolation (RI) and speciation. Through genomic studies of recombinant hybrids sampled in the wild, genetic variation associated with RI can be observed in situ, because selection against incompatible genotypes will leave detectable patterns of variation in the hybrid genomes. To better understand the mechanisms directly involved in RI, we investigated three natural 'replicate' hybrid zones between two divergent Populus species via locus-specific patterns of ancestry across recombinant hybrid genomes. As expected, genomic patterns in hybrids and their parental species were consistent with the presence of underdominant selection at several genomic regions. Surprisingly, many loci displayed greatly increased between-species heterozygosity in recombinant hybrids despite striking genetic differentiation between the parental genomes, the opposite of what would be expected with selection against intermediate genotypes. Only a limited, reproducible set of genotypic combinations was present in hybrid genomes across localities. In the absence of clearly delimited 'hybrid habitats', our results suggest that complex epistatic interactions within genomes play an important role in advanced stages of RI between these ecologically divergent forest trees. This calls for more genomic studies that test for unusual patterns of genomic ancestry in hybridizing species.

Towards a quantitative description of landscape, demography and flowering phenology effects on realized hybridization potential.

In chemistry, the law of mass action describes how variations in the concentrations of chemical compounds lead to different chemical reaction outcomes. Does the extent of hybridization, or more particularly, the formation of hybrid offspring, likewise depend on the local abundance of pollen from compatible species in systems as complex as tall trees which depend on (insect) pollinators? In this issue of Molecular Ecology, Field et al. (2011a) present a study involving two ecologically divergent eucalypt species. By comparing several contrasting settings with different local densities and geographical arrangements of adult trees and by studying parentage in progeny arrays, they show that on top of pre-mating barriers like flowering time differences, local demography and varying scales of pollen dispersal, which in themselves depend on pollinator behaviour in reaction to flowering abundance, all interact in a somewhat predictable way. In other words, these factors can explain some of the variation in hybrid formation observed. In this way, the study introduces important progress towards a quantitative description of hybridization potential. Therefore, let me tell you about the birds and the bees and the flowers and the trees (Newman 1964).

Genomic admixture analysis in European Populus spp. reveals unexpected patterns of reproductive isolation and mating.

Admixture between genetically divergent populations facilitates genomic studies of the mechanisms involved in adaptation, reproductive isolation, and speciation, including mapping of the loci involved in these phenomena. Little is known about how pre- and postzygotic barriers will affect the prospects of "admixture mapping" in wild species. We have studied 93 mapped genetic markers (microsatellites, indels, and sequence polymorphisms, ∼60,000 data points) to address this topic in hybrid zones of Populus alba and P. tremula, two widespread, ecologically important forest trees. Using genotype and linkage information and recently developed analytical tools we show that (1) reproductive isolation between these species is much stronger than previously assumed but this cannot prevent the introgression of neutral or advantageous alleles, (2) unexpected genotypic gaps exist between recombinant hybrids and their parental taxa, (3) these conspicuous genotypic patterns are due to assortative mating and strong postzygotic barriers, rather than recent population history. We discuss possible evolutionary trajectories of hybrid lineages between these species and outline strategies for admixture mapping in hybrid zones between highly divergent populations. Datasets such as this one are still rare in studies of natural hybrid zones but should soon become more common as high throughput genotyping and resequencing become feasible in nonmodel species.

Admixture facilitates adaptation from standing variation in the European aspen (Populus tremula L.), a widespread forest tree.

Adaptation to new environments can start from new mutations or from standing variation already present in natural populations. Whether admixture constrains or facilitates adaptation from standing variation is largely unknown, especially in ecological keystone or foundation species. We examined patterns of neutral and adaptive population divergence in Populus tremula L., a widespread forest tree, using mapped molecular genetic markers. We detected the genetic signature of postglacial admixture between a Western and an Eastern lineage of P. tremula in Scandinavia, an area suspected to represent a zone of postglacial contact for many species of animals and plants. Stringent divergence-based neutrality tests provided clear indications for locally varying selection at the European scale. Six of 12 polymorphisms under selection were located less than 1 kb away from the nearest gene predicted by the Populus trichocarpa genome sequence. Few of these loci exhibited a signature of 'selective sweeps' in diversity-based tests, which is to be expected if adaptation occurs primarily from standing variation. In Scandinavia, admixture explained genomic patterns of ancestry and the nature of clinal variation and strength of selection for bud set, a phenological trait of great adaptive significance in temperate trees, measured in a common garden trial. Our data provide a hitherto missing direct link between past range shifts because of climatic oscillations, and levels of standing variation currently available for selection and adaptation in a terrestrial foundation species.

Clonality and spatial genetic structure in Populus x canescens and its sympatric backcross parent P. alba in a Central European hybrid zone.

Spatial genetic structure (SGS) holds the key to understanding the role of clonality in hybrid persistence, but multilocus SGS in hybrid zones has rarely been quantified. Here, the aim was to fill this gap for natural hybrids between two diploid, ecologically divergent European tree species with mixed sexual/asexual reproduction, Populus alba and P. tremula. Nuclear microsatellites were used to quantify clonality, SGS, and historical gene dispersal distances in up to 407 trees from an extensive Central European hybrid zone including three subpopulation replicates. The focus was on P. x canescens and its backcross parent P. alba, as these two genotypic classes co-occur and interact directly. Sexual recombination in both taxa was more prominent than previously thought, but P. x canescens hybrids tended to build larger clones extending over larger areas than P. alba. The 3.4 times stronger SGS in the P. x canescens genet population was best explained by a combination of interspecific gene flow, assortative mating, and increased clonality in hybrids. Clonality potentially contributes to the maintenance of hybrid zones of P. alba and P. tremula in time and space. Both clonality and SGS need to be taken into account explicitly when designing population genomics studies of locus-specific effects in hybrid zones.

Somatic mutations as a useful tool for studying clonal dynamics in trees.

The seemingly eternal cycles of clonal growth in many tree species, with members of Populus (aspen, poplars, cottonwoods and the like) featuring most prominently, provoke a number of questions on the interface between ecology, genetics and forestry. In this issue, two groups present their approaches to clonal dynamics (Ally et al. 2008 and Mock et al. 2008), using microsatellite (or simple sequence repeat, SSR) variation in P. tremuloides. Ally et al. developed and applied a model for using microsatellites to estimate clone age and infer other community characteristics. Mock et al. used fewer microsatellites but in more individuals, to examine clone size and distribution across the landscape.

A database of PCR primers for the chloroplast genomes of higher plants.

BACKGROUND: Chloroplast genomes evolve slowly and many primers for PCR amplification and analysis of chloroplast sequences can be used across a wide array of genera. In some cases 'universal' primers have been designed for the purpose of working across species boundaries. However, the essential information on these primer sequences is scattered throughout the literature. RESULTS: A database is presented here which assembles published primer information for chloroplast DNA. Additional primers were designed to fill gaps where little or no primer information could be found. Amplicons are either the genes themselves (typically useful in studies of sequence variation in higher-order phylogeny) or they are spacers, introns, and intergenic regions (for studies of phylogeographic patterns within and among species). The current list of 'generic' primers consists of more than 700 sequences. Wherever possible, we give the locations of the primers in the thirteen fully sequenced chloroplast genomes (Nicotiana tabacum, Atropa belladonna, Spinacia oleracea, Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, Pinus thunbergii, Marchantia polymorpha, Zea mays, Oenothera elata, Acorus calamus, Eucalyptus globulus, Medicago trunculata). CONCLUSION: The database described here is designed to serve as a resource for researchers who are venturing into the study of poorly described chloroplast genomes, whether for large- or small-scale DNA sequencing projects, to study molecular variation or to investigate chloroplast evolution.

Hybrid zones as a tool for identifying adaptive genetic variation in outbreeding forest trees: lessons from wild annual sunflowers (Helianthus spp.).

The identification and study of adaptively important genes in forest trees represents a formidable challenge because of their long generation spans. In annual or perennial herbs, formal genetic studies can be employed to identify the quantitative trait loci (QTLs) and/or candidate genes that underlie important traits, and the segregating populations can be transplanted into natural populations to measure the strength and direction of selection. However, the application of these methods to forest trees is difficult, because the creation of appropriate genetic material is extremely time-consuming in long-lived, woody plants, and lifetime fitness estimates are difficult or impossible to obtain. Although QTL mapping should in principle be feasible in wild intraspecific populations (as an alternative to artificial crosses), this approach is less likely to be successful in trees because LD (linkage disequilibrium) will decay quickly in large outbreeding plant populations. Within the present paper, we discuss a modified approach based on natural hybrid zones. We describe the use of wild annual sunflowers (Helianthus spp.) as a model for exploring the hybrid zone approach. Transplanted experimental hybrids allowed us to assess the adaptive value of individual chromosomal blocks in nature, and data on natural Helianthus hybrids suggest that similar approaches are possible in natural hybrid zones. Our results allowed us to test the role of hybridization in the origin of ecological divergence in wild sunflowers. In addition, they have practical implications for identifying adaptively important genes or QTLs in trees. This is exemplified by three temperate forest taxa, Populus (poplars, aspens, cottonwoods), Fraxinus (ash), and Quercus (oak). All three are diploid and important genomic tools are under development. Moreover, all three offer extensive hybrid zones whose likely age can be inferred from fossil data. Age data enables estimates of the size and frequency of chromosomal blocks in hybrids, thereby providing guidance in designing marker-based experiments. We predict that natural hybrid zones will be valuable tools for identifying the QTLs and/or candidate genes responsible for adaptive traits in forest trees.