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The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics., Trends in Genetics, 39 (7), ISSN:0168-9525

BackgroundDespite 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.ResultsWe 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 offsprings’ 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.ConclusionsGenome-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 bio-markers 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.

DNA copy numbers have recently emerged as an important new marker system. In the absence of a contiguous reference genome, alternative detection systems such as the comparative hybridization method have been used to detect copy number variations (CNVs). With the advent of chromosome-level resolved reference genomes based on the incorporation of long-read sequencing and powerful bioinformatics pipelines, comprehensive detection of all structural variations (SVs) in the poplar genome is now within reach. Gene CNVs and their inheritance are important because they can cause dosage effects in phenotypic variations. These are potent genetic markers that should be considered in complex trait variation such as growth and adaptation in poplar. SVs such as CNVs could be used in future genomic selection studies for poplar, especially in cases when heterosis increases hybrid performance (hybrid vigor). This Chapter reports recent findings on SVs in natural populations ofPopulusspp. as well as on artificially induced SVs in poplar to understand their potential importance in generating a considerable amount of phenotypic improvement. The Chapter concludes with an outlook on the future implementation of knowledge on SVs in poplar crop breeding.

The European black poplar (Populus nigra L.) is a riparian tree that dominates the early successional stages of alluvial floodplain forests throughout Continental Europe. River management and hybrid poplar plantations have contributed to the loss of this foundational species, and measures to conserve and restore the ecologically rich and diverse natural woodlands must sustain poplar reproduction. P. nigra is able to reproduce through either generative (sexual) or vegetative (asexual or clonal) processes and this enhances its chances for preservation in future forests. This research was undertaken to quantify the dominant reproductive strategy of natural black poplar stands, and to determine how this influenced woodland structure and successional processes within the floodplains along the lower Allier River. Two approaches were integrated: (1) analyses of the degree of clonality of stands based on DNA genotyping, sapling excavations, and the uniformity of poplar height and spatial distributions; and (2) retrospective analyses of successional processes to investigate spatiotemporal correspondences with the reproductive strategies. Structural stand patterns indicated the dominant reproductive strategy, with vertical heterogenity indicating clonality, while generative stands were more uniform. One half (50.2%, or 164 ha) of the floodplain study area supported woodlands with P. nigra stands or individuals. Of this area, 42% had apparently reproduced generatively with limited clonality; 43% had dominantly vegetative regeneration with high clonality; and 15% included generatively established, solitary old trees. The generatively and vegetatively reproduced stands displayed generally similar establishment rates but differed in their spatial patterns, with generative seedling bands or patches largely limited to barren streamside sites, while vegetative stands were more diversely distributed. The reproductive types also differed in successional dynamics, with generative stands providing more persistent successional phases with longer residence times. Thus, sexual reproduction enables woodland colonization and introduces genetic diversity, while clonal reproduction expands the population and structural diversity. Both reproductive strategies should be encouraged to provide healthy, diverse and resilient riparian woodlands.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics., Trends in Ecology & Evolution, 37 (3), ISSN:0169-5347, ISSN:1872-8383

Models to predict the effects of different silvicultural treatments on future forest development are the best available tools to demonstrate and test possible climate-smart pathways of mountain forestry. This chapter reviews the state of the art in modelling approaches to predict the future growth of European mountain forests under changing environmental and management conditions. Growth models, both mechanistic and empirical, which are currently available to predict forest growth are reviewed. The chapter also discusses the potential of integrating the effects of genetic origin, species mixture and new silvicultural prescriptions on biomass production into the growth models. The potential of growth simulations to quantify indicators of climate-smart forestry (CSF) is evaluated as well. We conclude that available forest growth models largely differ from each other in many ways, and so they provide a large range of future growth estimates. However, the fast development of computing capacity allows and will allow a wide range of growth simulations and multi-model averaging to produce robust estimates. Still, great attention is required to evaluate the performance of the models. Remote sensing measurements will allow the use of growth models across ecological gradients.

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.

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.

Over the last several decades, several lines of evidence have shown that epigenetic modifications modulate phenotype and mediate an organism&rsquo, 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.

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. Electronic supplementary material The online version of this article (10.1007/s11033-020-05527-y) contains supplementary material, which is available to authorized users.

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.

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.

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. The populations are fragmented and can be classified into three distinct clusters in the SMO of the two States (south, center and north), each group separated by a distance of about 300 km. The total area covered P. chihuahuana trees 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 also 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 vitality and genetic diversity of populations, which affect vigour, evolution and adaptability of the species, must also be considered. In this study, we examined the P. chihuahuana tree community growing in fourteen different locations, with the overall aim of discriminating the populations and clusters of this species using 22 climatic, 27 edaphic and 15 dasometric variables and three genetic diversity indices. Methods. Each location was represented by one 50 x 50 m plot established in the center of the location in which was measured the climate, soil, dasometric and genetic variables. The putative neutral and adaptive AFLP were used to calculate genetic diversity. Multivariate discriminant analysis including cross-validation was considered to test for significant differences in variables in the southern, central and northern populations and locations of the P. chihuahuana tree community. Spearman's correlation test was used to analyze the relationships between genetic diversity, population size, and the climatic, soil and dasometric variables. Results. The discriminant analysis revealed 22 highly significant variables, which separated the southern, central and northern populations. The mean genetic diversity of P. chihuahuana was significantly correlated with the mean temperature in the warmest month. Genetic diversity of P. chihuahuana calculated with putative adaptive AFLP was not statistically significantly correlated with any environmental factor. Finally, no significant correlations were observed between any of the three genetic diversity indices and population size. Discussion. At least three different ecotypes of P. chihuahuana probably exist, as local adaptation may take place because of the different environmental conditions. Therefore, future reforestation programs should take into account these different ecotypes and environmental conditions.

Only a few studies to date have conducted comparative genomics in the Myrtaceae family. Here, we report the complete sequence and bioinformatics analysis of the chloroplast genome of Syzygium cumini (L.), one of the family members. The size of S. cumini cp genome was within the range of reported angiosperm chloroplast genomes. Comparison of S. cumini cpDNA sequence with previously reported partial sequences of S. cumini revealed several SNPs that resulted in non-synonymous mutations in maturase K and NADH-plastoquinone oxidoreductase subunit-5. These polymorphic characters might serve as intra-specific markers to address whether lineage sorting from polymorphic ancestry has occurred. Comparison of the S. cumini chloroplast genome with related dicots revealed an expansion in the intergenic spacer located between IRA/large single copy (LSC) border and the first gene of LSC region, driven by sequence of 54 bp. This type of variation in the intergenic regions can be utilized in the development of species-specific vectors for chloroplast genetic engineering. Several of the longer (30–40 bp) repeats were found to be conserved in other dicot species, suggesting that they might be widespread in angiosperm chloroplast genomes.

International audience; This paper provides a review of theoretical and practical aspects related to genetic management of forest trees. The implementation of international commitments on forest genetic diversity has been slow and partly neglected. Conservation of forest genetic diversity is still riddled with problems, and complexities of national legal and administrative structures. Europe is an example of a complex region where the dis- tribution ranges of tree species extend across large geographical areas with profound environmental dif- ferences, and include many countries. Conservation of forest genetic diversity in Europe has been hampered by a lack of common understanding on the management requirements for genetic conserva- tion units of forest trees. The challenge resides in integrating scientific knowledge on conservation genet- ics into management of tree populations so that recommendations are feasible to implement across different countries. Here, we present pan-European minimum requirements for dynamic conservation units of forest genetic diversity. The units are natural or man-made tree populations which are managed for maintaining evolutionary processes and adaptive potential across generations. Each unit should have a designated status and a management plan, and one or more tree species recognized as target species for genetic conservation. The minimum sizes of the units are set at 500, 50 or 15 reproducing individuals depending on tree species and conservation objectives. Furthermore, silvicultural interventions should be allowed to enhance genetic processes, as needed, and field inventories carried out to monitor regen- eration and the population size. These minimum requirements are now used by 36 countries to improve management of forest genetic diversity.

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.

Date palm (Phoenix dactylifera L.) is an economically important and widely cultivated palm of the family Arecaceae. We sequenced the complete date palm chloroplast genome (cpDNA) from Pakistani cv. ‘Aseel’, using a combination of Sanger-based and next-generation sequencing technologies. Being very similar to a sequence from a Saudi Arabian date palm cultivar ‘Khalas’ published recently, the size of the genome was 158,458 bp with a pair of inverted repeat (IR) regions of 27,276 bp that were separated by a large single-copy (LSC) region of 86,195 bp and a small single-copy (SSC) region of 17,711 bp. Genome annotation demonstrated a total of 138 genes, of which 89 were protein coding, 39 were tRNA, and eight were rRNA genes. Comparison of cpDNA sequences of cultivars ‘Aseel’ and ‘Khalas’ showed following intervarietal variations in the LSC region; (a) two SNPs in intergenic spacers and one SNP in the rpoc1 gene, (b) polymorphism in two mono-nucleotide simple sequence repeats (SSR), and (c) a 4-bp indel in the accD-psaI intergenic spacer. The SSC region has a polymorphic site in the mono-nucleotide SSR located at position 120,710. We also compared cv. ‘Aseel’ cpDNA sequence with partial P. dactylifera cpDNA sequence entries deposited in Genbank and identified a number of potentially useful polymorphisms in this species. Analysis of date palm cpDNA sequences revealed a close relationship with Typha latifolia. Occurrence of small numbers of forward and inverted repeats in date palm cpDNA indicated conserved genome arrangement.

The nativity of Populus alba in the Mediterranean has only been confirmed in the last decade, following the discovery of 8,000-year-old leaf imprints in Southern France. Recent evidence has even emerged from molecular studies suggesting that the species is native to some of the islands, and these populations may be relicts of a native flora that arrived there much earlier than previously thought. In view of this, samples obtained from the Central Mediterranean archipelago of Malta and other neighbouring regions were analysed to determine the native status of the Maltese populations and possibly trace their origins. All 38 samples were investigated in order to assess the genetic diversity and origin of Maltese trees. Nuclear microsatellite analysis revealed that all 28 trees sampled from the two islands of Malta belonged to one clone. Chloroplast data suggested relatedness of the Maltese clone to Italian P. alba samples. However, nuclear data suggested additional admixture through pollen from North Africa. Existing archival and palaeontological records were also examined for any supporting evidence. On considering the latter records in combination with molecular evidence, we arrived to the conclusion that arrival of this clone in Malta through human introduction in the sixteenth century is the most likely explanation, since alternative scenarios like autovegetative propagation or arrival by seed seem highly unlikely.

inter-individual component for symmetric aspects and an important intra-individual component for asymmetric aspects of variation in leaf outlines. Symmetric traits displayed a striking variety of phenotypes in hybrids compared to their parental species, consistent with the notion of the phenotypic mosaic. Linear discriminant analysis of these morphometric traits revealed (1) clear differentiation between parental species and (2) divergence between recombinant hybrids and their sympatric backcross parent P. alba, mediated primarily by two P. tremula–like and two transgressive traits. Our results demonstrate the usefulness of geometric morphometrics to interrogate multiple independent phenotypic characters and detect individual traits affected by introgression and divergence in taxa with porous genomes. Digital image-based morphometrics holds great promise for large-scale studies of relationships between DNA polymorphism and phenotypes in evolutionary biology.

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. × canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. × canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P. × canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.

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.

Ten cpSCAR markers that show polymorphism in Prunus avium were used to fingerprint sweet cherry cultivars. The purpose of the study was also to contribute to identification and to help determine their genetic interrelationships. Samples of '0900 Ziraat', a superior Turkish variety, which were collected in several locations all over Turkey, had identical cpSCAR patterns, and they resembled a common European haplotype, A. 'Sweetheart', 'Summit' and 'Canada Giant' and their haplotype are intermediate between the previously described haplotypes A and B, which were originally found in Central and Eastern European sweet and wild cherries, and those from Northern Turkey, respectively. The data therefore suggests a local maternal descent (within Europe and Asia Minor) of the cultivars analysed. Our results show that chloroplast DNA analysis is a straightforward way to classify cherry cultivars. We compare our results to others previously reported for sweet cherry cultivars, and conclude that cpSCAR diversity data could be considered for phylogenetic studies in this group.

The renewed interest in the use of hybrid zones for studying speciation calls for the identification and study of hybrid zones across a wide range of organisms, especially in long-lived taxa for which it is often difficult to generate interpopulation variation through controlled crosses. Here, we report on the extent and direction of introgression between two members of the ‘model tree’ genus Populus : Populus alba (white poplar) and Populus tremula (European aspen), across a large zone of sympatry located in the Danube valley. We genotyped 93 hybrid morphotypes and samples from four parental reference populations from within and outside the zone of sympatry for a genome-wide set of 20 nuclear microsatellites and eight plastid DNA restriction site polymorphisms. Our results indicate that introgression occurs preferentially from P. tremula to P. alba via P. tremula pollen. This unidirectional pattern is facilitated by high levels of pollen vs. seed dispersal in P. tremula (pollen/seed flow = 23.9) and by great ecological opportunity in the lowland floodplain forest in proximity to P. alba seed parents, which maintains gene flow in the direction of P. alba despite smaller effective population sizes ( N e ) in this species ( P. alba N e c. 500–550; P. tremula N e c. 550–700). Our results indicate that hybrid zones will be valuable tools for studying the genetic architecture of the barrier to gene flow between these two ecologically divergent Populus species.

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).

Summary 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.

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.

Conserved chloroplast (cp) DNA primer pairs are useful in plant molecular genetics, evolution and ecology. We have designed 20 conserved cpDNA primer pairs that, in combination with 18 previously described ones, amplify overlapping fragments (mean size of 2.5 kb) spanning the large single copy (LSC) region from Eudicots. These 38 primer pairs as well as eight primer pairs flanking cpDNA microsatellites were tested on 20 plant species belonging to 13 families. At least 79% and up to 100% of the LSC (> 86 kb) can be amplified. Many primer pairs are robust and work with all species.

Microsatellites were used for genetic analysis of maternal half-sib families of Quercus robur, pedunculate oak, a highly outcrossing tree species. A model half-sib family including the mother tree and 28 offspring individuals as well as samples from six single tree harvests from a forestry company, 4–8 individuals each, were genotyped at 9 microsatellite loci. No prior information about the genotypes of the mother trees were available for these six seedlot samples. Analysis of the model half-sib family revealed that the maternal genotypes can be inferred from the offspring genotypes due to codominant Mendelian inheritance of the microsatellites. Analysis of the single tree harvests, supplied as six maternal half-sib families, revealed contaminations with unrelated seedlings in four out of six cases. Average relatedness between the remaining individuals indicated that they were indeed half-sibs, probably with a proportion of full-sibs among them. For five samples the genotypes of the mother trees were partially inferred from the offspring genotypes. The supposed number of five different mother trees was confirmed by direct comparison of the maternal genotypes and by pairwise FST calculations between families. We show that correct genotype reconstruction can be confirmed by monitoring recombination events between linked markers. Our results demonstrate that microsatellite analysis is a suitable means to approach two key problems of legal regulations on the marketing of seed material from pedunculate oak: the number of trees included in seed harvests and the detection of seed contaminations.

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.

“Active learning” is a concept for ensuring student attention during long lectures. This concept was used in a Molecular Genetics evening lecture class of the first author at a university of applied sciences. The purpose of this paper is to compare exam results in relation to whether a certain subject question was supported by an active learning element during the lecture, or not. The results of the final exams, while giving strong hints for the positive effect of the active learning elements, were not simple to interpret, but the authors' general conclusion is that active learning elements can greatly enhance a lecture class under certain conditions - they should be creative, thought-provoking and unexpected, but also meet the students where the actually are in terms of their basic knowledge and current situation. Moderately frequent, interesting elements will leave a positive impression that may even become manifest in exam results.

European larch (Larix decidua) is a native species from the Alps and from several mountainous ranges in Central Europe. Its early introduction outside its native range, especially in lowlands western and northern Europe has been problematic for ecological (phenology) and sanitary (canker) reasons due to the lack of knowledge of its genetic variability. As a substitute, Japanese larch (Larix kaempferi) and then hybrids between European and Japanese larches have been preferred in these regions together with Siberian larches in northern Scandinavia. Results from intensive provenances testing (as early as in the 1940s) have been the corner stone on which breeding programmes have developed both at the intra- (mostly in Central and Eastern Europe) and inter-specific (mostly in North-Western Europe) levels. Inter-specific hybridization is probably the most emblematic and promising breeding strategy for larch, taking benefit of both species trait complementarity, hybrid vigour (heterosis) and hybrid homeostasis. While seed orchards are now the main source for dissemination of genetic progress for European larch, mass-production of hybrid larch remains problematic either through sexual (hybridization seed orchard) or asexual (vegetative propagation by cutting) ways and still impedes large deployment of hybrids in Europe. While progress is expected from current research on this topic (coupling of somatic embryogenesis with bulk vegetative propagation by cutting, 2nd generation hybridization orchard), other hot issues include decoding of heterosis, evaluation of genetic variability for the most original larch wood properties (heartwood formation and durability), adaptation to climate, etc. Joined efforts at European level are expected for genetic resource conservation, breeding and deployment, and tackling of several scientific and technical bottlenecks.

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.

As opposed to standard polymerase chain reaction (PCR) using specific primers, genome analysis involving short random primers, for example RAPD, may yield inconsistent results if crude plant DNA preparations are used as the template. When RNase A, a thermostable enzyme, was added to such reactions, highly repeatable banding patterns were obtained from crude plant DNA, thus speeding up analyses substantially.

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.

International audience; Conservation of Populus genetic resources can be defined at three levels: conservation of all Populus species, conservation of the variation present within species, and the conservation of the key evolutionary processes that will enhance further evolution of Populus species. In this context, genomics, the study of an organism’ entire genome, can be considered as a powerful tool that would allow us to reach an integrated view of the genetic variation to be conserved. In particular, the application of principles and methodologies of population genomics to Populus hold great potential to answer some of the most relevant questions in poplar, aspen and cottonwood conservation genetics, such as how much functional variation is currently present and how to conserve it. Here, we review recent progress in applied conservation genetics in Populus and we sketch the potential for conservation genomics to transform this field in the very near future.

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.

Summary • 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. × 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. × canescens hybrids tended to build larger clones extending over larger areas than P. alba. The 3.4 times stronger SGS in the P. × 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.

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.

The use of admixed human populations to scan the genome for chromosomal segments affecting complex phenotypic traits has proved a powerful analytical tool. However, its potential in other organisms has not yet been evaluated. Here, we use DNA microsatellites to assess the feasibility of this approach in hybrid zones between two members of the 'model tree' genus Populus: Populus alba (white poplar) and Populus tremula (European aspen). We analyzed samples of both species and a Central European hybrid zone (N=544 chromosomes) for a genome-wide set of 19 polymorphic DNA microsatellites. Our results indicate that allele frequency differentials between the two species are substantial (mean delta=0.619+/-0.067). Background linkage disequilibrium (LD) in samples of the parental gene pools is moderate and should respond to sampling schemes that minimize drift and account for rare alleles. LD in hybrids decays with increasing number of backcross generations as expected from theory and approaches background levels of the parental gene pools in advanced generation backcrosses. Introgression from P. tremula into P. alba varies strongly across marker loci. For several markers, alleles from P. tremula are slightly over-represented relative to neutral expectations, whereas a single locus exhibits evidence of selection against P. tremula genotypes. We interpret our results in terms of the potential for admixture mapping in these two ecologically divergent Populus species, and we validate a modified approach of studying genotypic clines in 'mosaic' hybrid zones.

We report the draft genome of the black cottonwood tree, Populus trichocarpa . Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis , ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

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 life-time 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. © 2004 Elsevier B.V. All rights reserved.