Aspen Genotypes Research Articles

Forest defoliation by an invasive outbreak insect: Catastrophic consequences for a charismatic mega moth.

Earth is now experiencing declines in insect abundance and diversity unparalleled in human history. The drivers underlying those declines are many, complex, and incompletely known. Here, using a natural experiment, we report the first test of the hypothesis that forest defoliation by an invasive outbreak insect compromises the fitness of a native insect via damage-induced increases in toxicity of the forest canopy. We demonstrate that defoliation by the invasive spongy moth (Lymantria dispar) elicits an average 8.4-fold increase in foliar defense expression among aspen (Populus tremuloides) genotypes. In turn, elevated defense dramatically reduces survivorship, feeding, and growth of a charismatic mega moth (Anthereae polyphemus). This work suggests that changes to the phytochemical landscape of forests, mediated by invasive outbreak insects, are likely to negatively impact native insects, with potential repercussions for community diversity and ecosystem function across expansive scales.

Growth ranking of hybrid aspen genotypes and its linkage to leaf gas exchange

BackgroundAfforestation of non-forestland is a new measure by the European Union to enhance climate mitigation and biodiversity. Hybrid aspen (Populus tremula L. × P. tremuloides Michx.) is among the suitable tree species for afforestation to produce woody biomass. However, the best performing genotypic material for intensive biomass production and its physiological adaptation capacity is still unclear. We compared 22 hybrid aspen genotypes growth and leaf physiological characteristics (stomatal conductance, net photosynthesis, intrinsic water-use efficiency) according to their geographical north- or southward transfer (European P. tremula parent from 51° to 60° N and North American P. tremuloides parent from 45° to 54° N) to hemiboreal Estonia (58° N) in a completely randomized design progeny trial. We tested whether the growth ranking of genotypes of different geographical origin has changed from young (3-year-old) to mid-rotation age (13-year-old). The gas exchange parameters were measured in excised shoots in 2021 summer, which was characterised with warmer (+ 4 °C) and drier (17% precipitation from normal) June and July than the long-term average.ResultsWe found that the northward transfer of hybrid aspen genotypes resulted in a significant gain in growth (two-fold greater diameter at breast height) in comparison with the southward transfer. The early selection of genotypes was generally in good accordance with the middle-aged genotype ranking, while some of the northward transferred genotypes showed improved growth at the middle-age period in comparison with their ranking during the early phase. The genotypes of southward transfer demonstrated higher stomatal conductance, which resulted in higher net photosynthesis, and lower intrinsic water-use efficiency (iWUE) compared with northward transfer genotypes. However, higher photosynthesis did not translate into higher growth rate. The higher physiological activity of southern transferred genotypes was likely related to a better water supply of smaller and consequently more shaded trees under drought. Leaf nitrogen concentration did not have any significant relation with tree growth.ConclusionsWe conclude that the final selection of hybrid aspen genotypes for commercial use should be done in 10–15 years after planting. Physiological traits acquired during periods of droughty conditions may not fully capture the growth potential. Nonetheless, we advocate for a broader integration of physiological measurements alongside traditional traits (such as height and diameter) in genotype field testing to facilitate the selection of climate-adapted planting material for resilient forests.

Salicylic acid metabolism and signalling coordinate senescence initiation in aspen in nature

Deciduous trees exhibit a spectacular phenomenon of autumn senescence driven by the seasonality of their growth environment, yet there is no consensus which external or internal cues trigger it. Senescence starts at different times in European aspen (Populus tremula L.) genotypes grown in same location. By integrating omics studies, we demonstrate that aspen genotypes utilize similar transcriptional cascades and metabolic cues to initiate senescence, but at different times during autumn. The timing of autumn senescence initiation appeared to be controlled by two consecutive “switches”; 1) first the environmental variation induced the rewiring of the transcriptional network, stress signalling pathways and metabolic perturbations and 2) the start of senescence process was defined by the ability of the genotype to activate and sustain stress tolerance mechanisms mediated by salicylic acid. We propose that salicylic acid represses the onset of leaf senescence in stressful natural conditions, rather than promoting it as often observed in annual plants.

Environment and Genotype Influence Quantitative and Qualitative Variation in Condensed Tannins in Aspen.

Condensed tannins (CTs) are abundant, ecologically-relevant secondary metabolites in many plants, which respond to variables associated with anthropogenic environmental change. While many studies have reported how genetic and environmental factors affect CT concentrations, few have explored how they influence CT molecular structure. Here, using trembling aspen (Populus tremuloides) as a model organism, we report how foliar CT concentrations, polymer sizes, representation of procyanidins and prodelphinidins, and stereochemistry vary in response to changes in air temperature (warming and freeze damage), air composition (elevated CO2 and O3), soil quality (nutrients and microbiome), and herbivory (mammal and lepidopteran). Use of multiple aspen genotypes enabled assessment of genetic influences on aspen CTs. CT concentration and composition were analyzed by thiolysis-ultra high performance liquid chromatography/mass spectrometry in archived leaf samples from prior experiments. All environmental variables explored except for soil microbiome influenced both CT quantity and quality, with climate factors appearing to have larger effect magnitudes than herbivory. Climate, soil, and herbivory effects varied among genotypes, while air composition effects were consistent across genotypes. Considering that CT properties (concentrations and molecular structures) mediate functions at the organismal through ecosystem scales, intraspecific variation in responses of CT properties to environmental factors could provide a pathway through which environmental change exerts selective pressure on Populus populations. Future studies are needed to identify the molecular-level mechanisms by which environmental factors influence CT concentrations and structures, and to establish their ecological and evolutionary significance.

Genetic markers and tree properties predicting wood biorefining potential in aspen (Populus tremula) bioenergy feedstock

BackgroundWood represents the majority of the biomass on land and constitutes a renewable source of biofuels and other bioproducts. However, wood is recalcitrant to bioconversion, raising a need for feedstock improvement in production of, for instance, biofuels. We investigated the properties of wood that affect bioconversion, as well as the underlying genetics, to help identify superior tree feedstocks for biorefining.ResultsWe recorded 65 wood-related and growth traits in a population of 113 natural aspen genotypes from Sweden (https://doi.org/10.5061/dryad.gtht76hrd). These traits included three growth and field performance traits, 20 traits for wood chemical composition, 17 traits for wood anatomy and structure, and 25 wood saccharification traits as indicators of bioconversion potential. Glucose release after saccharification with acidic pretreatment correlated positively with tree stem height and diameter and the carbohydrate content of the wood, and negatively with the content of lignin and the hemicellulose sugar units. Most of these traits displayed extensive natural variation within the aspen population and high broad-sense heritability, supporting their potential in genetic improvement of feedstocks towards improved bioconversion. Finally, a genome-wide association study (GWAS) revealed 13 genetic loci for saccharification yield (on a whole-tree-biomass basis), with six of them intersecting with associations for either height or stem diameter of the trees.ConclusionsThe simple growth traits of stem height and diameter were identified as good predictors of wood saccharification yield in aspen trees. GWAS elucidated the underlying genetics, revealing putative genetic markers for bioconversion of bioenergy tree feedstocks.

Whole genome expression analysis of trembling aspen (Populus tremuloides) genotypes exposed to increasing concentrations of nickel

Excess metals in the soil are a known type of abiotic stress and many plant species have evolved specific metal resistance mechanisms to cope and avoid or reduce toxicity symptoms. Studies on P. tremuloides genetic response to nickel are limited. This present study aims to 1) Assess gene expression dynamics in P. tremuloides seedlings treated with varying concentrations of nickel salts and, 2) Compare gene expression profiles among the different treatment groups. Trembling aspen (Populus tremuloides) seedlings were treated with varying concentrations of nickel nitrates (150 mg Ni / 1 kg of dry soil, 800 mg / kg, and 1, 600 mg / kg). The whole genome expression was analyzed using Illumina sequencing. Overall, 52,987 genes were identified from which 36,770 genes were selected as differently expressed. In general, there was an increase in number of differentially expressed genes as the nickel concentration increased when compared to water. The number of downregulated (439–600) and upregulated (123–560) genes increases with the nickel concentration increase. A detailed analysis suggested that the 800 mg / kg nickel concentration is the threshold at which an early abiotic stress response may be triggered as seen by the highly upregulated LEA protein and two calcium binding proteins when compared to water. For the highest nickel concentration, 7-deoxyloganetin glucosyltransferase was highly upregulated while an auxin response factor, Flavonol 3-sulfotransferase, and a predicted ABC transporter family protein were downregulated. The heatmap showed that the genes were grouped into six clusters based on the changes in expression as nickel concentration increased. The cluster of genes that had increased gene expression with increasing nickel concentration also had multiple enriched Gene ontology (GO) terms related to heavy metal and abiotic stress including metal ion transport, antioxidant activity, photosynthesis, and ribosomal activity. GO terms that decreased in expression with increasing nickel concentrations include ATP and ADP binding, protein kinase activity, integral component of membrane, protein phosphorylation and transmembrane transport.

Molecular studies of rust on European aspen suggest an autochthonous relationship shaped by genotype.

Forests are at increasing risk from pathogen outbreak. Climate change for example enhance the risk of local disease outbreaks, and naturalization of exotic pathogens may follow human activities, warranting robust pest surveillance routines to support forest management. Melampsora pinitorqua (pine twisting rust) is of concern in Swedish forestry, and here we evaluate the use of visible rust scores (VRS) on its obligate summer host, European aspen (Populus tremula) as a tool for quantification of the pathogen. With use of species-specific primers, we could detect the native rust, but we failed to detect two exotic rusts (M. medusae and M. larici-populina). We found that aspen genotype determined the presence of fungal genetic markers (amplifying the ITS2 region of the fungal rDNA sequence) as well as DNA sequences specific to M. pinitorqua. We correlated VRS with the amount of fungal DNA in the same leaf, and we related the findings to aspen genotype-specific parameters such as the ability to synthesize and store leaf condensed tannins (CT). At the genotype level both positive and negative relationships were observed between CTs, fungal markers, and rust infestations. However, at the population level, foliar CT concentrations correlated negatively with general fungal- and rust-specific marker abundances. Our results, therefore, do not support the use of VRS to assess Melampsora infestation in Aspen. They do, however, suggest that the relationship between European aspen and rust infestation may be characterized as autochthonous in northern Sweden.

Genotypic variation rather than ploidy level determines functional trait expression in a foundation tree species in the presence and absence of environmental stress.

At the population level, genetic diversity is a key determinant of a tree species' capacity to cope with stress. However, little is known about the relative importance of the different components of genetic diversity for tree stress responses. We compared how two sources of genetic diversity, genotype and cytotype (i.e. differences in ploidy levels), influence growth, phytochemical and physiological traits of Populus tremuloides in the presence and absence of environmental stress. In a series of field studies, we first assessed variation in traits across diploid and triploid aspen genotypes from Utah and Wisconsin under non-stressed conditions. In two follow-up experiments, we exposed diploid and triploid aspen genotypes from Wisconsin to individual and interactive drought stress and defoliation treatments and quantified trait variations under stress. We found that (1) tree growth and associated traits did not differ significantly between ploidy levels under non-stressed conditions. Instead, variation in tree growth and most other traits was driven by genotypic and population differences. (2) Genotypic differences were critical for explaining variation of most functional traits and their responses to stress. (3) Ploidy level played a subtle role in shaping traits and trait stress responses, as its influence was typically obscured by genotypic differences. (4) As an exception to the third conclusion, we showed that triploid trees expressed 17 % higher foliar defence (tremulacin) levels, 11 % higher photosynthesis levels and 23 % higher rubisco activity under well-watered conditions. Moreover, triploid trees displayed greater drought resilience than diploids as they produced 35 % more new tissue than diploids when recovering from drought stress. Although ploidy level can strongly influence the ecology of tree species, those effects may be relatively small in contrast to the effects of genotypic variation in highly diverse species.

Microclonal reproduction of common aspen (Populus tremula L.) genotypes in the Republic of Tatarstan

The research presents the results of an experiment on propagation of core rot-resistant aspen clones (Populus tremula L.) of diploid and triploid genotypes using microclonal propagation method and their introduction into forestry production in the Republic of Tatarstan. The expediency of using this method in the conditions of the Republic for obtaining healthy aspen planting material is proved, and the prospects of reproduction of clone No.35 with a triploid genotype are shown. The reliable difference of test tube regenerating plants of triploid forms in the height of the shoot and in the development of root systems is proved

Impact of Plant-Associated Bacteria on the In Vitro Growth and Pathogenic Resistance against Phellinus tremulae of Different Aspen (Populus) Genotypes.

Aspens (Populus tremula and its hybrids), economically and ecologically important fast-growing trees, are often damaged by Phellinus tremulae, a rot-causing fungus. Plant-associated bacteria can be used to increase plant growth and resistance; however, no systematic studies relating the activity of symbiotic bacteria to aspen resistance against Phellinus tremulae have been conducted so far. The present pioneer study investigated the responses of two Populus tremula and two P. tremula × P. tremuloides genotypes to in vitro inoculations with, first, either Pseudomonas sp. or Paenibacillus sp. bacteria (isolated originally from hybrid aspen tissue cultures and being most closely related to Pseudomonas oryzihabitans and Paenibacillus tundrae, respectively) and, in the subsequent stage, with Phellinus tremulae. Both morphological parameters of in vitro-grown plants and biochemical content of their leaves, including photosynthesis pigments and secondary metabolites, were analyzed. It was found that both Populus tremula × P. tremuloides genotypes, whose development in vitro was significantly damaged by Phellinus tremulae, were characterized by certain responses to the studied bacteria: decreased shoot development by both Paenibacillus sp. and Pseudomonas sp. and increased phenol content by Pseudomonas sp. In turn, these responses were lacking in both Populus tremula genotypes that showed in vitro resistance to the fungus. Moreover, these genotypes showed positive long-term growth responses to bacterial inoculation, even synergistic with the subsequent fungal inoculation. Hence, the studied bacteria were demonstrated as a potential tool for the improved in vitro propagation of fungus-resistant aspen genotypes.

Response of aspen genotypes to browsing damage is not influenced by soil community diversity

Both environmental and genetic factors influence plant growth and foliar chemistry. The purpose of this experiment was to investigate the independent and interactive effects of soil community (and soil community diversity), tree genotype, and simulated browsing damage on the growth, biomass allocation and foliar chemistry of Populus tremuloides. We employed a factorial randomized complete block design with four soil treatments of varying diversity, five aspen genotypes, and a simulated ungulate browsing treatment. In the second year of growth, half the trees were subjected to simulated ungulate browsing, and 2 mo later trees were harvested and analyzed for biomass, biomass allocation, and foliar chemistry. Tree genotype was the strongest driver of foliar chemistry, but simulated browsing was the strongest driver of plant growth. Soil community influenced plant growth but not foliar chemistry, and soil community effects were rarely modified by tree genotype or simulated browsing. While environment and genotype had relatively equal influences on plant growth, tree genotype had a stronger influence than environmental effects (soil and simulated browsing) on foliar chemistry. We also documented negative plant–soil feedbacks for tree biomass. Our results highlight future opportunities for research on the potential effects of soil community diversity on trees.

Полевые испытания размноженных in vitro клонов осины (Populus tremula L.): рост, продуктивность, качество древесины, генетическая стабильность

The widespread introduction of highly productive and rot-resistant forms and hybrids of aspen (Populus tremula L.) into the forest production is constrained by the lack of effective methods of their vegetative reproduction. Achievements of modern forest biotechnology enable in vitro cloning of economically valuable aspen genotypes and obtain planting material for plantation forest growing. The study of the manifestation features of valuable traits and genetic stability of in vitro clones propagated in the field (ex vitro) is important for the development of effective and reliable propagation methods that guarantee the preservation of the genetic and economic value of parent trees for their sustainable and targeted reproduction. The results of genetic stability evaluation, growth dynamics, productivity and wood quality of three in vitro propagated aspen clones (6/3, 15/01, and 20/4) in the field are presented. Micropropagation was carried out by direct organogenesis using hormone-free nutrient media. According to the results of 17-year field trials, clones demonstrate good growth and sufficiently high productivity of stands; preserve the growth characteristics of parent trees, showing no signs of somaclonal variability. At the age of 17, the average height of the trees was 18–20 m; the strunk diameter was 25–30 cm; the trunk volume was 0.391–0.553 m3; the timber volume was 111–227 m3/ha at trees placing 4×4 and survival rate 44–71 %. High genetic stability of clones in vitro and ex vitro was revealed based on the results of analysis of microsatellite loci, ploidy level and mixoploidy. It is shown that the triploid clone 15/01, which has high myxoploidy level, is characterized by a higher preservation and better wood quality attributes (wooddensity and fiber length) than diploid clones. This is probably due to the optimal balanced ratio of cells with different ploidy levels (triploid, diploid, and aneuploid), which provides the most effective expression of genes in specific environmental conditions.For citation: Mashkina O.S., Shabanova E.A., Varivodina I.N., Grodetskaia T.A. Field Trials of in vitro Propagated Aspen Clones (Populus tremula L.): Growth, Productivity, Wood Quality, and Genetic Stability. Lesnoy Zhurnal [Russian Forestry Journal], 2019, no. 6, pp. 25–38. DOI: 10.17238/issn0536-1036.2019.6.25

Chemical defense over decadal scales: Ontogenetic allocation trajectories and consequences for fitness in a foundation tree species

Abstract Expression of herbivore defense traits can change dramatically during the course of plant development. Little is known, however, about the degree of genetic or sexual variation in these ontogenetic defense trajectories or whether the trajectories themselves are adaptive, especially in long‐lived species. We used a 13‐year dataset of chemical defense traits, growth and survivorship from a common garden of trembling aspen (Populus tremuloides) genotypes to document long‐term defense trajectories and their relationship to tree fitness during juvenile and early mature stages. Overall, concentrations of the two principal classes of aspen defense compounds (salicinoid phenolic glycosides [SPGs] and condensed tannins [CTs]) decreased to differing degrees in foliage of juvenile trees and then remained relatively constant in maturity. Initial values, juvenile rates of change and average mature values all exhibited significant genetic variation for both SPGs and CTs. Relationships between defense trajectory parameters and metrics of tree fitness (growth and survivorship) depended on compound type and tree sex. Females with higher‐allocation SPG trajectories (high initial juvenile concentrations, slow juvenile declines, high mature concentrations) grew more slowly relative to females with lower‐allocation trajectories. In males, higher‐allocation SPG trajectories had a lesser effect on growth but were associated with reduced mortality. Juvenile CT trajectories were not correlated with tree fitness, but average CT concentration in maturity was positively related to growth in females. These results suggest that ontogenetic defense trajectories are adaptive and subject to natural selection. Genotypic variation and ontogeny shape tree defensive chemistry, both independently and interactively. These patterns of defense expression have the potential to structure trophic interactions and the genetic composition of forests in both space and time. A free Plain Language Summary can be found within the Supporting Information of this article.

Storage lipid accumulation is controlled by photoperiodic signal acting via regulators of growth cessation and dormancy in hybrid aspen.

The signalling pathways that control seasonal modulation of carbon metabolism in perennial plants are poorly understood. Using genetic, metabolic and natural variation approaches, we identify factors mediating photoperiodic control of storage lipid accumulation in the model tree hybrid aspen (Populus tremula×tremuloides). We characterized lipid accumulation in transgenic hybrid aspen with impaired photoperiodic and hormonal responses. Genome-wide association mapping was performed in Swedish aspen (P.tremula) genotypes to determine genetic loci associated with genotype variation in lipid content. Our data show that the storage lipid triacylglycerol (TAG) accumulates in cambial meristem and pith rays of aspen in response to photoperiodic signal controlling growth cessation and dormancy induction. We show that photoperiodic control of TAG accumulation is mediated by the FLOWERING LOCUS T/CONSTANS module, which also controls the induction of growth cessation. Hormonal and chromatin remodelling pathways also contribute to TAG accumulation by photoperiodic signal. Natural variation exists in lipid accumulation that is controlled by input from multiple loci. Our data shed light on how the control of storage metabolism is temporally coordinated with growth cessation and dormancy by photoperiodic signal, and reveals that storage lipid accumulation between seeds and perennating organs of trees may involve distinct regulatory circuits.

Genetic, morphological, and spectral characterization of relictual Niobrara River hybrid aspens (Populus × smithii).

Aspen groves along the Niobrara River in Nebraska have long been a biogeographic curiosity due to morphological differences from nearby remnant Populus tremuloides populations. Pleistocene hybridization between P. tremuloides and P. grandidentata has been proposed, but the nearest P. grandidentata populations are currently several hundred kilometers east. We tested the hybrid-origin hypothesis using genetic data and characterized putative hybrids phenotypically. We compared nuclear microsatellite loci and chloroplast sequences of Niobrara River aspens to their putative parental species. Parental species and putative hybrids were also grown in a common garden for phenotypic comparison. On the common garden plants, we measured leaf morphological traits and leaf-level spectral reflectance profiles, from which chemical traits were derived. The genetic composition of the three unique Niobrara aspen genotypes is consistent with the hybridization hypothesis and with maternal chloroplast inheritance from P. grandidentata. Leaf margin dentition and abaxial pubescence differentiated taxa, with the hybrids showing intermediate values. Spectral profiles allowed statistical separation of taxa in short-wave infrared wavelengths, with hybrids showing intermediate values, indicating that traits associated with internal structure of leaves and water absorption may vary among taxa. However, reflectance values in the visible region did not differentiate taxa, indicating that traits related to pigments are not differentiated. Both genetic and phenotypic results support the hypothesis of a hybrid origin for these genetically unique aspens. However, low genetic diversity and ongoing ecological and climatic threats to the hybrid taxon present a challenge for conservation of these relictual boreal communities.

Autumn senescence in aspen is not triggered by day length.

Autumn senescence in mature aspens, grown under natural conditions, is initiated at almost the same date every year. The mechanism of such precise timing is not understood but we have previously shown that the signal must be derived from light. We studied variation in bud set and autumn senescence in a collection of 116 natural Eurasian aspen (Populus tremula) genotypes, from 12 populations in Sweden and planted in one northern and one southern common garden, to test the hypothesis that onset of autumn senescence is triggered by day length. We confirmed that, although bud set seemed to be triggered by a critical photoperiod/day length, other factors may influence it. The data on initiation of autumn senescence, on the other hand, were incompatible with the trigger being the day length per se, hence the trigger must be some other light-dependent factor.

Spring and autumn phenology of hybrid aspen (Populus tremula L. × P. tremuloides Michx.) genotypes of different geographic origin in hemiboreal Estonia§

Increasing demand for renewable energy resources and the need to mitigate climate change have raised interest in short-rotation forestry with fast-growing deciduous trees like hybrid aspen (Populus tremula L. × P. tremuloides Michx.) in northern Europe. Given that climate warming has already considerably extended the growing season in this region, northward transfer of genotypes could improve forest plantation productivity and enable more efficient mitigation of climate change. We studied the spring and autumn phenology of hybrid aspen genotypes of different geographic origin (European P. tremula parent from 51° to 60° N and North American P. tremuloides parent from 45° to 54° N) 3 and 6 years after planting in a progeny trial established in Estonia at 58° N. The effect of geographic origin on spring and autumn phenology of hybrid aspen was evident at the age of 3 and 6 years. Geographic origin did not affect spring phenology. However, hybrids with P. tremula parents of northern origin, with bud-burst occurring some days later, were able to unfold and develop full-sized leaves faster than genotypes with early bud-burst. The main differences between different geographic origins appeared in the autumn of year 6, when genotypes of northern origin (60° N) started autumn defoliation significantly earlier than those of southern origin (51° to 57° N). The genotypes of southern origin (55° 53′ to 57° 31′ N) had a period from bud-burst to defoliation 27 days longer than that of genotypes of northern origin (60° 22′ N). The interval between spring and autumn phenological processes showed significant positive correlation with current annual height growth for both study years. Hybrid aspen genotypes from 55° to 57° N responded well to northward transfer, having a longer leafy period and greater height increment than southward transferred genotypes. Northward-transferred genotypes were apparently better adapted to climate-change-induced extension of the growing season at higher latitudes.

Testing Transgenic Aspen Plants with bar Gene for Herbicide Resistance under Semi-natural Conditions

Obtaining herbicide resistant plants is an important task in the genetic engineering of forest trees. Transgenic European aspen plants (Populus tremula L.) expressing the bar gene for phosphinothricin resistance have been produced using Agrobacterium tumefaciens-mediated transformation. Successful genetic transformation was confirmed by PCR analysis for thirteen lines derived from two elite genotypes. In 2014-2015, six lines were evaluated for resistance to herbicide treatment under semi-natural conditions. All selected transgenic lines were resistant to the herbicide Basta at doses equivalent to 10 l/ha (twofold normal field dosage) whereas the control plants died at 2.5 l/ha. Foliar NH4-N concentrations in transgenic plants did not change after treatment. Extremely low temperatures in the third ten-day period of October 2014 revealed differences in freeze tolerance between the lines obtained from Pt of f2 aspen genotypes. Stable expression of the bar gene after overwintering outdoors was confirmed by RT-PCR. On the basis of the tests, four transgenic aspen lines were selected. The bar gene could be used for retransformation of transgenic forest trees expressing valuable traits, such as increased productivity.

Phytochemical traits underlie genotypic variation in susceptibility of quaking aspen (Populus tremuloides) to browsing by a keystone forest ungulate

Summary Overbrowsing by ungulates is a major cause of poor aspen stand regeneration across North America and Eurasia. In general, factors driving ungulate browser preferences include concentrations of plant secondary compounds and the nutritional composition (non‐structural carbohydrates, protein and minerals) of foliage. While each of these phytochemical factors has been shown to independently influence ungulate preference, the relative impact of each factor is unknown, as no study to date has examined them simultaneously. Plant fitness depends not only on the capacity of plants to resist browsing, but also on their capacity to tolerate browsing once it has occurred. Little is known of aspen tolerance to browsing, which inflicts a different form of damage than insect herbivory. We employed multiple aspen genotypes, replicate trees of which were subjected to different soil nutrient treatments, to investigate: (i) the effects of aspen genotype, nutrient treatment and genotype × nutrient interactions on susceptibility to browsing by white‐tailed deer, (ii) the phytochemical basis for the patterns observed in (i), and (iii) the effects of genotype, soil nutrients and their interaction on short‐term tolerance to deer browsing. Aspen genotypes varied markedly in their vernal susceptibility to deer browsing. Genetic variation in early season levels of non‐structural carbohydrates (sugars), protein and multiple macro‐ and trace minerals had the strongest influence on tree susceptibility to browsing. In contrast, levels of phytochemical defences had minimal effects, although the range of levels expressed in this study was small. Soil nutrient availability did not significantly influence deer preference. The extent of browsing affected post‐browse tolerance across genotypes. Soil nutrient treatment had little differential effect on tolerance, and, for the most part, genotypes did not display differential tolerance to browsing, regardless of which soil nutrient treatment they experienced. Synthesis. Genetic variation for susceptibility to browsing indicates that ungulate browsers have the potential to be agents of selection in aspen populations. In contrast with previous studies in aspen highlighting the importance of phytochemical defences in shaping preferences of browsing mammals, our results indicate that the nutritional composition of foliage (sugars, protein and mineral concentrations) can have sizable effects on preference. The observed lack of influence of soil nutrient availability on tree browsing tolerance contrasts with predictions of the limiting resource model, the prevailing model for plant tolerance.

Application of Microsatellite Loci for Molecular Identification of Elite Genotypes, Analysis of Clonality, and Genetic Diversity in Aspen Populus tremula L. (Salicaceae)

Testing systems for molecular identification of micropropagated elite aspen (Populus tremula L.) genotypes were developed on the base on microsatellite (SSR) loci. Out of 33 tested microsatellite loci, 14 were selected due to sustainable PCR amplification and substantial variability in elite clones of aspen aimed for establishment of fast-rotated forest plantations. All eight tested clones had different multilocus genotypes. Among 114 trees from three reference native stands located near the established plantations, 80 haplotypes were identified while some repeated genotypes were attributed to natural clones which appeared as a result of sprouting. The selected set of SSR markers showed reliable individual identification with low probability of appearance of identical aspen genotypes (a minimum of 4.8 · 10−10 and 1 × 10−4 for unrelated and related individuals, resp.). Case studies demonstrating practical applications of the test system are described including analysis of clonal structure and levels of genetic diversity in three natural aspen stands growing in the regions where plantations made of elite clones were established.