Natural hybridisation between <Emphasis Type="Italic">Populus nigra</Emphasis> L. and <Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">x canadensis</Emphasis> Moench. Hybrid offspring competes for niches along the Rhine river in the Netherlands

Black poplar (Populus nigra L.) is a major species for European riparian forests but its abundance has decreased over the decades due to human influences. For restoration of floodplain woodlands, the remaining black poplar stands may act as source population. A potential problem is that P. nigra and Populus deltoides have contributed to many interspecific hybrids, which have been planted in large numbers. As these Populus x canadensis clones have the possibility to intercross with wild P. nigra trees, their offspring could establish themselves along European rivers. In this study, we have sampled 44 poplar seedlings and young trees that occurred spontaneously along the Rhine river and its tributaries in the Netherlands. Along these rivers, only a few native P. nigra L. populations exist in combination with many planted cultivated P. x canadensis trees. By comparison to reference material from P. nigra, P. deltoides and P. x canadensis, species-specific AFLP bands and microsatellite alleles indicated that nearly half of the sampled trees were not pure P. nigra but progeny of natural hybridisation that had colonised the Rhine river banks. The posterior probability method as implemented in NewHybrids using microsatellite data was the superior method in establishing the most likely parentage. The results of this study indicate that offspring of hybrid cultivated poplars compete for the same ecological niche as native black poplars. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Fitness dynamics within a poplar hybrid zone: II. Impact of exotic sex on native poplars in an urban jungle

Trees bearing novel or exotic gene components are poised to contribute to the bioeconomy for a variety of purposes such as bioenergy production, phytoremediation, and carbon sequestration within the forestry sector, but sustainable release of trees with novel traits in large‐scale plantations requires the quantification of risks posed to native tree populations. Over the last century, exotic hybrid poplars produced through artificial crosses were planted throughout eastern Canada as ornamentals or windbreaks and these exotics provide a proxy by which to examine the fitness of exotic poplar traits within the natural environment to assess risk of exotic gene escape, establishment, and spread into native gene pools. We assessed postzygotic fitness traits of native and exotic poplars within a naturally regenerated stand in eastern Canada (Quebec City, QC). Pure natives (P. balsamifera and P. deltoides spp. deltoides), native hybrids (P. deltoides × P. balsamifera), and exotic hybrids (trees bearing Populus nigra and P. maximowiczii genetic components) were screened for reproductive biomass, yield, seed germination, and fungal disease susceptibility. Exotic hybrids expressed fitness traits intermediate to pure species and were not significantly different from native hybrids. They formed fully viable seed and backcrossed predominantly with P. balsamifera. These data show that exotic hybrids were not unfit and were capable of establishing and competing within the native stand. Future research will seek to examine the impact of exotic gene regions on associated biotic communities to fully quantify the risk exotic poplars pose to native poplar forests.     

A large scale survey of Populus nigra presence and genetic introgression from non-native poplars in Switzerland based on molecular identification

Populus nigra is considered a rare and threatened tree species in Switzerland because of dramatic habitat loss owing to river regulations during the last two centuries and because of potential gene introgression from non-indigenous P. deltoides through planted P. x canadensis hybrids. The significance of introgression as an endangerment to P. nigra, however, is controversial. The aims of the present study were (1) to assess how abundant P. nigra trees are in Switzerland and (2) to assess potential gene introgression. We present data from a molecular survey of 1372 putative P. nigra trees from Switzerland, using both chloroplast DNA and nuclear DNA markers. The results show that P. nigra is more abundant in Switzerland than hitherto thought. Furthermore, we detected a low frequency of gene introgression.     

Relationship between genotype and soil environment during colonization of poplar roots by mycorrhizal and endophytic fungi

Poplars are among the few tree genera that can develop both ectomycorrhizal (ECM) and arbuscular (AM) associations; however, variable ratios of ECM/AM in dual mycorrhizal colonizations were observed in the roots of a variety of poplar species and hybrids. The objective of our study was to analyze the effect of internal and external factors on growth and dual AM and ECM colonization of poplar roots in three 12–15-year-old common gardens in Poland. We also analyzed the abundance of nonmycorrhizal fungal endophytes in the poplar roots. The Populus clones comprised black poplars (Populus deltoides and P. deltoides × Populus nigra), balsam poplars (Populus maximowiczii × Populus trichocarpa), and a hybrid of black and balsam poplars (P. deltoides × P. trichocarpa). Of the three sites that we studied, one was located in the vicinity of a copper smelter, where soil was contaminated with copper and lead. Poplar root tip abundance, mycorrhizal colonization, and soil fungi biomass were lower at this heavily polluted site. The total mycorrhizal colonization and the ratio of ECM and AM colonization differed among the study sites and according to soil depth. The influence of Populus genotype was significantly pronounced only within the individual study sites. The contribution of nonmycorrhizal fungal endophytes differed among the poplar clones and was higher at the polluted site than at the sites free of pollution. Our results indicate that poplar fine root abundance and AM and ECM symbiosis are influenced by environmental conditions. Further studies of different site conditions are required to characterize the utility of poplars for purposes such as the phytoremediation of polluted sites.     

Genetic linkage maps of Populus nigra L. including AFLPs, SSRs, SNPs, and sex trait

Black poplar (Populus nigra L.) is a tree of ecological and economic interest. A better knowledge of P. nigra genome is needed for an effective protection and use of its genetic resources. The main objective of this study is the construction of a highly informative genetic map of P. nigra species including genes of adaptive and economic interest. Two genotypes originated from contrasted natural Italian populations were crossed to generate a F₁ mapping pedigree of 165 individuals. Amplification fragment length polymorphism (AFLP), simple sequence repeat (SSR), and single nucleotide polymorphism (SNP) markers were used to genotype 92 F₁ individuals, and the pseudo-test-cross strategy was applied for linkage analysis. The female parent map included 368 markers (274 AFLPs, 91 SSRs, and 3 SNPs) and spanned 2,104 cM with 20 linkage groups, and the male parent map, including 317 markers (205 AFLPs, 106 SSRs, 5 SNPs, and sex trait), spanned 2,453 cM with 23 main linkage groups. The sex, as morphological trait, was mapped on the linkage group XIX of the male parent map. The generated maps are among the most informative in SSRs when compared to the Populus maps published so far and allow a complete alignment with the 19 haploid chromosomes of Populus sequence genome. These genetic maps provide informative tools for a better understanding of P. nigra genome structure and genetic improvement of this ecologically and economically important European tree species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genomic regions involved in productivity of two interspecific poplar families in Europe. 1. Stem height, circumference and volume

Interspecific hybrids of Populus species are known for their superior growth. In this study, we examined the effect of the genetic background and contrasting environmental conditions on growth and searched for quantitative trait loci (QTL) affecting growth traits. To this end, two hybrid poplar families resulting from controlled crosses, Populus deltoides ‘S9-2’ × P. nigra ‘Ghoy’ (D × N, 180 F₁) and P. deltoides ‘S9-2’ × P. trichocarpa ‘V24’ (D × T, 182 F₁), were grown at two contrasting sites, Northern Italy and Central France. At the end of the second growing season, tree dimensions (stem height, circumference, and volume) were assessed. The performances of both families significantly differed within and between sites. Tree volume was significantly larger at the Italian site as compared to the French site. Genotype by environment interactions were significant but low for both families and for all growth traits. Tight correlations among the individual growth traits indicated that there may be a common genetic mechanism with pleiotropic effects on these growth traits. In line with previous studies, linkage groups I, VII, IX, X, XVI, XVII, and XIX appeared to have genomic regions with the largest effects on growth traits. This study revealed that (1) both families have high potential for selection of superior poplar hybrids due to the pronounced heterosis (hybrid vigor) and the large genetic variability in terms of growth and (2) the choice of site is crucial for poplar cultivation. Dillen and Storme contributed equally to the work. An erratum to this article can be found at     

Application of genetic markers to the discrimination of European Black Poplar (Populus nigra) from American Black Poplar (P. deltoides) and Hybrid Poplars (P. x canadensis) in Switzerland

European Black Poplar (Populus nigra) is considered a rare and endangered tree species because of severe reduction of its natural riverine habitat and potential hybridisation with the related non-indigenous taxa P. deltoides and P. x canadensis. As it is difficult to distinguish these taxa solely based on their morphology, we applied a PCR-based assay with an easy-to-use and robust molecular marker set (cpDNA trnL-trnF/RsaI RFLP, nDNA win3 and nDNA POPX/MspI RFLP) in order to identify pure P. nigra. Different plant tissues could be used for fast and standardised DNA extraction. The application of the three marker types was tested on a number of different Populus taxa, and they were also used for the verification of pure P. nigra in a sample of 304 putative P. nigra individuals from Switzerland. Cross-checking of the DNA data with those using a traditional allozyme approach resulted in complete agreement. The availability of molecular identification methods is an important prerequisite for the conservation of European Black Poplar, because pure, non-introgressed plant material can then be used in restoration projects of European floodplains.     

Studies on the Polyphenolic Composition and the Antioxidant Properties of the Leaves of Poplar (Populus spp.) Various Species and Hybrids

The chemical composition in terms of flavonoid and salicylic compounds of leaves from 6 species and 3 hybrids of poplars (Populus) was identified with the use of TLC and HPLC-DAD/ESI-MS methods. Chromatographic analyses were carried out with 21 standard compounds including salicylic compounds (2), phenolic acids (3) and flavonoids (16). Moreover, on the basis of the obtained chromatographic data from the HPLC-DAD/ESI-MS and TLC separations, the presence of salicortin, tremulacin and chlorogenic acid was confirmed, depending on the analyzed poplar species or hybrid. The content of salicylic compounds was determined by HPLC-UV method and expressed on salicin as free and total fraction. Total flavonoid content was determined by spectroscopic method as quercetin equivalent. Significant qualitative and quantitative differences in the chemical composition of the analyzed leaves were demonstrated. The highest concentration of flavonoids (8.02 mg/g) was found in the leaves of Populus nigra, while the highest content of salicylic compounds (47.14 mg/g) was found in the leaves of P.×berolinensis. The antioxidant and xanthine oxidase inhibition properties of extracts from poplar leaves were investigated by TLC bioautography. It has been shown that the richest set of compounds with antioxidant properties are present in the leaves of P. alba, P.×candicans and P. nigra.     

EVOLUTION OF POPULUS NIGRA (SECT. AIGEIROS):INTROGRESSIVE HYBRIDIZATION AND THE CHLOROPLAST CONTRIBUTION OF POPULUS ALBA (SECT. POPULUS)

Restriction site variation in chloroplast DNA and nuclear ribosomal DNA was examined in 16 accessions from the Salicaceae comprising ten species of Populus and one outgroup species of Salix. Forty-nine restriction site mutations in the chloroplast DNAs were used to generate one most parsimonious phylogenetic tree. This tree indicates that all varieties of P. nigra (black poplars of sect. Aigeiros) have a chloroplast genome, maternally inherited, derived from the clade including the white poplars (P. alba and segregate species of sect. Populus) and divergent from the American cottonwoods of their own section. Twenty-one restriction site mutations in the nuclear ribosomal DNAs generated a single most parsimonious phylogenetic tree that indicates that the nuclear genome ofP. nigra is distinct from both the white poplars and American cottonwoods. The incongruity of these independent molecular phylogenies provides evidence for an unusual origin of the black poplars. Populus alba or its immediate ancestor acted as the maternal parent in a hybridization event with the paternal lineage of P. nigra. Subsequent backcrosses to the paternal species gave rise to the extant P. nigra with a chloroplast genome of P. alba and the nuclear genome of the paternal species. These hybridization and introgression events must have pre-dated the divergence of the black poplar varieties. The biphyletic nature of the P. nigra genomes suggests that dependency on one class of molecular or morphological markers or the merging of the two kinds of data sets to derive accurate estimates of true phylogenies could be misleading in plants.     

Short-distance gene flow in <Emphasis Type="Italic">Populus nigra</Emphasis> L. accounts for small-scale spatial genetic structures: implications for in situ conservation measures

The European black poplar (Populus nigra L.) is a major species of riparian softwood forests. Due to human influences, it is one of the most threatened tree species in Europe. For restoration purposes, remaining stands may act as source populations. We analysed a natural population of P. nigra for genetic diversity and spatial genetic structure using seven microsatellite markers. For the first time, paternity analysis of seedlings as well as juveniles from a restricted area of natural regeneration was used to quantify pollen- and seed-mediated gene flow, respectively. In both cases, cultivated P. x canadensis trees in vicinity could act as potential parents. Spatial genetic patterns of the adult tree population suggest small-scale isolation by distance due to short-distance gene flow, the major part of which (i.e. 70%) takes place within distances of less than 1 km. This helps to explain the reduced diversity in the juveniles. It has implications for the spatial management of natural regeneration areas within in situ conservation measures.     

Retrospective identification of hybridogenic walnut plants by SSR fingerprinting and parentage analysis

Juglans × intermedia (Juglans nigra × Juglans regia) is considered the prototype walnut for quality wood production in Europe. Hybridization between the parental species is rare under natural conditions and difficult using controlled pollination because of phenological and genetic incompatibilities. The identification of hybridogenic parents is the first step toward obtaining hybrid progeny. We report the application of microsatellite markers for DNA fingerprinting and parentage analysis of half-sib families collected in a natural mixed population for which no phenological and morphological data were available. Ten nuclear, neutral, simple sequence repeat markers were used to analyse 600 samples. The high levels of polymorphism detected positively influenced the exclusion and identity probabilities. The assignment analysis revealed the presence of 198 diploid J. × intermedia hybrids among the seedling progeny. Maternity checks were performed on all individuals and few errors of sampling were found. Four distinct hybridogenic mother trees were identified, each showing different reproductive success rates. The 198 diploid hybrids belonged to four open-pollinated families based on an analysis of paternity using a likelihood approach. Differential male reproductive success was observed among pollen donors within the research site. Forty-nine of the 198 diploid hybrids detected in four progenies were sired by only three J. regia genotypes. Backward selection might be used to establish new seed orchards for inter-specific F₁ hybrid production using genotypes with demonstrated compatibility.     

Effects of flooding on leaf development,transpiration, and photosynthesis in narrowleaf cottonwood,a willow-like poplar

The narrowleaf cottonwood, Populus angustifolia, occurs in occasionally flooded, low elevation zones along river valleys near the North American Rocky Mountains. This small poplar has narrow leaves and fine branching and thus resembles willows, which are commonly flood-tolerant. We investigated the flood response of narrowleaf cottonwoods and a related native hybrid, jackii cottonwood (P. × jackii = P. balsamifera × P. deltoides), by studying saplings of 24 clones in a greenhouse, with some pots being inundated to provide the flood treatment. Flooding slightly reduced leaf numbers (−10%), and leaf sizes were reduced by about 21% in female P. angustifolia versus a 50% reduction in the female hybrids. Flooding-reduced stomatal conductance and net photosynthetic rate, and reduced transpiration particularly in P. × jackii. The effects on foliar gas exchange declined over a 5-week interval, suggesting compensation. The moderate impact of flooding supports the hypothesis that narrowleaf cottonwoods are flood-tolerant, and we anticipate that these trees could provide traits to increase the flood tolerance of fast-growing hybrid poplars. The results further indicate that female cottonwoods may be more flood-tolerant than males, and females could be more successful in lower, flood-prone sites.     

Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry

Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the β-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4–10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1–2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra × P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.     

Low level expression of prokaryotic tzs gene enhances growth performance of transgenic poplars

We modulated the level of a hormone gene expression in poplars using either 35S promoter (p35S) of cauliflower mosaic virus (CaMV) or aux promoter (pAUX) of A. rhizogenes. The transgenic poplars (Populus alba × P. tremula var. glandulosa), in which the bacterial trans-zeatin secretion (tzs) gene was attached either to the 35S promoter or to the aux promoter, were compared for their performance in tissue culture as well as in nursery. Northern blot analysis of total RNA probed with tzs coding region showed that the total tzs mRNA expression by p35S was approximately 200–300-fold higher than that driven by pAUX. In contrast, the cellular zeatin content of p35S-tzs transgenic poplars was merely 13-fold of those found in pAUX-tzs plants. Due to different levels of cellular zeatin levels, the two types of transgenic poplars showed different morphogenetic as well as growth responses. The p35S-tzs transgenic plants showed morphological characteristics typical of those treated with cytokinin in culture. These include multiple axillary shoot formation, thick stems, narrow leaves and absence of roots. In contrast, the pAUX-tzs plants had slightly higher cellular cytokinin levels than did control plants and showed a lower degree of cytokinin-related phenotypes, including a few axillary shoots in root-inducing media. Since p35S-tzs did not develop roots, only pAUX-tzs transgenic poplars could be transplanted to the nursery where they resumed a close-to-normal growth. Nevertheless, pAUX-tzs plants transferred to the nursery developed cytokinin-related phenotypes, including greater number of shoots, smaller leaves and slightly retarded growth in height, but with a high total biomass.     

Natural hybridization between <Emphasis Type="Italic">Phlomis lycia</Emphasis> D. Don × <Emphasis Type="Italic">P. bourgaei</Emphasis> Boiss., (Lamiaceae) revealed by RAPD markers

Randomly Amplified Polymorphic DNA markers (RAPD) were used to assess the hybrid identity of individuals sampled as Phlomis × termessi Davis. Out of 95 primers screened, 11 primers produced reproducible amplification patterns used for discrimination of P. × termessi and their parents. Eleven primers produced 81 bands. Forty two percent of the RAPD bands existed in parents. Of the 54 bands found in P. lycia, 19 were found only in this species and 7 of these were monomorphic. Similarly, of 57 RAPD bands observed in P. bourgaei, 18 were found only in P. bourgaei and 6 of these were monomorphic. Among hybrid individuals, 35 of the 73 markers were monomorphic. Fifteen of these existed in individual parents showing that parents were homozygous for these markers. Of the 35 monomorphic bands observed among hybrid individuals, 5 were present in the samples of one of the parents and completely absent from the samples of the other; therefore, additive inheritance is indicated. Of the 5 additive bands, 1 was inherited from P. bourgaei and 4 were inherited from P. lycia. Among 38 polymorhic markers observed in hybrid individuals, 9 were new and hybrid-specific. Pollen fertility was also investigated. Mean pollen fertility for P. lycia and P. bourgaei was 93% and 97% respectively. However, mean pollen fertility for hybrids was 65% (±10.5).     

Chloroplast DNA inheritance in Populus

Summary The inheritance of chloroplast (cp) DNA was examined in F₁ hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F₁ offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.     

Brassica naponigra,a somatic hybrid resistant to Phoma lingam

Summary Brassica napus and B. nigra were combined via protoplast fusion into the novel hybrid Brassica naponigra. The heterokaryons were identified by fluorescent markers and selected by flow sorting. Thirty hybrid plants were confirmed by isozyme analysis to contain both B. nigra and B. napus chromosomes; of these, 20 plants had the sum of the parental chromosome numbers. A non-random segregation of the chloroplasts was found in the hybrids. Of 14 hybrid plants investigated, all had the B. napus type of chloroplast. The resistance to Phoma lingam found in the B. nigra cultivar used in the fusion experiments was expressed in 26 of the hybrid plants. The hybrids obtained in this study contain all of the three Brassica genomes (A, B and C) and have thus created unique possibilities for genetic exchanges between the genomes. Since most of the plants were fertile as well as resistant to P. lingam, they have been incorporated into conventional rapeseed breeding programs.     

Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.