Phylogeny of Populus (Salicaceae) based on nucleotide sequences of chloroplast TRNT-TRNF region and nuclear rDNA

The species of the genus Populus, collectively known as poplars, are widely distributed over the northern hemisphere and well known for their ecological, economical, and evolutionary importance. The extensive interspecific hybridization and high morphological diversity in this group pose difficulties in identifying taxonomic units for comparative evolutionary studies and systematics. To understand the evolutionary relationships among poplars and to provide a framework for biosystematic classification, we reconstructed a phylogeny of the genus Populus based on nucleotide sequences of three noncoding regions of the chloroplast DNA (intron of trnL and intergenic regions of trnT-trnL and trnL-trnF) and ITS1 and ITS2 of the nuclear rDNA. The resulting phylogenetic trees showed polyphyletic relationships among species in the sections Tacamahaca and Aigeiros. Based on chloroplast DNA sequence data, P. nigra had a close affinity to species of section Populus, whereas nuclear DNA sequence data suggested a close relationship between P. nigra and species of the section Aigeiros, suggesting a possible hybrid origin for P. nigra. Similarly, the chloroplast DNA sequences of P. tristis and P. szechuanica were similar to that of the species of section Aigeiros, while the nuclear sequences revealed a close affinity to species of the section Tacamahaca, suggesting a hybrid origin for these two Asiatic balsam poplars. The incongruence between phylogenetic trees based on nuclear- and chloroplast-DNA sequence data suggests a reticulate evolution in the genus Populus.     

Pleistocene speciation in the genus Populus (salicaceae)

The macroevolutionary consequences of recent climate change remain controversial, and there is little paleobotanical or morphological evidence that Pleistocene (1.8-0.12 Ma) glacial cycles acted as drivers of speciation, especially among lineages with long generation times, such as trees. We combined genetic and ecogeographic data from 2 closely related North American tree species, Populus balsamifera and P. trichocarpa (Salicacaeae), to determine if their divergence coincided with and was possibly caused by Pleistocene climatic events. We analyzed 32 nuclear loci from individuals of P. balsamifera and P. trichocarpa to produce coalescent-based estimates of the divergence time between the 2 species. We coupled the coalescent analyses with paleodistribution models to assess the influence of climate change on species' range. Furthermore, measures of niche overlap were used to investigate patterns of ecological differentiation between species. We estimated the divergence date of P. balsamifera and P. trichocarpa at approximately 75 Ka, which corresponds closely with the onset of Marine Isotope Stage 4 (～76 Ka) and a rapid increase in global ice volume. Significance tests of niche overlap, in conjunction with genetic estimates of migration, suggested that speciation occurred in allopatry, possibly resulting from the environmental effects of Pleistocene glacial cycles. Our results indicate that the divergence of keystone tree species, which have shaped community diversity in northern North American ecosystems, was recent and may have been a consequence of Pleistocene-era glaciation and climate change.     

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules

The ectomycorrhizal fungus Tricholoma populinum is host-specific with Populus species. T. populinum has wind-dispersed progagules and may be capable of long-distance dispersal. In this study, we tested the hypothesis of a panmictic population between Scandinavia and North America. DNA sequences from five nuclear loci were used to assess phylogeographic structure and nucleotide divergence between continents. Tricholoma populinum was composed of Scandinavian and North American lineages with complete absence of shared haplotypes and only one shared nucleotide mutation. Divergence of these lineages was estimated at approx. 1.7-1.0 million yr ago (Ma), which occurred after the estimated divergence of host species Populus tremula and Populus balsamifera/Populus trichocarpa at 5 Ma. Phylogeographic structure was not observed within Scandinavian or North American lineages of T. populinum. Intercontinental divergence appears to have resulted from either allopatric isolation; a recent, rare long-distance dispersal founding event followed by genetic drift; or the response in an obligate mycorrhizal fungus with a narrow host range to contractions and expansion of host distribution during glacial and interglacial episodes within continents. Understanding present genetic variation in populations is important for predicting how obligate symbiotic fungi will adapt to present and future changing climatic conditions.     

Comparative genome mapping among Populus adenopoda, P. alba, P. deltoides, P. euramericana and P. trichocarpa

Among the genus Populus, the sections Populus (white poplar), Aigeiros Duby (black poplar) and Tacamahaca Spach contain many tree species of economical and ecological important properties. Two parental maps for the inter-specific hybrid population of Populus adenopoda × P. alba (two species of Populus section) were constructed based on SSR and SRAP markers by means of a two-way pseudo-test cross mapping strategy. The same set of SSR markers developed from the P. trichocarpa (belonging to Tacamahaca section) genome which were used to construct the maps of P. deltoides and P. euramericana (two species of Aigeiros section) was chosen to analyze the genotype of the experimental population of P. adenopoda × P. alba. Using the mapped SSR markers as allelic bridges, the alignment of the white and black poplar maps to each other and to the P. trichocarpa physical map was conducted. The alignment showed high degree of marker synteny and colinearity and the closer relationship between Aigeiros and Tacamahaca sections than that of Populus and Tacamahaca. Moreover, there was evidence for the chromosomal duplication and inter-chromosomal reorganization involving some poplar linkage groups, suggesting a complicated course of fission or fusion in one of the lineages. A poplar consensus map based on the comparisons could be constructed will be useful in practical applications including marker assisted selection.     

Microsatellite DNA fingerprinting, differentiation, and genetic relationships of clones, cultivars, and varieties of six poplar species from three sections of the genus Populus.

Accurate identification of Populus clones and cultivars is essential for effective selection, breeding, and genetic resource management programs. The unit of cultivation and breeding in poplars is a clone, and individual cultivars are normally represented by a single clone. Microsatellite DNA markers of 10 simple sequence repeat loci were used for genetic fingerprinting and differentiation of 96 clones/cultivars and varieties belonging to six Populus species (P. deltoides, P. nigra, P. balsamifera, P. trichocarpa, P. grandidentata, and P maximowiczii) from three sections of the genus. All 96 clones/cultivars could be uniquely fingerprinted based on their single- or multilocus microsatellite genotypes. The five P. grandidentata clones could be differentiated based on their single-locus genotypes, while six clones of P. trichocarpa and 11 clones of P. maximowiczii could be identified by their two-locus genotypes. Twenty clones of P. deltoides and 25 clones of P. nigra could be differentiated by their multilocus genotypes employing three loci, and 29 clones of P. balsamifera required the use of multilocus genotypes at five loci for their genetic fingerprinting and differentiation. The loci PTR3, PTR5, and PTR7 were found to be the most informative for genetic fingerprinting and differentiation of the clones. The mean number of alleles per locus ranged from 2.9 in P. trichocarpa or P. grandidentata to 6.0 in P. balsamifera and 11.2 in 96 clones of the six species. The mean number of observed genotypes per locus ranged from 2.4 in P. grandidentata to 7.4 in P. balsamifera and 19.6 in 96 clones of the six species. The mean number of unique genotypes per locus ranged from 1.3 in P. grandidentata to 3.9 in P. deltoides and 8.8 in 96 clones of the six species. The power of discrimination of the microsatellite DNA markers in the 96 clones ranged from 0.726 for PTR4 to 0.939 for PTR7, with a mean of 0.832 over the 10 simple sequence repeat loci. Clones/cultivars from the same species showed higher microsatellite DNA similarities than the clones from the different species. A UPGMA cluster plot constructed from the microsatellite genotypic similarities separated the 96 clones into six major groups corresponding to their species. Populus nigra var. italica clones were genetically differentiated from the P. nigra var. nigra clones. Microsatellite DNA markers could be useful in genetic fingerprinting, identification, classification, certification, and registration of clones, clultivars, and varieties as well as genetic resource management and protection of plant breeders' rights in Populus.     

Genetic consequences of glacial survival: the late Quaternary history of balsam poplar (Populus balsamifera L.) in North America

Aim Beringia, the unglaciated region encompassing the former Bering land bridge, as well as the land between the Lena and Mackenzie rivers, is recognized as an important refugium for arctic plants during the last ice age. Compelling palaeobotanical evidence also supports the presence of small populations of boreal trees within Beringia during the Last Glacial Maximum. The occurrence of balsam poplar (Populus balsamifera) in Beringia provides a unique opportunity to assess the implications of persistence in a refugium on present‐day genetic diversity for this boreal tree species. Location North America. Methods We sequenced three variable non‐coding regions of the chloroplast genome (cpDNA) from 40 widely distributed populations of balsam poplar across its North American range. We assessed patterns of genetic diversity, geographic structure and historical demography between glaciated and unglaciated regions of the balsam poplar’s range. We also utilized a coalescent model to test for divergence between regions. Results Levels of genetic diversity were consistently greater for populations at the southern margin (θ_W = 0.00122) than in the central (θ_W = 0.00086) or northern (θ_W = 0.00034) regions of the current distribution of balsam poplar, and diversity decreased with increasing latitude (R² = 0.49, P < 0.01). We detected low, but significant, structure (F_CT = 0.05, P = 0.05), among regions of P. balsamifera’s distribution. The cpDNA genealogy was shallow, however, showing an absence of highly differentiated chloroplast haplotypes. Coalescent analyses supported a model of divergence between the southern ice margin and the northern unglaciated region of balsam poplar’s distribution, but analyses of other regional comparisons did not converge. Main conclusions The palaeobotanical record supports the presence of a Beringian refugium for balsam poplar, but we were unable to definitively identify the presence of known refugial populations based on genetic data alone. Balsam poplar populations from Beringia are not a significant reservoir of cpDNA diversity today. Unique alleles that may have been present in the small, isolated populations that survived within Beringia were probably lost through genetic drift or swamped by post‐glacial, northward migration from populations south of the ice sheets.     

Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa

Abstract Ecotilling was used as a simple nucleotide polymorphism (SNP) discovery tool to examine DNA variation in natural populations of the western black cottonwood, Populus trichocarpa, and was found to be more efficient than sequencing for large-scale studies of genetic variation in this tree. A publicly available, live reference collection of P. trichocarpa from the University of British Columbia Botanical Garden was used in this study to survey variation in nine different genes among individuals from 41 different populations. A large amount of genetic variation was detected, but the level of variation appears to be less than in the related species, Populus tremula, based on reported statistics for that tree. Genes examined varied considerably in their level of variation, from PoptrTB1 which had a single SNP, to PoptrLFY which had more than 23 in the 1000-bp region examined. Overall nucleotide diversity, measured as (Total), was relatively low at 0.00184. Linkage disequilibrium, on the other hand, was higher than reported for some woody plant species, with mean r2 equal to 0.34. This study reveals the potential of Ecotilling as a rapid genotype discovery method to explore and utilize the large pool of genetic variation in tree species.     

Admixture in European Populus hybrid zones makes feasible the mapping of loci that contribute to reproductive isolation and trait differences

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.     

Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies

ABSTRACT: BACKGROUND: Quantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene expression are important towards advancing knowledge of this important developmental and physiological trait. The objective of this study was to evaluate reference gene expression stability in various tissues and growth conditions in two important Populus genotypes (P. trichocarpa "Nisqually 1" and P. tremula X P. alba 717 1-B4) following MIQE guidelines. RESULTS: We evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063, TIP4-like, UBQ7, PT1 and ANT using two software packages, geNormPLUS and BestKeeper. GeNormPLUS ranked TIP4-like and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and ACT2 among the most stable genes. CONCLUSIONS: This is the first comprehensive evaluation of reference genes in two important Populus genotypes and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to be suitable for either genotype considered here and may potentially be suitable for other Populus species and genotypes. These results provide a valuable resource for the Populus research community.     

Stomatal factors and vulnerability of stem xylem to cavitation in poplars

The relationships between the vulnerability of stem xylem to cavitation, stomatal conductance, stomatal density, and leaf and stem water potential were examined in six hybrid poplar (P38P38, Walker, Okanese, Northwest, Assiniboine and Berlin) and balsam poplar (Populus balsamifera) clones. Stem xylem cavitation resistance was examined with the Cavitron technique in well-watered plants grown in the greenhouse. To investigate stomatal responses to drought, plants were subjected to drought stress by withholding watering for 5 (mild drought) and 7 (severe drought) days and to stress recovery by rewatering severely stressed plants for 30 min and 2 days. The clones varied in stomatal sensitivity to drought and vulnerability to stem xylem cavitation. P38P38 reduced stomatal conductance in response to mild stress while the balsam poplar clone maintained high leaf stomatal conductance under more severe drought stress conditions. Differences between the severely stressed clones were also observed in leaf water potentials with no or relatively small decreases in Assiniboine, P38P38, Okanese and Walker. Vulnerability to drought-induced stem xylem embolism revealed that balsam poplar and Northwest clones reached loss of conductivity at lower stem water potentials compared with the remaining clones. There was a strong link between stem xylem resistance to cavitation and stomatal responsiveness to drought stress in balsam poplar and P38P38. However, the differences in stomatal responsiveness to mild drought suggest that other drought-resistant strategies may also play a key role in some clones of poplars exposed to drought stress.     

Hybrid poplar clones with Populus maximowiczii parentage demonstrate postoviposition antibiosis to Cryptorhynchus lapathi (Coleoptera: Curculionidae)

Damage caused by the poplar-and-willow borer, Cryptorhynchus lapathi (L.) (Coleoptera: Curculionidae), is reported to vary among hybrid poplar clones. We evaluated oviposition preferences and larval success in four hybrid poplars on potted and field-planted trees. Oviposition occurred somewhat less frequently and abundantly on two clones with Populus maximowiczii Henry parentage in field-planted and potted trees, and significantly fewer larvae survived to adulthood on those clones. No adults emerged from field-planted NM 6 (Populus nigra L. x P. maximowiczii) and four emerged from TM 256-28 (Populus trichocarpa Torrey & Gray x P. maximowiczii) on which male-female pairs of C. lapathi had been caged. In contrast, 50 and 140 adults emerged over the same 2-yr period from two susceptible clones (n = 20), TD 52-226 (P. trichocarpa x Populus deltoides Bartram ex Marshall) and TN 302-9 (P. trichocarpa x P. nigra), respectively. Thus, resistance expressed by clones with P. maximowiczii parentage partially involves decreased levels of oviposition, but more significantly, antibiosis in resistant clones prevents the development of larvae, probably in early spring.     

Genetic analysis of post-mating reproductive barriers in hybridizing European Populus species

Molecular genetic analyses of experimental crosses provide important information on the strength and nature of post-mating barriers to gene exchange between divergent populations, which are topics of great interest to evolutionary geneticists and breeders. Although not a trivial task in long-lived organisms such as trees, experimental interspecific recombinants can sometimes be created through controlled crosses involving natural F(1)'s. Here, we used this approach to understand the genetics of post-mating isolation and barriers to introgression in Populus alba and Populus tremula, two ecologically divergent, hybridizing forest trees. We studied 86 interspecific backcross (BC(1)) progeny and >350 individuals from natural populations of these species for up to 98 nuclear genetic markers, including microsatellites, indels and single nucleotide polymorphisms, and inferred the origin of the cytoplasm of the cross with plastid DNA. Genetic analysis of the BC(1) revealed extensive segregation distortions on six chromosomes, and >90% of these (12 out of 13) favored P. tremula donor alleles in the heterospecific genomic background. Since selection was documented during early diploid stages of the progeny, this surprising result was attributed to epistasis, cyto-nuclear coadaptation, heterozygote advantage at nuclear loci experiencing introgression or a combination of these. Our results indicate that gene flow across 'porous' species barriers affects these poplars and aspens beyond neutral, Mendelian expectations and suggests the mechanisms responsible. Contrary to expectations, the Populus sex determination region is not protected from introgression. Understanding the population dynamics of the Populus sex determination region will require tests based on natural interspecific hybrid zones.     

Reduced expression of the SHORT-ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis

SHORT-ROOT (SHR) is a well characterized regulator of cell division and cell fate determination in the Arabidopsis primary root. However, much less is known about the functions of SHR in the aerial parts of the plant. In this work, we cloned SHR gene from Populus trichocarpa (PtSHR1) as an AtSHR ortholog and down-regulated its expression in hybrid poplar (Populus tremula×P. tremuloides Michx-clone T89) in order to determine its physiological functions in shoot development. Sharing a 90% similarity to AtSHR at amino acid level, PtSHR1 was able to complement the Arabidopsis shr mutant. Down regulation of PtSHR1 led to a strong enhancement of primary (height) and secondary (girth) growth rates in the transgenic poplars. A similar approach in Arabidopsis showed a comparable accelerated growth and development phenotype. Our results suggest that the response to SHR could be dose-dependent and that a partial down-regulation of SHR could lead to enhanced meristem activity and a coordinated acceleration of plant growth in woody species. Therefore, SHR functions in plant growth and development as a regulator of cell division and meristem activity not only in the roots but also in the shoots. Reducing SHR expression in transgenic poplar was shown to lead to significant increases in primary and secondary growth rates. Given the current interest in bioenergy crops, SHR has a broader role as a key regulator of whole plant growth and development and SHR suppression has considerable potential for accelerating biomass accumulation in a variety of species.     

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.     

Clonal fingerprinting in the genus <Emphasis Type="Italic">Populus</Emphasis> L. by nuclear microsatellite loci regarding differences between sections,species and hybrids

Reliable methods for clone identification are desired to characterise and distinguish breeding products within the genus Populus L. (Salicaceae). Ten nuclear microsatellite loci (PMGC14, PMGC456, PMGC2163, PTR2, PTR7, WPMS05, WPMS09, WPMS14, WPMS15 and WPMS20) were applied on a clone collection with several species and hybrids belonging to the sections Tacamahaca (balsam poplars), Aigeiros (black poplars, cottonwoods) and Populus (white poplars and aspens) and intersectional hybrids between black and balsam poplars. The members of the different sections and species do not always share their allelic ladders. Some shifts of one or two nucleotides in allele length were observed for several loci. This could be explained by nucleotide sequence differences in the flanking regions of loci in diverse taxonomic groups. Such shifts of allelic ladders result in irregular patterns in hybrid genotypes. The set of loci should have a sufficient amount of variation for a differentiation between clones, even if they are full siblings originating from crossing experiments.     

Barrier to gene flow between two ecologically divergent Populus species, P. alba (white poplar) and P. tremula (European aspen): the role of ecology and life history in gene introgression

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.     

Host locating behaviour of Leschenaultia exul and Patelloa pachypyga: two tachinid parasitoids of the forest tent caterpillar, Malacosoma disstria

Leschenaultia exul (Townsend) and Patelloa pachypyga (Aldrich & Webber) (Diptera: Tachinidae) are the principal larval parasitoids of the forest tent caterpillar (FTC) Malacosoma disstria (Hübner) (Lepidoptera: Lasiocampidae) in Canada. The response of these two fly species to M. disstria differs depending on the tree species on which the host feeds. In wind tunnel experiments, L. exul spent more time on the side of the tunnel containing volatiles from FTC frass and was attracted to the FTC-aspen poplar (Populus tremuloides Michx.) complex preferentially to the FTC-balsam poplar (Populus balsamifera L.) complex. Field bioassays confirmed that this fly species was preferentially attracted to the herbivore-aspen poplar complex as compared to the herbivore-balsam poplar complex. In field bioassays, P. pachypyga was also attracted preferentially to aspen poplar trees containing FTC larvae, compared to balsam poplar trees with host larvae.     

A microarray-based genotyping and genetic mapping approach for highly heterozygous outcrossing species enables localization of a large fraction of the unassembled Populus trichocarpa genome sequence

Microarrays have demonstrated significant power for genome-wide analyses of gene expression, and recently have also revolutionized the genetic analysis of segregating populations by genotyping thousands of loci in a single assay. Although microarray-based genotyping approaches have been successfully applied in yeast and several inbred plant species, their power has not been proven in an outcrossing species with extensive genetic diversity. Here we have developed methods for high-throughput microarray-based genotyping in such species using a pseudo-backcross progeny of 154 individuals of Populus trichocarpa and P. deltoides analyzed with long-oligonucleotide in situ- synthesized microarray probes . Our analysis resulted in high-confidence genotypes for 719 single-feature polymorphism (SFP) and 1014 gene expression marker (GEM) candidates. Using these genotypes and an established microsatellite (SSR) framework map, we produced a high-density genetic map comprising over 600 SFPs, GEMs and SSRs. The abundance of gene-based markers allowed us to localize over 35 million base pairs of previously unplaced whole-genome shotgun (WGS) scaffold sequence to putative locations in the genome of P. trichocarpa . A high proportion of sampled scaffolds could be verified for their placement with independently mapped SSRs, demonstrating the previously un-utilized power that high-density genotyping can provide in the context of map-based WGS sequence reassembly. Our results provide a substantial contribution to the continued improvement of the Populus genome assembly, while demonstrating the feasibility of microarray-based genotyping in a highly heterozygous population. The strategies presented are applicable to genetic mapping efforts in all plant species with similarly high levels of genetic diversity.     

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.