Geographical barriers and climate influence demographic history in narrowleaf cottonwoods.

Studies of genetic variation can clarify the role of geography and spatio-temporal variation of climate in shaping demography, particularly in temperate zone tree species with large latitudinal ranges. Here, we examined genetic variation in narrowleaf cottonwood, Populus angustifolia, a dominant riparian tree. Using multi-locus surveys of polymorphism in 363 individuals across the species' 1800 km latitudinal range, we found that, first, P. angustifolia has stronger neutral genetic structure than many forest trees (simple sequence repeat (SSR) FST=0.21), with major genetic groups corresponding to large apparent geographical barriers to gene flow. Second, using SSRs and putatively neutral sequenced loci, coalescent simulations indicated that populations diverged before the last glacial maximum (LGM), suggesting the presence of population structure before the LGM. Third, the LGM and subsequent warming appear to have had different influences on each of these distinct populations, with effective population size reduction in the southern extent of the range but major expansion in the north. These results are consistent with the hypothesis that climate and geographic barriers have jointly affected the demographic history of P. angustifolia, and point the importance of both factors as being instrumental in shaping genetic variation and structure in widespread forest trees.

A 34K SNP genotyping array for Populus trichocarpa: design, application to the study of natural populations and transferability to other Populus species.

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost-effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.

Revisiting the sequencing of the first tree genome: Populus trichocarpa.

Ten years ago, it was announced that the Joint Genome Institute with funds provided by the Department of Energy, Office of Science, Biological and Environmental Research would sequence the black cottonwood (Populus trichocarpa Torr. & Gray) genome. This landmark decision was the culmination of work by the forest science community to develop Populus as a model system. Since its public release in late 2006, the availability of the Populus genome has spawned research in plant biology, morphology, genetics and ecology. Here we address how the tree physiologist has used this resource. More specifically, we revisit our earlier contention that the rewards of sequencing the Populus genome would depend on how quickly scientists working with woody perennials could adopt molecular approaches to investigate the mechanistic underpinnings of basic physiological processes. Several examples illustrate the integration of functional and comparative genomics into the forest sciences, especially in areas that target improved understanding of the developmental differences between woody perennials and herbaceous annuals (e.g., phase transitions). Sequencing the Populus genome and the availability of genetic and genomic resources has also been instrumental in identifying candidate genes that underlie physiological and morphological traits of interest. Genome-enabled research has advanced our understanding of how phenotype and genotype are related and provided insights into the genetic mechanisms whereby woody perennials adapt to environmental stress. In the future, we anticipate that low-cost, high-throughput sequencing will continue to facilitate research in tree physiology and enhance our understanding at scales of individual organisms and populations. A challenge remains, however, as to how genomic resources, including the Populus genome, can be used to understand ecosystem function. Although examples are limited, progress in this area is encouraging and will undoubtedly improve as future research targets the many unique aspects of Populus as a keystone species in terrestrial ecosystems.

Population substructure in continuous and fragmented stands of Populus trichocarpa.

Population substructure has important implications for both basic and applied genetic research. We used 10 microsatellite markers to characterize population substructure in two ecologically and demographically contrasting populations of the model tree Populus trichocarpa. The Marchel site was a continuous stand growing in a mesic habitat in western Oregon, whereas the Vinson site consisted of three disjunct and isolated stands in the high desert of eastern Oregon. A previous study revealed that pollen-mediated gene flow is extensive in both populations. Surprisingly, model-based clustering, principal components analysis and analyses of molecular variance provided overwhelming support for the existence of at least two intermingled sub-populations within the continuous Marchel population (F(ST)=0.026, P<0.001), which occupied an area with a radius of only about 250 m. Genets in these two sub-populations appeared to have different relative clone ages and phenologies, leading us to hypothesize that they correspond to different seedling cohorts, each established from seeds produced by relatively few mothers. As expected, substructure was stronger in the fragmented Vinson population (F(ST)=0.071, P=0.001), and this difference appeared to result from the more extensive family structure in this population. Using group-likelihood methods, we reconstructed multiple interconnected half-sib families in the Vinson population, with some genets having as many as eight putative siblings. Researchers involved in ongoing and future association studies in P. trichocarpa should account for the likely presence of subtle but practically significant substructure in populations throughout the range of this species.

Cytogenetic analysis of Populus trichocarpa--ribosomal DNA, telomere repeat sequence, and marker-selected BACs.

The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequence assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.

Extensive pollen flow in two ecologically contrasting populations of Populus trichocarpa.

Pollen-mediated gene flow was measured in two populations of black cottonwood using direct (paternity analysis) and indirect (correlated paternity) methods. The Marchel site was an area with an approximate radius of 250 m in a large continuous stand growing in a mesic habitat in western Oregon. In contrast, the Vinson site was an area with a radius of approximately 10 km and consisted of small, disjunct and isolated stands in the high desert of eastern Oregon. Pollen immigration was extensive in both populations, and was higher in the Marchel site (0.54 +/- 0.02) than in the substantially larger and more isolated Vinson site (0.32 +/- 0.02). Pollen pool differentiation among mothers was approximately five times stronger in the Vinson population (Phi FT = 0.253, N = 27 mothers) than in the Marchel population (Phi FT = 0.052, N = 5 mothers). Pollen dispersal was modelled using a mixed dispersal curve that incorporated pollen immigration. Predicted pollination frequencies generated based on this curve were substantially more accurate than those based on the widely used exponential power dispersal curve. Male neighbourhood sizes (sensu Wright 1946) estimated using paternity analysis and pollen pool differentiation were remarkably similar. They were three to five times smaller in the Vinson population, which reflected the substantial ecological and demographic differences between the two populations. When the same mathematical function was used, applying direct and indirect methods resulted in similar pollen dispersal curves, thus confirming the value of indirect methods as a viable lower-cost alternative to paternity analysis.

The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis.

Mycorrhizal symbioses--the union of roots and soil fungi--are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains approximately 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

A dense linkage map of hybrid cottonwood (Populus fremontii x P. angustifolia) contributes to long-term ecological research and comparison mapping in a model forest tree.

Cottonwoods are foundation riparian species, and hybridization among species is known to produce ecological effects at levels higher than the population, including effects on dependent species, communities and ecosystems. Because these patterns result from increased genetic variation in key cottonwood traits, novel applications of genetic tools (for example, QTL mapping) could be used to place broad-scale ecological research into a genomic perspective. In addition, linkage maps have been produced for numerous species within the genus, and, coupled with the recent publication of the Populus genome sequence, these maps present a unique opportunity for genome comparisons in a model system. Here, we conducted linkage analyses in order to (1) create a platform for QTL and candidate gene studies of ecologically important traits, (2) create a framework for chromosomal-scale perspectives of introgression in a natural population, and (3) enhance genome-wide comparisons using two previously unmapped species. We produced 246 backcross mapping (BC(1)) progeny by crossing a naturally occurring F(1) hybrid (Populus fremontii x P. angustifolia) to a pure P. angustifolia from the same population. Linkage analysis resulted in a dense linkage map of 541 AFLP and 111 SSR markers distributed across 19 linkage groups. These results compared favorably with other Populus linkage studies, and addition of SSR loci from the poplar genome project provided coarse alignment with the genome sequence. Preliminary applications of the data suggest that our map represents a useful framework for applying genomic research to ecological questions in a well-studied system, and has enhanced genome-wide comparisons in a model tree.

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).

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

Large-scale heterospecific segregation distortion in Populus revealed by a dense genetic map.

We report the most complete genetic map to have been constructed for the genus Populus. This map includes 544 markers mapped onto 19 linkage groups, equivalent to the Populus chromosome number, with all markers displaying internally consistent linkage patterns. We estimate the genome length to be between 2,300 and 2,500 cM, based both on the observed number of crossovers in the maternal haplotypes, as well as the total observed map length. Genome coverage was estimated to be greater than 99.9% at 20 cM per marker. We did not detect obvious recombination repression in the maternal tree (a hybrid of Populus trichocarpa Hooker x P. deltoides Marsh.) compared to the paternal tree (pure P. deltoides). Finally, most markers exhibiting segregation distortion were derived from the donor parent in this backcross, and generally occurred in large contiguous blocks on two linkage groups. We hypothesize that divergent selection has occurred on chromosomal scales among the parental species used to create this pedigree, and explore the evolutionary implications of this observation. This genetic linkage map provides the most comprehensive view of the Populus genome reported to date and will prove invaluable for future inquiries into the structural and functional genomics, evolutionary biology, and genetic improvement of this ecologically important model species.

Poplar genomics is getting popular: the impact of the poplar genome project on tree research.

Trees, due to their long life-span, have characteristics that distinguish them from annual, herbaceous plants. It is likely that many of these properties are based on a tree-specific genetic foundation. The U.S. Department of Energy initiated a genome-sequencing project for Populus, a model perennial plant. Through international collaboration and input to the sequencing effort, the annotated whole genome sequence of Populus trichocarpa will be released to the public in early 2004. This genomic resource will, for the first time, allow comparison between a perennial and an annual plant on a whole genome basis and therefore provide clues for molecular research on tree-specific questions like dormancy, development of a secondary cambium, juvenile-mature phase change, or long-term host-pest interactions. The approximately 520 Mbp of annotated genomic sequence will complement and expand the knowledge provided so far by the 125,000 ESTs from poplar that are available in public databases. This article introduces the international poplar research programmes and points out the significance of the poplar genome project for plant research.

Genetic and physical mapping of Melampsora rust resistance genes in Populus and characterization of linkage disequilibrium and flanking genomic sequence.

• In an attempt to elucidate the molecular mechanisms of Melampsora rust resistance in Populus trichocarpa, we have mapped two resistance loci, MXC3 and MER, and intensively characterized the flanking genomic sequence for the MXC3 locus and the level of linkage disequilibrium (LD) in natural populations. • We used an interspecific backcross pedigree and a genetic map that was highly saturated with AFLP and SSR markers, and assembled shotgun-sequence data in the region containing markers linked to MXC3. • The two loci were mapped to different linkage groups. Linkage disequilibrium for MXC3 was confined to two closely linked regions spanning 34 and 16 kb, respectively. The MXC3 region also contained six disease-resistance candidate genes. • The MER and MXC3 loci are clearly distinct, and may have different mechanisms of resistance, as different classes of putative resistance genes were present near each locus. The suppressed recombination previously observed in the MXC3 region was possibly caused by extensive hemizygous rearrangements confined to the original parent tree. The relatively low observed LD may facilitate association studies using candidate genes for rust resistance, but will probably inhibit marker-aided selection.

Genomics and the tree physiologist.

During the last decade, a strong case has been made for viewing trees as model systems in plant biology. More recently, forest biologists have argued for the sequencing of the genome of a forest tree. Now, the United States Department of Energy has announced plans for sequencing the genome of the Populus trichocarpa clone, "Nisqually-1." Thus, forest biology is poised to enter an exciting period of scientific discovery. Embracing new technology and new research paradigms, however, is never easy. It is, therefore, timely to ask how tree physiologists will take advantage of the Populus genome data. We contend that the most attractive opportunities will arise through genome-wide research designed to: (1). examine the differences between trees and herbaceous annuals; (2). explore questions relating to the temporal and spatial scales that characterize the life histories and growth of trees; and (3). investigate cause-and-effect relationships that are intractable to conventional research methodologies. To highlight the potential applications of genomics in tree physiology, we briefly discuss each of these approaches.

Structure and expression of duplicate AGAMOUS orthologues in poplar.

To investigate the homeotic systems underlying floral development in a dioecious tree, and to provide tools for the manipulation of floral development, we have isolated two Populus trichocarpa genes, PTAG1 and PTAG2, homologous to the Arabidopsis floral homeotic gene AGAMOUS (AG). PTAG1 and PTAG2 are located on separate linkage groups, but their non-coding regions are highly similar, consistent with a phylogenetically recent duplication. Intron/exon structure is conserved in relation to AG and the Antirrhinum AG orthologue, PLENA (PLE), and low-stringency Southern analysis demonstrated the absence of additional genes in the poplar genome with significant PTAG1/2 homology. PTAG1 and PTAG2 exhibit an AG-like floral expression pattern, and phylogenetic analysis of the AG subfamily strongly supports evolutionary orthology to C-class organ identity genes. The high degree of similarity shared by PTAG1 and PTAG2 in both sequence (89% amino acid identity) and expression indicates that they are unlikely to be functionally associated with specification of tree gender. Unexpectedly, PTAG transcripts were consistently detected in vegetative tissues.

Factors limiting seed production of Taxus brevifolia (Taxaceae) in Western Oregon.

Seed production of Pacific yew (Taxus brevifolia), an understory conifer, was studied at four sites in western Oregon over 2 yr. The effects of pollen supplementation, overstory canopy, and predator exclusion on ovule attrition were examined. Supplemental hand-pollination of ten trees at two sites resulted in significantly increased rates of ovule development and a doubling of seed efficiency (ratio of seeds to ovules). However, seed efficiency still averaged <15% on branches receiving supplemental pollen, so pollination was not a primary factor limiting seed production. The number of developing ovules was positively associated with overstory openness, but seed production was not. Seed efficiency was negatively associated with overstory openness. Branches bagged to exclude vertebrate seed predators had higher seed production than unbagged branches at three of four sites for 2 yr. In contrast to unbagged branches, seed production on bagged branches was positively associated with overstory openness, as was the effectiveness of bagging. Therefore, both vertebrate predation and overstory were important in limiting seed production, and these factors interacted. Factors limiting seed production varied in importance among the four sites and between years, illustrating the importance of examining multiple limiting factors over several sites and years.