Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2001. / Härtill 4 uppsatser.

The genus Populus consists of many ecologically and economically important forest tree species. Their rapid growth makes them one of the most productive hardwoods growing in temperate latitudes. Populus spp. frequently hybridize where their ranges overlap, and poplar hybrids are the most frequently planted genotypes for fiber production. To better understand the genomics of hybridization in Populus, we sampled and sequenced the genome of 574 poplar trees from six east-west transects across the hybrid zone between Populus trichocarpa and Populus balsamifera in western North America. I used these data to characterize population structure within and between transects, and hybridization between the species. There was a consistent transition from greater P. balsamifera ancestry in the north and east to greater P. trichocarpa ancestry in the south and west. Hybridization between the species was common across each of the six transects, though more common in colder climates. The results also showed that both latitude and longitude affect the genetic structure of this species complex, and that subtle introgression from P. balsamifera may facilitate adaptation of P. trichocarpa to colder climates. / Master of Science / The genus Populus has many ecologically and economically important forest tree species. Balsam poplar (Populus balsamifera) and black cottonwood (Populus trichocarpa) are two such species, both for fiber production and models for understanding tree biology and adaptation. Whereas black cottonwood is distributed close to the west coast of North America from California through Alaska, balsam poplar mostly occurs across the interior of Canada from Newfoundland through Alberta. Where their ranges overlap, the species often hybridize. In this study, we used genome sequencing of trees collected across six east-west transects from Washington state through British Columbia, Canada, and Alaska to understand genetic variation and the geography of hybridization. I found evidence of widespread hybridization across all transects. While the influence of P. balsamifera was extensive in northern populations, a large number of pure P. trichocarpa were found in southern populations. The transition from P. trichocarpa to P. balsamifera was also steeper in the south than the north, with a narrower hybrid zone in the south. Additionally, I found that gene flow among some populations was limited by temperature and geographical barriers. Taken together, my results suggest genetic structure and hybridization within and between these species is driven by climate variation, and that P. balsamifera ancestry may help northern P. trichocarpa populations adapt to their local environments.

No description available.

Wood has a wide variety of uses and is arguably the most important renewable raw material. The composition of xylem cell types in wood determines the utility of different types of wood for distinct commercial applications. Using expression profiling and phylogenetic analysis, we identified many xylem-associated regulatory genes that may control the differentiation of cells involved in wood formation in Arabidopsis and poplar. Prominent among these are NAC-domain transcription factors (NACs). In addition to their roles as regulators of xylem differentiation, NACs are regulators of meristem development, organ elongation and separation. We studied a subset of Populus and Arabidopsis NACs with putative involvement in xylem cell expansion and elongation (XND1/ANAC104, PopNAC118, PopNAC122, PopNAC128, PopNAC129), and secondary cell wall synthesis (ANAC073, PopNAC105, PopNAC154, PopNAC156, PopNAC157). Using quantitative Real-Time PCR, we evaluated expression of the selected Populus NACs in a developmental gradient and in response to bending stress. We prepared transgenic Arabidopsis and Populus plants with increased or decreased expression of select NAC genes. For dominant repression of target gene expression, we evaluated transgenic plants expressing translational fusions of NAC-EAR (ERF amphiphilic repressor) chimeras through chimeric repressor silencing-technology (CRES-T). XND1 overexpression in Populus and Arabidopsis resulted in severe stunting and suppression of xylem differentiation. Overexpression of PopNAC122, an XND1 ortholog, yielded an analogous phenotype in Arabidopsis. Populus XND1 overexpressors lacked phloem fibers and showed a reduction in cell size and number, vessel number and frequency of rays. Knowledge gained through characterization of these wood-associated regulatory genes can be used to optimize molecular breeding and genetic engineering strategies for improved wood quality and increased biomass. / Master of Science

Temperate tree species exhibit seasonal growth cycling, and the timing of such transition varies with local climate. Under anthropogenic climate change, the local pattern of growth and dormancy in tree populations is expected to become uncoupled with shifting seasonal environmental signals, particularly temperature. Thus, an understanding of the genetic underpinnings of local adaptation is key to predicting the fate of tree populations in the future. In this thesis, we coupled sampling of range-wide natural accessions of P. trichocarpa with adaptive trait phenotyping and genome-wide genotyping to uncover relationships between genotype, phenotype, and environment. We detected strong correlations between adaptive phenotypes, climate, and geography, which suggested climatic selection driving adaptation of these populations to local environments. We subsequently combined genotype-phenotype association tests with sliding window analysis and identified regions strongly associated with these adaptive traits. We also compared adaptive markers identified in two independent GWAS on samples across latitude and altitude transects and found a set of associated variants shared across both transects. We further scanned the genome with three selection tests to identify regions showing evidence of recent positive and divergent selection. By comparing candidate selection regions across altitude and latitude, we detected a set of overlapping regions showing differentiation across gradients of the same climate variables. We validated the functional imortance of these selection regions by combining GWAS and showed that selection regions contain a strong signature of phenotypic associations. We also studied the distribution of deleterious allels across genome and natural populations, and found that deleterious alleles preferentially accumulate in regions of low recombination and hithihking regions. Finally, marginal populations contained more deleterious alleles compared with central populations, which is likely due to ineffective selection in small populations and recent bottlenecks associated with postglacial recolonization. These findings provide new insights into the genomic architecture underlying climatic adaptation and how selection drives adaptive evolution of tree species. / Ph. D.

Forest tree breeding methods rank among the most efficient ways to increase productivity and quality of forests. With the advent of high-throughput genotyping technology, genome-enabled breeding has started to gain importance and may overcome some weaknesses of traditional tree breeding. Genomic Selection (GS), which involves using genome-wide markers to predict breeding values of individuals in a population, has been proposed for animal and plant breeding programs. GS enables very accurate selection decisions through estimation of genomic estimated breeding values (GEBVs). While the goal of GS is to predict phenotype from genotype, it does not identify the underlying genes that have important roles in a trait. Genome-Wide Association Studies (GWAS) approaches are therefore complementary to GS, enabling identification of these genes, which may be useful for marker-assisted selection in some traits. In this study, we first estimated heritability for several adaptive traits (cold hardiness, dbh, bud flush, height, and bud set) in a population of Populus trichocarpa and for height, diameter, and stem straightness in Pinus taeda. GEBVs accuracies were estimated using a ridge regression–best linear unbiased prediction (rrBLUP) model, and these accuracies were compared with estimated heritabilities. GWAS was also performed for the both imputed and non–imputed data of P. taeda population using TASSEL (Trait Analysis by aSSociation Evolution and Linkage) software, as well as rrBLUP and FFBSKAT (Fast Family-Based Sequence Kernel Association Test) packages in R. Heritabilities ranged from 0.34 to 0.56 for P. trichocarpa and 0.14 to 0.37 for P.taeda. GWAS identified 3244 associations for dbh, 4077 associations for stem straightness, and 5280 SNPs for height (p≤0.05) in TASSEL using the reduced model (marker data only), whereas 2729, 3272 and 3531 associations were found with the full model where we also included population structure as a covariate. FFBSKAT showed a similar number of SNP associations (2989, 3046 and 3058). There was an inflation of SNP associations (~20k) found in rrBLUP, which suggests population structure was not effectively controlled. The GEBVs accuracies ranged from 0.09 and 0.22 for P.trichocarpa and 0.09 to 0.23 for P.taeda using rrBLUP method. Testing the effect of repetation on the accuracy of GEBV for poplar showed that there was no significant difference between the number of cycles. Also, there was no significant difference the accuracy of GEBVs in pine between two different imputation methods, the marker mean value and Beagle software. / Master of Science

Forest trees are an important pool of biodiversity at the gene, individual and an ecosystem level. This variation is a result of complex environmental interactions, as well as neutral and selective forces acting on populations. Patterns of standing genetic variation are the result of adaption to past and contemporary climate change, but also historical demographic events, and disentangling the role of these forces is a central problem in population genomics. The overall goal of this study is to characterize the relative effects of demography and selection in the genome of Populus trichocarpa, a riparian deciduous tree species of North America. Specifically, I used a variety of methods to summarize patterns of genetic diversity and population structure in P. trichocarpa, and to reconstruct its demographic history. I subsequently incorporated these demographic insights to guide the application of several methods to identify genome-wide targets of natural selection within and among rangewide populations adapted to heterogeneous selection regimes. Results of this study provide insights into the history of divergence and differentiation in P. trichocarpa populations and help us identify the functional genetic variants contributing to phenotypic divergence and fitness of the individuals in it. / Ph. D.

Les glutathion transférases (GSTs) constituent une famille multigénique d’enzymes présentes dans les trois domaines du vivant. Cette présence ubiquitaire souligne l’origine sans doute très ancienne de ces enzymes ainsi que des fonctions fondamentales conservées au cours de l’évolution. Ces enzymes sont impliquées notamment dans la détoxication cellulaire de molécules toxiques et dans le métabolisme secondaire. Les analyses phylogénétiques regroupent les GSTs des organismes photosynthétiques au sein de quatorze classes qui peuvent être séparées en deux grands groupes selon le résidu catalytique : les GSTs à sérine catalytique qui possèdent des activités de conjugaison du glutathion (GSH) et/ou peroxydase tandis que les GSTs à cystéine catalytique présentent des activités thioltransférase, déshydroascorbate réductase et de déglutathionylation. Les GSTs à sérine catalytique de la classe Phi (GSTF) sont présentes chez les organismes photosynthétiques et certains basidiomycètes. Chez les plantes, cette classe comprend un nombre de gènes plus important que les autres classes de GSTs. Ceux-ci sont parmi les plus régulés en réponse à divers stress et ils ont été fortement étudiés chez les plantes céréalières en raison de l’activité de détoxication des herbicides des protéines correspondantes. Pourtant, à quelques exceptions près, les rôles physiologiques des GSTFs restent inconnus et la redondance d’isoformes dans cette classe reste incomprise. Par des approches moléculaires, biochimiques et structurales, l’analyse structure-fonction des huit GSTFs de l’arbre modèle Populus trichocarpa a été réalisée au cours de cette thèse. L’analyse phylogénétique des GSTFs chez les organismes photosynthétiques a montré que cette classe est apparue au moment de l’apparition terrestre des végétaux et que différents groupes pouvaient être identifiés avec des motifs catalytiques distincts. L’analyse transcriptionnelle a montré que les gènes relatifs aux GSTFs de peuplier sont principalement exprimés dans les fleurs femelles, les pétioles et les fruits. Certains aspects du mécanisme réactionnel ont été caractérisés en déterminant notamment les paramètres cinétiques et d’interaction des huit GSTFs et de plusieurs variants mutés pour des résidus clés vis-à-vis de substrats modèles. Les structures de cinq des huit GSTFs ont été résolues et ces protéines dimériques adoptent un repliement GST canonique et des spécificités structurales au niveau du site actif ont pu être observées. De plus, au regard de la capacité des orthologues des GSTFs à lier des hormones et des flavonoïdes ainsi que de l’expression récurrente des GSTFs de peuplier dans les fruits et les fleurs femelles, deux organes riches en ces molécules, il peut être supposé qu’elles ont aussi des propriétés de type ligandine. Des résultats préliminaires ont également été obtenus pour la recherche de substrats physiologiques à partir de métabolites extraits de différents organes de peuplier. A terme, l’identification de ces substrats permettra de déterminer le mode d’action (catalytique vs ligandine) de chaque enzyme et d’identifier clairement les fonctions in planta de ces enzymes / Glutathione transferases (GSTs) belong to a multigenic family whose presence in most eukaryotes, prokaryotes and archaea reflects their widespread nature and very likely important functions. These enzymes represent a major group of enzymes involved in xenobiotic detoxification and secondary metabolism. From the most recent genomic and phylogenetic analyses, the GST family is subdivided into 14 classes that can be separated into two main groups based on the catalytic residue which is either a serine (Ser-GST) or a cysteine (Cys-GST). Ser-GSTs usually catalyze glutathione (GSH) conjugation and/or peroxide reduction. On the other hand, Cys-GSTs cannot perform GSH-conjugation reactions but instead catalyze thiol-transferase, dehydroascorbate reductase and deglutathionylation reactions. Ser-GSTs from the Phi class (GSTF) are present in photosynthetic organisms and some basidiomycetes. This class is composed of a large number of genes compared to other GST classes which are amongst the most stress-inducible. The corresponding proteins have been extensively studied in crops with regard to their detoxification activities toward herbicides. However, with a few exceptions, very little is known about their roles in planta and it is not well understood why this class has expanded. By combining molecular, cellular, biochemical and structural approaches, the eight isoforms from the model tree Populus trichocarpa have been characterized during this PhD project. Phylogenetic analysis of GSTFs in the green lineage shows that the apparition of this class is concomitant with the appearance of terrestrial plants and that different groups can be distinguished based on the active site signature. RT-PCR analysis of the eight isoforms of GSTFs showed that transcripts mostly accumulate in female flowers, petioles and fruits. Some aspects of the reaction mechanism have been characterized by determining kinetic parameters of the eight poplar GSTFs and of several mutated variants for key residues towards model substrates. The structures of five GSTFs have been solved and these dimeric proteins display a typical GST fold but specificities have been observed at the catalytic site level. Moreover, considering the demonstrated capacity of GSTF orthologs to bind hormones, anthocyanins or flavonoids, and the consistent high expression of poplar GSTFs in female flowers and fruits, two organs rich in these molecules, we speculate that they may also possess ligandin properties. Preliminary results have been obtained regarding the nature of the substrates in various poplar organs by analyzing protein thermostability in the presence of putative ligands. In order to assess whether a functional redundancy between poplar Phi GSTs exists and to identify their mode of action (catalytic vs ligandin functions), we started to isolate and identify physiological substrates

We are using the yeast two-hybrid (Y2H) system to identify novel protein-protein interactions (PPI) relevant to wood formation. Bait proteins for Y2H binary assays and screening against a xylem cDNA prey library were selected from approximately 400 Populus trichocarpa genes that are at least 8-fold more highly expressed in differentiating secondary xylem versus phloem-cambium, and designated here as poplar biomass (PB) genes. Here we report some of the interactions involving selected PB proteins and efforts to characterize their functions in Populus and Arabidopsis. Members of the ROP GTPase family, PB15 in poplar and ROP11 in Arabidopsis, interact with the domain of unknown function (DUF) 620 (DUF620) proteins (e.g., PB129 in poplar). Ectopic co-expression of PB15 and PB129 in Arabidopsis caused outgrowths at the base of flower pedicels and altered leaf morphology. Interestingly, the co-expression phenotype could not be observed in transgenic plants that are only expressing either one of the interacting partners separately. Transgenics altered in expression of PB15 and/or PB129 were prepared in Populus and characterization of transgenic trees will be performed in greenhouse and field. In addition to DUF620 family proteins, ROP11 also interacts with the COP9 subunit CSN5A in Arabidopsis. We confirmed the interaction of ROP11 and CSN5A in Y2H and employed available mutants for ROP11 and CSN5A in Arabidopsis to genetically characterize this interaction. Surprisingly, loss of ROP11 was found to rescue the csn5a-2 pleiotropic phenotype. Ectopic expression of a ROP11 dominant negative mutant in the csn5a-2 background also complemented the stunted growth phenotype. Transcript analysis and gel blot assays showed that CSN5A transcript levels remained unchanged in all rescue lines, whereas CSN5A protein levels increased relative to WT. Taken together, we concluded that ROP11 negatively regulate CSN5A protein level in plant by some as yet unknown mechanism. / Ph. D.

Local adaptation to climate in temperate forest trees involves the integration of multiple physiological, morphological, and phenological traits. Latitudinal clines for the relevant component traits are frequently observed for species that have a north-south distribution, but these relationships do not account for climatic variation within a given latitudinal band, which may be reflected in adaptive traits. We used black cottonwood (Populus trichocarpa) as a model to characterize the interplay between geography, climate, and adaptation to abiotic factors. Twelve traits (height, diameter, volume index, crown diameter, number of branches, number of sylleptic branches, relative number of branches, Relative canopy depth, Bud set, Bud flush, cold index of injury, carbon isotope ratio) were measured in a range-wide sample of 124 P. trichocarpa genotypes grown in a common garden. Heritability's were moderate to high (0.24 to 0.55) and significant population differentiation (QST > 0.3) suggested adaptive divergence. When climate variables were taken as predictors and the 12 traits as response variables in a multivariate regression tree analysis, aridity (Eref) explained the most variation, with subsequent splits grouping individuals according to mean temperature of the warmest month, frost-free period (FFP), and mean annual precipitation (MAP). This grouping matches relatively well the splits using geographic variables as predictors: the northernmost groups (short FFP and low Eref) had the lowest growth performance, and the highest cold hardiness. The groups spanning the south of British Columbia (low Eref and intermediate temperatures) displayed an average growth and cold hardiness. The group from the coast of California and Oregon (high Eref and FFP) had the best growth performance and the lowest cold hardiness. The southernmost and high-elevated group (with High Eref and low FFP) performed poorly, had a low cold hardiness and a significantly lower WUE. / Master of Science