The Douglas-Fir Genome Sequence Reveals Specialization of the Photosynthetic Apparatus in Pinaceae.

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp). Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms.

Comparative chloroplast genomics reveals the evolution of Pinaceae genera and subfamilies.

As the largest and the basal-most family of conifers, Pinaceae provides key insights into the evolutionary history of conifers. We present comparative chloroplast genomics and analysis of concatenated 49 chloroplast protein-coding genes common to 19 gymnosperms, including 15 species from 8 Pinaceous genera, to address the long-standing controversy about Pinaceae phylogeny. The complete cpDNAs of Cathaya argyrophylla and Cedrus deodara (Abitoideae) and draft cpDNAs of Larix decidua, Picea morrisonicola, and Pseudotsuga wilsoniana are reported. We found 21- and 42-kb inversions in congeneric species and different populations of Pinaceous species, which indicates that structural polymorphics may be common and ancient in Pinaceae. Our phylogenetic analyses reveal that Cedrus is clustered with Abies-Keteleeria rather than the basal-most genus of Pinaceae and that Cathaya is closer to Pinus than to Picea or Larix-Pseudotsuga. Topology and structural change tests and indel-distribution comparisons lend further evidence to our phylogenetic finding. Our molecular datings suggest that Pinaceae first evolved during Early Jurassic, and diversification of Pinaceous subfamilies and genera took place during Mid-Jurassic and Lower Cretaceous, respectively. Using different maximum-likelihood divergences as thresholds, we conclude that 2 (Abietoideae and Larix-Pseudotsuga-Piceae-Cathaya-Pinus), 4 (Cedrus, non-Cedrus Abietoideae, Larix-Pseudotsuga, and Piceae-Cathaya-Pinus), or 5 (Cedrus, non-Cedrus Abietoideae, Larix-Pseudotsuga, Picea, and Cathaya-Pinus) groups/subfamilies are more reasonable delimitations for Pinaceae. Specifically, our views on subfamilial classifications differ from previous studies in terms of the rank of Cedrus and with recognition of more than two subfamilies.

Phylogeography of Douglas-fir based on mitochondrial and chloroplast DNA sequences: testing hypotheses from the fossil record.

The integration of fossil and molecular data can provide a synthetic understanding of the ecological and evolutionary history of an organism. We analysed range-wide maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA sequence data with coalescent simulations and traditional population genetic methods to test hypotheses of population divergence generated from the fossil record of Douglas-fir (Pseudotsuga menziesii), an ecologically and economically important western North American conifer. Specifically, we tested (i) the hypothesis that the Pliocene orogeny of the Cascades and Sierra Nevada caused the divergence of coastal and Rocky Mountain Douglas-fir varieties; and (ii) the hypothesis that multiple glacial refugia existed on the coast and in the Rocky Mountains. We found that Douglas-fir varieties diverged about 2.11 Ma (4.37 Ma-755 ka), which could be consistent with a Pliocene divergence. Rocky Mountain Douglas-fir probably resided in three or more glacial refugia. More variable molecular markers would be required to detect the two coastal refugia suggested in the fossil record. Comparison of mitochondrial DNA and chloroplast DNA variation revealed that gene flow via pollen linked populations isolated from seed exchange. Postglacial colonization of Canada from coastal and Rocky Mountain refugia near the ice margin at the Last Glacial Maximum produced a wide hybrid zone among varieties that formed almost exclusively by pollen exchange and chloroplast DNA introgression, not seed exchange. Postglacial migration rates were 50-165 m/year, insufficient to track projected 21st century warming in some regions. Although fossil and genetic data largely agree, each provides unique insights.

Metabolite profiling of Douglas-fir (Pseudotsuga menziesii) field trials reveals strong environmental and weak genetic variation.

The primary objective of this study was to assess metabolomics for its capacity to discern biological variation among 10 full-sib families of a Douglas-fir tree breeding population, replicated on two sites. The differential accumulation of small metabolites in developing xylem was examined through metabolite profiles (139 metabolites common to 181 individual trees) generated by gas chromatography mass spectrometry and a series of statistical analyses that incorporated family, site, and tree growth and quantitative phenotypic wood traits (wood density, microfibril angle, wood chemistry and fiber morphology). Multivariate discriminant, canonical discriminant and factor analyses and broad-sense heritabilities revealed that metabolic and phenotypic traits alike were strongly related to site, while similar associations relating to genetic (family) structure were weak in comparison. Canonical correlation analysis subsequently identified correlations between specific phenotypic traits (i.e. tree growth, fibre morphology and wood chemistry) and metabolic traits (i.e. carbohydrate and lignin biosynthetic metabolites), demonstrating a coherent relationship between genetics, metabolism, environmental and phenotypic expression in wood-forming tissue. The association between cambial metabolites and tree phenotype, as revealed by metabolite profiling, demonstrates the value of metabolomics for systems biology approaches to understanding tree growth and secondary cell wall biosynthesis in plants.