BACKGROUND: Forest geneticists typically use provenances to account for population differences in their improvement schemes; however, the historical records of the imported materials might not be very precise or well-aligned with the genetic clusters derived from advanced molecular techniques. The main objective of this study was to assess the impact of marker-based population structure on genetic parameter estimates related to growth and wood properties and their trade-offs in Norway spruce, by either incorporating it as a fixed effect (model-A) or excluding it entirely from the analysis (model-B).
RESULTS: Our results indicate that models incorporating population structure significantly reduce estimates of additive genetic variance, resulting in substantial reduction of narrow-sense heritability. However, these models considerably improve prediction accuracies. This was particularly significant for growth and solid-wood properties, which showed to have the highest population genetic differentiation (QST) among the studied traits. Additionally, although the pattern of correlations remained similar across the models, their magnitude was slightly lower for models that included population structure as a fixed effect. This suggests that selection, consistently performed within populations, might be less affected by unfavourable genetic correlations compared to mass selection conducted without pedigree restrictions.
CONCLUSIONS: We conclude that the results of models properly accounting for population structure are more accurate and less biased compared to those neglecting this effect. This might have practical implications for breeders and forest managers where, decisions based on imprecise selections can pose a high risk to economic efficiency.

Despite its small share of total forest biomass, ground vegetation plays an important role in biogeochemical cycles, being able to modify carbon (C) and nutrients fluxes. Global climate warming may affect plant nutrient uptake and the carbon:nitrogen:phosphorus (C:N:P) stoichiometry, the release of nutrients from the soil and soil organic matter, as well as significantly influence the tree stand nutrient supply. In this context, the response of Norway spruce (Picea abies (L.) H.Karst) stands\' ground vegetation to warming is uncertain. An open-top chamber soil-warming simulation, lasting two growing seasons, was conducted in a spruce forest. At the end of each of the two growing seasons, before leaf senescence, European blueberry (Vaccinium myrtillus L.) aboveground biomass (leaves and stems) and mineral topsoil samples were collected from the plots. The C, N, P, micronutrient, and macronutrient concentrations were estimated in the samples. Warming caused significant decreases in C, N, and P in the soil. Warming also decreased the C:P and N:P stoichiometric ratios in the soil and increased the C:P ratio in plant stems. Significant increase in foliar C and decrease in foliar P in warmed plots were observed. The most evident effect was reduction of N and P in the soil, which directly affected the plant C:P and soil N:P stoichiometry. Our results show that warming has caused a significant decrease in the content of some nutrients in the aboveground plant tissues of blueberries. Given that N is a limiting factor of ecosystems productivity, its reduction in the soil caused by warming may be a serious threat to proper nutrient uptake and cause disruption of biogeochemical cycles. The decrease in nutrient content in aboveground tissues due to warming can result in disruptions to physiological processes.

BACKGROUND: Conserved polycomb repressive complex 2 (PRC2) mediates H3K27me3 to direct transcriptional repression and has a key role in cell fate determination and cell differentiation in both animals and plants. PRC2 subunits have undergone independent multiplication and functional divergence in higher plants. However, relevant information is still absent in gymnosperms.
RESULTS: To launch gymnosperm PRC2 research, we identified and cloned the PRC2 core component genes in the conifer model species Picea abies, including one Esc/FIE homolog PaFIE, two p55/MSI homologs PaMSI1a and PaMSI1b, two E(z) homologs PaKMT6A2 and PaKMT6A4, a Su(z)12 homolog PaEMF2 and a PaEMF2-like fragment. Phylogenetic and protein domain analyses were conducted. The Esc/FIE homologs were highly conserved in the land plant, except the monocots. The other gymnospermous PRC2 subunits underwent independent evolution with angiospermous species to different extents. The relative transcript levels of these genes were measured in endosperm and zygotic and somatic embryos at different developmental stages. The obtained results proposed the involvement of PaMSI1b and PaKMT6A4 in embryogenesis and PaKMT6A2 and PaEMF2 in the transition from embryos to seedlings. The PaEMF2-like fragment was predominantly expressed in the endosperm but not in the embryo. In addition, immunohistochemistry assay showed that H3K27me3 deposits were generally enriched at meristem regions during seed development in P. abies.
CONCLUSIONS: This study reports the first characterization of the PRC2 core component genes in the coniferous species P. abies. Our work may enable a deeper understanding of the cell reprogramming process during seed and embryo development and may guide further research on embryonic potential and development in conifers.

Genome-wide association studies (GWAS) identify loci underlying the variation of complex traits. One of the main limitations of GWAS is the availability of reliable phenotypic data, particularly for long-lived tree species. Although an extensive amount of phenotypic data already exists in breeding programs, accounting for its high heterogeneity is a great challenge. We combine spatial and factor-analytics analyses to standardize the heterogeneous data from 120 field experiments of 483,424 progenies of Norway spruce to implement the largest reported GWAS for trees using 134 605 SNPs from exome sequencing of 5056 parental trees.
We identify 55 novel quantitative trait loci (QTLs) that are associated with phenotypic variation. The largest number of QTLs is associated with the budburst stage, followed by diameter at breast height, wood quality, and frost damage. Two QTLs with the largest effect have a pleiotropic effect for budburst stage, frost damage, and diameter and are associated with MAP3K genes. Genotype data called from exome capture, recently developed SNP array and gene expression data indirectly support this discovery.
Several important QTLs associated with growth and frost damage have been verified in several southern and northern progeny plantations, indicating that these loci can be used in QTL-assisted genomic selection. Our study also demonstrates that existing heterogeneous phenotypic data from breeding programs, collected over several decades, is an important source for GWAS and that such integration into GWAS should be a major area of inquiry in the future.

Brassinosteroids (BRs) are essential plant hormones. In angiosperms, brassinolide and castasterone, the first and second most active BRs, respectively, are synthesised by CYP85A2 and CYP85A/A1, respectively. BRs in angiosperms function through an essential receptor, BR Insensitive 1 (BRI1). In addition, some angiosperms also have non-essential BRI1-like 1/3 (BRL1/3). In conifers, BRs promote seed germination under drought stress; however, how BRs function in gymnosperms is unknown. In this study, we performed functional complementation of BR biosynthesis and receptor genes from Picea abies with respective Arabidopsis mutants. We found that P. abies possessed functional PaCYP85A and PaBRL1 but not PaCYP85A2 or PaBRI1, and this results in weak BR signaling, and both PaCYP85A and PaBRL1 were abundantly expressed. However, neither BR treatment of P. abies seedlings nor expression of PaBRL1 in the Arabidopsis Atbri1 mutant promoted plant height, despite the fact that BR-responsive genes were activated. Importantly, chimeric AtBRI1 replaced with the BR-binding domain of PaBRL1 complemented the Atbri1 phenotypes. Furthermore, PaBRL1 had less kinase activity than BRI1 in vitro. Overall, P. abies had weak but still active BR signaling, explaining aspects of its slow growth and high stress tolerance. Our study sheds light on the functional and evolutionary significance of distinct BR signaling that is independent of BRI1 and brassinolide.

Norway spruce (Picea abies L. Karst) is one of the most important forest tree species with significant economic and ecological impact in Europe. For decades, genomic and genetic studies on Norway spruce have been challenging due to the large and repetitive genome (19.6 Gb with more than 70% being repetitive). To accelerate genomic studies, including population genetics, genome-wide association studies (GWAS) and genomic selection (GS), in Norway spruce and related species, we here report on the design and performance of a 50K single nucleotide polymorphism (SNP) genotyping array for Norway spruce. The array is developed based on whole genome resequencing (WGS), making it the first WGS-based SNP array in any conifer species so far. After identifying SNPs using genome resequencing data from 29 trees collected in northern Europe, we adopted a two-step approach to design the array. First, we built a 450K screening array and used this to genotype a population of 480 trees sampled from both natural and breeding populations across the Norway spruce distribution range. These samples were then used to select high-confidence probes that were put on the final 50K array. The SNPs selected are distributed over 45,552 scaffolds from the P. abies version 1.0 genome assembly and target 19,954 unique gene models with an even coverage of the 12 linkage groups in Norway spruce. We show that the array has a 99.5% probe specificity, >98% Mendelian allelic inheritance concordance, an average sample call rate of 96.30% and an SNP call rate of 98.90% in family trios and haploid tissues. We also observed that 23,797 probes (50%) could be identified with high confidence in three other spruce species (white spruce [Picea glauca], black spruce [P. mariana] and Sitka spruce [P. sitchensis]). The high-quality genotyping array will be a valuable resource for genetic and genomic studies in Norway spruce as well as in other conifer species of the same genus.

Somatic embryogenesis in Norway spruce combined with reverse genetics can be used as a model to study the regulation of embryo development in conifers. The somatic embryo system includes a sequence of developmental stages, which are similar in morphology to their zygotic counterparts. The system can be sufficiently synchronized to enable the collection and study of a large number of somatic embryos at each developmental stage.Here we describe a protocol for establishing transgenic cell lines in which genes of interest are upregulated or downregulated. Furthermore, we present methods for comparing embryo morphology and development in transgenic and control cell lines, including phenotyping the embryos, histological analysis, and tracking embryo development. The expression pattern of different genes is determined by GUS reporter assays.

During winter, timberline trees are exposed to drought and frost, factors known to induce embolism. Studies indicated that conifers cope with winter embolism by xylem refilling. We analysed the loss of hydraulic conductivity (LC) in Picea abies branch xylem over 10 years, and correlated winter embolism to climate parameters. LC was investigated by direct X-ray micro-computer tomography (micro-CT) observations and potential cavitation fatigue by Cavitron measurements. Trees showed up to 100% winter embolism, whereby LC was highest, when climate variables indicated frost drought and likely freeze-thaw stress further increased LC. During summer, LC never exceeded 16%, due to hydraulic recovery. Micro-CT revealed homogenous embolism during winter and that recovery was based on xylem refilling. Summer samples exhibited lower LC in outermost compared to older tree rings, although no cavitation fatigue was detected. Long-term data and micro-CT observations demonstrate that timberline trees can survive annual cycles of pronounced winter-embolism followed by xylem refilling. Only a small portion of the xylem conductivity cannot be restored during the first year, while remaining conduits are refilled without fatigue during consecutive years. We identify important research topics to better understand the complex induction and repair of embolism at the timberline and its relevance to general plant hydraulics.

BACKGROUND: Root and butt rot of conifer trees caused by fungi belonging to the Heterobasidion annosum species complex is one of the most economically important fungal diseases in commercial conifer plantations throughout the Northern hemisphere. We investigated the interactions between Heterobasidion fungi and their host by conducting dual RNA-seq and chemical analysis on Norway spruce trees naturally infected by Heterobasidion spp. We analyzed host and pathogen transcriptome and phenolic and terpenoid contents of the spruce trees.
RESULTS: Presented results emphasize the role of the phenylpropanoid and flavonoid pathways in the chemical defense of Norway spruce trees. Accumulation of lignans was observed in trees displaying symptoms of wood decay. A number of candidate genes with a predicted role in the higher level regulation of spruce defense responses were identified. Our data indicate a possible role of abscisic acid (ABA) signaling in the spruce defense against Heterobasidion infection. Fungal transcripts corresponding to genes encoding carbohydrate- and lignin-degrading enzymes, secondary metabolism genes and effector-like genes were expressed during the host colonization.
CONCLUSIONS: Our results provide additional insight into defense strategies employed by Norway spruce trees against Heterobasidion infection. The potential applications of the identified candidate genes as markers for higher resistance against root and butt rot deserve further evaluation.

Plants live in close association with microbial symbionts, which may affect the host fitness, productivity, and tolerance against biotic and abiotic stressors. The composition of plant microbial communities is influenced by many biotic and abiotic factors, but little is known about the effect of plant pathogens on the structure of these communities. In this study, we investigated the structure of bacterial communities associated with different tissues of asymptomatic and symptomatic (Heterobasidion-rotten) Norway spruce (Picea abies (L.) Karst.) trees. Our results demonstrated that each of the investigated anatomic tissues (root, bark, down stem, upper stem, and needles) harbored a unique bacterial assemblage. However, the health status of the host trees had little effect on the structure of bacterial communities, as the only significant differences among asymptomatic and symptomatic trees were found in the composition of the bacterial communities of needles. Proteobacteria was predominant in all anatomic regions with the highest abundance in needles (86.7%), whereas Actinobacteria showed an opposite trend, being more abundant in the woody tissues than in needles. Additionally, we performed profiling of terpenoid compounds present in spruce xylem and phloem. Total concentrations of monoterpenes and sesquiterpenes were considerably higher in asymptomatic trees. However, we found no significant correlations between terpenoid profiles of spruce trees and the composition of their bacterial communities. Our results provide an insight into the diversity of bacteria associated with Norway spruce tree tissues. At the same time, the health status and terpenoid content of host trees had a limited effect on the composition of bacterial communities in our survey.