Future carbon sequestration potential in a widespread transcontinental boreal tree species: Standing genetic variation matters!

Climate change (CC) necessitates reforestation/afforestation programs to mitigate its impacts and maximize carbon sequestration. But comprehending how tree growth, a proxy for fitness and resilience, responds to CC is critical to maximize these programs' effectiveness. Variability in tree response to CC across populations can notably be influenced by the standing genetic variation encompassing both neutral and adaptive genetic diversity. Here, a framework is proposed to assess tree growth potential at the population scale while accounting for standing genetic variation. We applied this framework to black spruce (BS, Picea mariana [Mill] B.S.P.), with the objectives to (1) determine the key climate variables having impacted BS growth response from 1974 to 2019, (2) examine the relative roles of local adaptation and the phylogeographic structure in this response, and (3) project BS growth under two Shared Socioeconomic Pathways while taking standing genetic variation into account. We modeled growth using a machine learning algorithm trained with dendroecological and genetic data obtained from over 2600 trees (62 populations divided in three genetic clusters) in four 48-year-old common gardens, and simulated growth until year 2100 at the common garden locations. Our study revealed that high summer and autumn temperatures negatively impacted BS growth. As a consequence of warming, this species is projected to experience a decline in growth by the end of the century, suggesting maladaptation to anticipated CC and a potential threat to its carbon sequestration capacity. This being said, we observed a clear difference in response to CC within and among genetic clusters, with the western cluster being more impacted than the central and eastern clusters. Our results show that intraspecific genetic variation, notably associated with the phylogeographic structure, must be considered when estimating the response of widespread species to CC.

Contrasting physiological strategies explain heterogeneous responses to severe drought conditions within local populations of a widespread conifer.

Understanding how trees prioritize carbon gain at the cost of drought vulnerability under severe drought conditions is crucial for predicting which genetic groups and individuals will be resilient to future climate conditions. In this study, we investigated variations in growth, tree-ring anatomy as well as carbon and oxygen isotope ratios to assess the sensitivity and the xylem formation process in response to an episode of severe drought in 29 mature white spruce (Picea glauca [Moench] Voss) families grown in a common garden trial. During the drought episode, the majority of families displayed decreased growth and exhibited either sustained or increased intrinsic water-use efficiency (iWUE), which was largely influenced by reduced stomatal conductance as revealed by the dual carbon­oxygen isotope approach. Different water-use strategies were detected within white spruce populations in response to drought conditions. Our results revealed intraspecific variation in the prevailing physiological mechanisms underlying drought response within and among populations of Picea glauca. The presence of different genetic groups reflecting diverse water-use strategies within this largely-distributed conifer is likely to lessen the negative effects of drought and decrease the overall forest ecosystems' sensitivity to it.

Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay.

BACKGROUND: Bacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh ß-sheet of sfGFP, which can complement a truncated sfGFP that lacks this ß-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress. RESULTS: The biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SPPel, SPEpr and SPBsn obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels. CONCLUSION: Our results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.

Increasing genomic prediction accuracy for unphenotyped full-sib families by modeling additive and dominance effects with large datasets in white spruce.

Introduction: Genomic selection is becoming a standard technique in plant breeding and is now being introduced into forest tree breeding. Despite promising results to predict the genetic merit of superior material based on their additive breeding values, many studies and operational programs still neglect non-additive effects and their potential for enhancing genetic gains. Methods: Using two large comprehensive datasets totaling 4,066 trees from 146 full-sib families of white spruce (Picea glauca (Moench) Voss), we evaluated the effect of the inclusion of dominance on the precision of genetic parameter estimates and on the accuracy of conventional pedigree-based (ABLUP-AD) and genomic-based (GBLUP-AD) models. Results: While wood quality traits were mostly additively inherited, considerable non-additive effects and lower heritabilities were detected for growth traits. For growth, GBLUP-AD better partitioned the additive and dominance effects into roughly equal variances, while ABLUP-AD strongly overestimated dominance. The predictive abilities of breeding and total genetic value estimates were similar between ABLUP-AD and GBLUP-AD when predicting individuals from the same families as those included in the training dataset. However, GBLUP-AD outperformed ABLUP-AD when predicting for new unphenotyped families that were not represented in the training dataset, with, on average, 22% and 53% higher predictive ability of breeding and genetic values, respectively. Resampling simulations showed that GBLUP-AD required smaller sample sizes than ABLUP-AD to produce precise estimates of genetic variances and accurate predictions of genetic values. Still, regardless of the method used, large training datasets were needed to estimate additive and non-additive genetic variances precisely. Discussion: This study highlights the different quantitative genetic architectures between growth and wood traits. Furthermore, the usefulness of genomic additive-dominance models for predicting new families should allow practicing mating allocation to maximize the total genetic values for the propagation of elite material.

The phenotypic and genetic effects of drought-induced stress on wood specific conductivity and anatomical properties in white spruce seedlings, and relationships with growth and wood density.

Drought frequency and intensity are projected to increase with climate change, thus amplifying stress on forest trees. Resilience to drought implicates physiological traits such as xylem conductivity and wood anatomical traits, which are related to growth and wood density. Integrating drought-stress response traits at the juvenile stage into breeding criteria could help promote the survival of planted seedlings under current and future climate and thus, improve plantation success. We assessed in greenhouse the influence of drought-induced stress on 600 two-year-old white spruce (Picea glauca) seedlings from 25 clonal lines after two consecutive growing seasons. Three levels of drought-induced stress were applied: control, moderate and severe. Seedlings were also planted at a 45° angle to clearly separate compression from normal wood. We looked at the phenotypic and genetic effects of drought stress on xylem specific hydraulic conductivity, lumen diameter, tracheid diameter and length, and the number of pits per tracheid in the normal wood. We detected no significant effects of drought stress except for tracheid length, which decreased with increasing drought stress. We found low to high estimates of trait heritability, which generally decreased with increasing drought stress. Genetic correlations were higher than phenotypic correlations for all treatments. Specific conductivity was genetically highly correlated positively with lumen diameter and tracheid length under all treatments. Tracheid length and diameter were always negatively correlated genetically, indicating a trade-off in resource allocation. Moderate to high genetic correlations sometimes in opposite direction were observed between physico-anatomical and productivity traits, also indicating trade-offs. A large variation was observed among clones for all physico-anatomical traits, but clonal ranks were generally stable between control and drought-induced treatments. Our results indicate the possibility of early screening of genetic material for desirable wood anatomical attributes under normal growing conditions, thus allowing to improve the drought resilience of young trees.

Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation.

Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain. KEY POINTS: • Automated workflows for strain construction and screening of protein secretion • Comparison of spacer, signal peptide, and host combinations for cutinase secretion • Signal peptide screening for secretion by C. glutamicum using the split GFP assay.

Breeding for adaptation to climate change: genomic selection for drought response in a white spruce multi-site polycross test.

With climate change, increasingly intense and frequent drought episodes will be affecting water availability for boreal tree species, prompting tree breeders and forest managers to consider adaptation to drought stress as a priority in their reforestation efforts. We used a 19-year-old polycross progeny test of the model conifer white spruce (Picea glauca) replicated on two sites affected by distinct drought episodes at different ages to estimate the genetic control and the potential for improvement of drought response in addition to conventional cumulative growth and wood quality traits. Drought response components were measured from dendrochronological signatures matching drought episodes in wood ring increment cores. We found that trees with more vigorous growth during their lifespan resisted better during the current year of a drought episode when the drought had more severe effects. Phenotypic data were also analyzed using genomic prediction (GBLUP) relying on the genomic relationship matrix of multi-locus gene SNP marker information, and conventional analysis (ABLUP) based on validated pedigree information. The accuracy of predicted breeding values for drought response components was marginally lower than that for conventional traits and comparable between GBLUP and ABLUP. Genetic correlations were generally low and nonsignificant between drought response components and conventional traits, except for resistance which was positively correlated to tree height. Heritability estimates for the components of drought response were slightly lower than for conventional traits, but similar single-trait genetic gains could be obtained. Multi-trait genomic selection simulations indicated that it was possible to improve simultaneously for all traits on both sites while sacrificing little on gain in tree height. In a context of rapid climate change, our results suggest that with careful phenotypic assessment, drought response may be considered in multi-trait improvement of white spruce, with accelerated screening of large numbers of candidates and selection at young age with genomic selection.

Metadata analysis indicates biased estimation of genetic parameters and gains using conventional pedigree information instead of genomic-based approaches in tree breeding.

Forest tree improvement helps provide adapted planting stock to ensure growth productivity, fibre quality and carbon sequestration through reforestation and afforestation activities. However, there is increasing doubt that conventional pedigree provides the most accurate estimates for selection and prediction of performance of improved planting stock. When the additive genetic relationships among relatives is estimated using pedigree information, it is not possible to take account of Mendelian sampling due to the random segregation of parental alleles. The use of DNA markers distributed genome-wide (multi-locus genotypes) makes it possible to estimate the realized additive genomic relationships, which takes account of the Mendelian sampling and possible pedigree errors. We reviewed a series of papers on conifer and broadleaf tree species in which both pedigree-based and marker-based estimates of genetic parameters have been reported. Using metadata analyses, we show that for heritability and genetic gains, the estimates obtained using only the pedigree information are generally biased upward compared to those obtained using DNA markers distributed genome-wide, and that genotype-by-environment (GxE) interaction can be underestimated for low to moderate heritability traits. As high-throughput genotyping becomes economically affordable, we recommend expanding the use of genomic selection to obtain more accurate estimates of genetic parameters and gains.

Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum.

The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis.

BACKGROUND: Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh ß-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. RESULTS: As proof of concept, the GFP11-tag was fused C-terminally to the E. coli ß-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4-12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. CONCLUSION: Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level.

Connecting tree-ring phenotypes, genetic associations and transcriptomics to decipher the genomic architecture of drought adaptation in a widespread conifer.

As boreal forests face significant threats from climate change, understanding evolutionary trajectories of coniferous species has become fundamental to adapting management and conservation to a drying climate. We examined the genomic architecture underlying adaptive variation related to drought tolerance in 43 populations of a widespread boreal conifer, white spruce (Picea glauca [Moench] Voss), by combining genotype-environment associations, genotype-phenotype associations, and transcriptomics. Adaptive genetic variation was identified by correlating allele frequencies for 6,153 single nucleotide polymorphisms from 2,606 candidate genes with temperature, precipitation and aridity gradients, and testing for significant associations between genotypes and 11 dendrometric and drought-related traits (i.e., anatomical, growth response and climate-sensitivity traits) using a polygenic model. We identified a set of 285 genes significantly associated with a climatic factor or a phenotypic trait, including 110 that were differentially expressed in response to drought under greenhouse-controlled conditions. The interlinked phenotype-genotype-environment network revealed eight high-confidence genes involved in white spruce adaptation to drought, of which four were drought-responsive in the expression analysis. Our findings represent a significant step toward the characterization of the genomic basis of drought tolerance and adaptation to climate in conifers, which is essential to enable the establishment of resilient forests in view of new climate conditions.

Annual aboveground carbon uptake enhancements from assisted gene flow in boreal black spruce forests are not long-lasting.

Assisted gene flow between populations has been proposed as an adaptive forest management strategy that could contribute to the sequestration of carbon. Here we provide an assessment of the mitigation potential of assisted gene flow in 46 populations of the widespread boreal conifer Picea mariana, grown in two 42-year-old common garden experiments and established in contrasting Canadian boreal regions. We use a dendroecological approach taking into account phylogeographic structure to retrospectively analyse population phenotypic variability in annual aboveground net primary productivity (NPP). We compare population NPP phenotypes to detect signals of adaptive variation and/or the presence of phenotypic clines across tree lifespans, and assess genotype-by-environment interactions by evaluating climate and NPP relationships. Our results show a positive effect of assisted gene flow for a period of approximately 15 years following planting, after which there was little to no effect. Although not long lasting, well-informed assisted gene flow could accelerate the transition from carbon source to carbon sink after disturbance.

Genomic selection for resistance to spruce budworm in white spruce and relationships with growth and wood quality traits.

With climate change, the pressure on tree breeding to provide varieties with improved resilience to biotic and abiotic stress is increasing. As such, pest resistance is of high priority but has been neglected in most tree breeding programs, given the complexity of phenotyping for these traits and delays to assess mature trees. In addition, the existing genetic variation of resistance and its relationship with productivity should be better understood for their consideration in multitrait breeding. In this study, we evaluated the prospects for genetic improvement of the levels of acetophenone aglycones (AAs) in white spruce needles, which have been shown to be tightly linked to resistance to spruce budworm. Furthermore, we estimated the accuracy of genomic selection (GS) for these traits, allowing selection at a very early stage to accelerate breeding. A total of 1,516 progeny trees established on five sites and belonging to 136 full-sib families from a mature breeding population in New Brunswick were measured for height growth and genotyped for 4,148 high-quality SNPs belonging to as many genes along the white spruce genome. In addition, 598 trees were assessed for levels of AAs piceol and pungenol in needles, and 578 for wood stiffness. GS models were developed with the phenotyped trees and then applied to predict the trait values of unphenotyped trees. AAs were under moderate-to-high genetic control (h 2: 0.43-0.57) with null or marginally negative genetic correlations with other traits. The prediction accuracy of GS models (GBLUP) for AAs was high (PAAC: 0.63-0.67) and comparable or slightly higher than pedigree-based (ABLUP) or BayesCπ models. We show that AA traits can be improved and that GS speeds up the selection of improved trees for insect resistance and for growth and wood quality traits. Various selection strategies were tested to optimize multitrait gains.

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis.

BACKGROUND: Bacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins. RESULTS: The shuttle vector pBSMul1 containing the strong constitutive promoter PHpaII and the optimal Shine Dalgarno sequence TAAGGAGG was used as a template to construct a series of vectors with spacer lengths varying from 4 to 12 adenosines. For the intracellular proteins GFPmut3 and ß-glucuronidase, an increase of spacer lengths from 4 to 7-9 nucleotides resulted in a gradual increase of product yields up to 27-fold reaching a plateau for even longer spacers. The production of secreted proteins was tested with cutinase Cut and swollenin EXLX1 which were N-terminally fused to one of the Sec-dependent signal peptides SPPel, SPEpr or SPBsn. Again, longer spacer sequences resulted in up to tenfold increased yields of extracellular proteins. Fusions with signal peptides SPPel or SPBsn revealed the highest production yields with spacers of 7-10nt length. Remarkably, fusions with SPEpr resulted in a twofold lower production yield with 6 or 7nt spacers reaching a maximum with 10-12nt spacers. This pattern was observed for both secreted proteins fused to SPEpr indicating a dominant role also of the nucleotide sequence encoding the respective signal peptide for translation initiation. This conclusion was corroborated by RT qPCR revealing only slightly different amounts of transcript. Also, the effect of a putative alternative translation initiation site could be ruled out. CONCLUSION: Our results confirm the importance of the 5' end sequence of a target gene for translation initiation. Optimizing production yields thus may require screenings for optimal spacer sequence lengths. In case of secreted proteins, the 5' sequence encoding the signal peptide for Sec-depended secretion should also be considered.

Adaptive genetic variation to drought in a widely distributed conifer suggests a potential for increasing forest resilience in a drying climate.

Drought intensity and frequency are increasing under global warming, with soil water availability now being a major factor limiting tree growth in circumboreal forests. Still, the adaptive capacity of trees in the face of future climatic regimes remains poorly documented. Using 1481 annually resolved tree-ring series from 29-yr-old trees, we evaluated the drought sensitivity of 43 white spruce (Picea glauca (Moench) Voss) populations established in a common garden experiment. We show that genetic variation among populations in response to drought plays a significant role in growth resilience. Local genetic adaptation allowed populations from drier geographical origins to grow better, as indicated by higher resilience to extreme drought events, compared with populations from more humid geographical origins. The substantial genetic variation found for growth resilience highlights the possibility of selecting for drought resilience in boreal conifers. As a major research outcome, we showed that adaptive genetic variation in response to changing local conditions can shape drought vulnerability at the intraspecific level. Our findings have wide implications for forest ecosystem management and conservation.

Genomic prediction for hastening and improving efficiency of forward selection in conifer polycross mating designs: an example from white spruce.

Genomic selection (GS) has a large potential for improving the prediction accuracy of breeding values and significantly reducing the length of breeding cycles. In this context, the choice of mating designs becomes critical to improve the efficiency of breeding operations and to obtain the largest genetic gains per time unit. Polycross mating designs have been traditionally used in tree and plant breeding to perform backward selection of the female parents. The possibility to use genetic markers for paternity identification and for building genomic prediction models should allow for a broader use of polycross tests in forward selection schemes. We compared the accuracies of genomic predictions of offspring's breeding values from a polycross and a full-sib (partial diallel) mating design with similar genetic background in white spruce (Picea glauca). Trees were phenotyped for growth and wood quality traits, and genotyped for 4092 SNPs representing as many gene loci distributed across the 12 spruce chromosomes. For the polycross progeny test, heritability estimates were smaller, but more precise using the genomic BLUP (GBLUP) model as compared with pedigree-based models accounting for the maternal pedigree or for the reconstructed full pedigree. Cross-validations showed that GBLUP predictions were 22-52% more accurate than predictions based on the maternal pedigree, and 5-7% more accurate than predictions using the reconstructed full pedigree. The accuracies of GBLUP predictions were high and in the same range for most traits between the polycross (0.61-0.70) and full-sib progeny tests (0.61-0.74). However, higher genetic gains per time unit were expected from the polycross mating design given the shorter time needed to conduct crosses. Considering the operational advantages of the polycross design in terms of easier handling of crosses and lower associated costs for test establishment, we believe that this mating scheme offers great opportunities for the development and operational application of forward GS.

Multi-trait genomic selection for weevil resistance, growth, and wood quality in Norway spruce.

Plantation-grown trees have to cope with an increasing pressure of pest and disease in the context of climate change, and breeding approaches using genomics may offer efficient and flexible tools to face this pressure. In the present study, we targeted genetic improvement of resistance of an introduced conifer species in Canada, Norway spruce (Picea abies (L.) Karst.), to the native white pine weevil (Pissodes strobi Peck). We developed single- and multi-trait genomic selection (GS) models and selection indices considering the relationships between weevil resistance, intrinsic wood quality, and growth traits. Weevil resistance, acoustic velocity as a proxy for mechanical wood stiffness, and average wood density showed moderate-to-high heritability and low genotype-by-environment interactions. Weevil resistance was genetically positively correlated with tree height, height-to-diameter at breast height (DBH) ratio, and acoustic velocity. The accuracy of the different GS models tested (GBLUP, threshold GBLUP, Bayesian ridge regression, BayesCπ) was high and did not differ among each other. Multi-trait models performed similarly as single-trait models when all trees were phenotyped. However, when weevil attack data were not available for all trees, weevil resistance was more accurately predicted by integrating genetically correlated growth traits into multi-trait GS models. A GS index that corresponded to the breeders' priorities achieved near maximum gains for weevil resistance, acoustic velocity, and height growth, but a small decrease for DBH. The results of this study indicate that it is possible to breed for high-quality, weevil-resistant Norway spruce reforestation stock with high accuracy achieved from single-trait or multi-trait GS.

Genetic control and evolutionary potential of a constitutive resistance mechanism against the spruce budworm (Choristoneura fumiferana) in white spruce (Picea glauca).

Insect herbivory may drive evolution by selecting for trees with heritable resistance against defoliation. The spruce budworm (Choristoneura fumiferana, SBW) is a highly damaging forest insect pest that can affect population structure of white spruce (Picea glauca) in North America. Resistance against SBW was recently described in white spruce and was linked to three constitutive resistance biomarkers: the phenolic compounds piceol and pungenol, and expression of a beta-glucosidase encoding gene (Pgßglu-1). We investigated the phenotypic variability and heritability of these resistance biomarkers and of picein, the precursor of piceol, in the foliage of 874 trees belonging to 33 full-sib families and 71 clonal lines under evaluation in seven field locations in Eastern Canada. We aimed to (i) determine their genetic control, (ii) estimate the genetic and phenotypic correlations among defense biomarkers, and (iii) determine whether their constitutive levels are associated with detrimental trade-offs on growth. Quantitative genetics analyses indicated that all four traits are moderately to highly heritable. The full-sib and clonal analyses showed that additive and non-additive genetic effects play major and minor roles, respectively. Positive genetic and phenotypic correlations between resistance biomarkers and primary growth indicated that there is no trade-off between total height and height increment and resistance traits, contradicting the GDBH (Growth Differentiation Balance Hypothesis). Our findings about the predominant additive genetic basis of the resistance biomarkers show that adaptive evolution of white spruce natural populations to resist to SBW is possible and that potentially important gains could also be expected from artificial selection.

Tree rings provide a new class of phenotypes for genetic associations that foster insights into adaptation of conifers to climate change.

Local adaptation in tree species has been documented through a long history of common garden experiments where functional traits (height, bud phenology) are used as proxies for fitness. However, the ability to identify genes or genomic regions related to adaptation to climate requires the evaluation of traits that precisely reflect how and when climate exerts selective constraints. We combine dendroecology with association genetics to establish a link between genotypes, phenotypes and interannual climatic fluctuations. We illustrate this approach by examining individual tree responses embedded in the annual rings of 233 Pinus strobus trees growing in a common garden experiment representing 38 populations from the majority of its range. We found that interannual variability in growth was affected by low temperatures during spring and autumn, and by summer heat and drought. Among-population variation in climatic sensitivity was significantly correlated with the mean annual temperature of the provenance, suggesting local adaptation. Genotype-phenotype associations using these new tree-ring phenotypes validated nine candidate genes identified in a previous genetic-environment association study. Combining dendroecology with association genetics allowed us to assess tree vulnerability to past climate at fine temporal scales and provides avenues for future genomic studies on functional adaptation in forest trees.