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The maintenance of genetic diversity across seed orchard generations is an important management objective. Here, we used Pinus tabuliformis Carrière as a model to explore the extent of genetic diversity across the species’ breeding activities through their corresponding seed orchard generations. We utilized a large number of simple sequence repeat (SSR) markers selected from P. tabuliformis transcriptomic data, and then assessed the effect of marker number on genetic diversity and genetic relationships of individuals across orchard generations. In total, we designed 125 SSR markers, of which 39 were polymorphic and used in the present study. The genetic diversity and genetic distance parameters tended to increase with an increase in loci number and a stable trend was reached at 24 SSRs. The selected optimal 24 SSR markers were further used to assess the genetic diversity across seed orchard generations, and a decreasing trend was detected with the advancement of orchard’s generations. Genetic distance analysis indicated that individuals in the 2nd generation orchard were more closely related compared with those of the 1st generation and the 1.5 generation. This study provided valuable information on the effect of selection and breeding on genetic diversity and highlighted its role for effective seed orchard management.

Prior to Ginkgo leaf harvest, we implemented four exogenous applications of salicylic acid (SA: 50, 80, and 100 mg·L−1) or methyl jasmonate (MJ: 20, 40, and 80 mg·L−1) as leaf spray to investigate their effect on flavonoids content and related enzyme activity. Compared with the control, a significant increase of 12% and 26% in flavonoid content was observed 7 days after the first application of 100 mg·L−1 SA and 20 mg·L−1 MJ, respectively; both were determined to be the best treatments. Operationally, administering either elicitor (SA or MJ) 1 week prior to leaf harvest is deemed to be the most practical and economic option. Additionally, enzymes activity involved in flavonoid metabolism (phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and 4-coumarate:coenzyme A ligase) and antioxidants (peroxidase, superoxide dismutase, and catalase) significantly increased in most SA and MJ treatments. The results strongly support Ginkgo leaf response to SA- or MJ-induced stress through increased nonenzymatic antioxidant compounds (flavonoids) and activity of antioxidant enzymes.

Salicylic acid (SA) is a vital hormone for adaptive responses to biotic and abiotic stresses, which facilitates growth-immunity trade-offs in plants. However, the genetic regulatory networks underlying the metabolic pathway of SA biosynthesis in perennial species remain unclear. Here, we integrated genome-wide association study (GWAS) with metabolite and expression profiling methodologies to dissect the genetic architecture of SA biosynthesis in Populus. First, we quantified nine intermediate metabolites of SA biosynthesis in 300 unrelated Populus tomentosa Carr. individuals. Then, we used a systematic genetic strategy to identify candidate genes for constructing the genetic regulatory network of SA biosynthesis. We focused on WRKY70, an efficient transcription factor, as the key causal gene in the regulatory network, and combined the novel genes coordinating the accumulation of SA. Finally, we identified eight GWAS signals and eight expression quantitative trait loci situated in a selective sweep, and showed the presence of large allele frequency differences among the three geographic populations, revealing that candidate genes subject to selection were involved in SA biosynthesis. This study provides an integrated strategy for dissecting the genetic architecture of the metabolic pathway of SA biosynthesis in Populus, thereby enhancing our understanding of genetic regulation of SA biosynthesis in trees, and accelerating marker-assisted breeding efforts toward high-resistance elite varieties of Populus.

We conducted an integrative system biology of metabolome and transcriptome profile analyses during pomegranate (Punica granatum L.) seed germination and utilized a weighted gene co-expression network analysis (WGCNA) to describe the functionality and complexity of the physiological and morphogenetic processes as well as gene expression and metabolic differences during seed germination stages. In total, 489 metabolites were detected, including 40 differentially accumulated metabolites. The transcriptomic analysis showed the expression of 6 984 genes changed significantly throughout the whole germination process. Using WGCNA, we identified modules related to the various seed germination stages and hub genes. In the initial imbibition stage (stage 1), the pivotal genes involved in RNA transduction and the glycolytic pathway were most active, while in the sprouting stage (stage 4), the pivotal genes were involved in multiple metabolic pathways. In terms of secondary metabolic pathways, we found flavonoid 4-reductase genes of anthocyanin biosynthesis pathway are most significantly affected during pomegranate seed germination, while the flavonol synthase gene was mainly involved in the regulation of isoflavonoid biosynthesis.

Background Inbreeding in seed orchards is expected to increase with the advancement of breeding cycles, which results in the delivery of crops with suboptimal genetic gain, reduced genetic diversity, and lower seed set. Here, a genetic distance-dependent method for clonal spatial deployment in seed orchards was developed and demonstrated, which reduced the inbreeding levels. The method’s main evaluation parameter of inbreeding is the genetic distance among individuals and the deployment method used an improved adaptive parallel genetic algorithm (IAPGA) based on Python language. Using inbreeding-prone Chinese Mongolian pine breeding population material originating from a single natural population, the proposed method was compared to a traditional orchard design and a distance-based design; namely, complete randomized block (RCB) and optimum neighborhood (ONA) designs, respectively. Results With the advancement of selective breeding cycles, group separation among orchard related individuals is expected to increase. Based on the genetic distance among individuals, the IAPGA design was superior in significantly reducing the inbreeding level as compared to the two existing designs, confirming its suitability to advanced-generation orchards where relatedness among parents is common. In the 1st, 2nd, and mixed generations clonal deployment schemes, the IAPGA design produced lower inbreeding with 87.22%, 81.49%, and 87.23% of RCB, and 92.78%, 91.30%, and 91.67% of ONA designs, respectively. Conclusions The IAPGA clonal deployment proposed in this study has the obvious advantage of controlling inbreeding, and it is expected to be used in clonal deployment in seed orchards on a large-scale. Further studies are needed to focus on the actual states of pollen dispersal and mating in seed orchards, and more assumptions should be taken into account for the optimized deployment method.

Marker-assisted selection (MAS) is implemented to improve Camellia oleifera yield and fruit attributes for meeting China’s increased demand for new varieties development. We conducted three-generational (G0, G1, and G2) hybridization (diallel mating) and selection experiment and used Sequence-Related Amplified Polymorphism (SRAP). SRAP markers to investigate their utility in a MAS framework. The utilized SRAP markers were instrumental in hybrid authenticity and the identification of matroclinal or patroclinal inheritance presence, thus guiding mating pair selection and direction (their role as male or females). Across the studied 3 generations, estimates of genetic diversity parameters showed steady increase with percentage increase of ((G0 to G1 and G1 to G2) 9.25 and 9.05: observed number of alleles; 3.12 and 7.80: means effective number of alleles; 12.35 and 22.34: Nei‘s gene diversity; and 14.21 and 21.77: Shannon‘s index), indicating lack of diversity reduction associated with selection. Estimates of genetic distance and their correlation with heterosis were useful in guiding selection of mating pairs for achieving the desired yield and fruit attributes (fruit diameter, height, weight, and index, peel thickness, number of seeds per fruit, seed weight per fruit, and seed rate). Most yield and fruit attributes exhibited high broad-sense heritability with increasing trend over generation intervals, indicating the increased potential of hybrid breeding for this species.

Through stochastic simulations, accuracies of breeding values and response to selection were assessed under traditional pedigree- (BLUP) and genomic-based evaluation methods (GBLUP) in forest tree breeding. The latter provides a methodological foundation for genomic selection. We evaluated the impact of clonal replication in progeny testing on the response to selection realized in seed orchards under variable marker density and target effective population sizes. We found that clonal replication in progeny trials boosted selection accuracy, thus provided additional genetic gains under BLUP. While a similar trend was observed for GBLUP, however, the added gains did not surpass those under BLUP. Therefore, breeding programs deploying extensive progeny testing with clonal propagation might not benefit from the deployment of genomic information. These findings could be helpful in the context of operational breeding programs.

To study the effect of N-P-K fertilization on Phoebe bournei seedlings’ organs dry biomass, and nutrients accumulation and allocation, and to further uncover how nutrients regulating dry biomass formation through fertilization, we utilized the “3414” experiment design. The results showed that N, P, and K fertilizer promoted dry biomass accumulation, and root, stem, and total plant N, P, and K content and accumulation in seedlings. The dry biomass accumulations of root, stem, and total plant increased first and then decreased with the increase of N, P, and K application rates, which was basically consistent with the change in dry biomass allocations and N, P, and K contents, accumulations, and allocations. Root N accumulation, root P accumulation, and total plant K accumulation were the key indicators for seedlings growth. N fertilizer had the greatest effect on total dry biomass and root N accumulation, was the most important fertilizer for the growth of Phoebe bournei seedlings, can regulate the growth of root and leaves, is beneficial to root growth at medium-low N fertilizer levels (N: 0.266–0.532 g·plant−1), and leaves growth at high N fertilizer level (N: 0.798 g·plant−1). P fertilizer rate can regulate the seedling stem growth, reaching the maximum at the medium level P application (P2O5: 0.1332 g·plant−1). K fertilizer had the greatest effect on the root P accumulation and total K accumulation, promoting K transport from leaves to root, improved root and stem growth, and inhibited leaves growth. The N, P, and K fertilizer three-factor application can better promote nutrient uptake than double-factor and single-factor fertilization, with highest dry biomass accumulation at the medium level of N, P, K fertilizer (N: 0.532 g·plant−1; P2O5: 0.1232 g·plant−1; K2O: 0.356 g·plant−1). In conclusion, N, P, and K fertilization promoted the N, P, and K absorption, increased root, stem, and leaves N, P, and K content and accumulation, and promoted the seedling dry biomass accumulation, but reversed under excessive application of N, P, and K fertilizer; and N fertilizer was beneficial to root and leaves growth, P fertilizer to stem growth, and K fertilizer to material transfer, which provided a theoretical basis for robust Phoebe bournei seedling cultivation.

We investigated the genetic structure of three phenotypically distinct ecotypic groups of Norway spruce (Picea abies) belonging to three elevational classes; namely, low- (acuminata), medium- (europaea), and high-elevation (obovata) form, each represented by 150 trees. After rigorous filtering, we used 1916 Genotyping-by-Sequencing generated SNPs for analysis. Outputs from three multivariate analysis methods (Bayesian clustering algorithm implemented in STRUCTURE, Principal Component Analysis, and the Discriminant Analysis of Principal Components) indicated the presence of a distinct genetic cluster representing the high-elevation ecotypic group. Our findings bring a vital message to forestry practice affirming that artificial transfer of forest reproductive material, especially for stands under harsh climate conditions, should be considered with caution.

Background In angiosperms, phenotypic variation of floral organs is often considered as the traditional basis for the evolutionary relationship of different taxonomic groups above the species level. However, little is known about that at or below the species level. Here, we experimentally tested the phenotypic variation of Malus floral organs using combined methods of intraspecific uniformity test, interspecific distinctness analysis, principal component analysis, Pearson correlation analysis, and Q-type cluster analysis. The ancestor-inclined distribution characteristic analysis of Malus species and cultivars floral attributes was also carried out, so as to explore its taxonomic significance. Results 15/44 phenotypic traits (e.g., flower shape, flower type, flower diameter, ...) were highly consistent, distinguishable, and independent and could be used as the basis for Malus germplasm taxonomy. The studied 142 taxa were divided into two groups (A, B) and five sub-groups (A1, A2, B1, B2, B3), with significantly variable floral phenotypic attributes between groups and within sub-groups. Malus natural species were relatively clustered in the same section (series) while homologous cultivars showed evidence of ancestor-inclined distribution characteristics. However, no significant correlation between the evolutionary order of sections (Sect. Docyniopsis → Sect. Chloromeles → Sect. Sorbomalus → Sect. Eumalus) and group/sub-groups (B3 → B2 → B1 → A). Conclusions Phenotypic variation of floral organs could better explore the genetic relationship between Malus taxa. The findings improved our cognition of floral phenotypic variation taxonomic significance under the species level.

Background: Melia azedarach L. is a globally distributed tree species of economic importance; however, it is unclear how the species distribution will respond to future climate changes.Methods: We aimed to select the most accurate one among seven data mining models to predict the species suitable contemporary and future habitats. These models include: maximum entropy (MaxEnt), support vector machine (SVM), generalized linear model (GLM), random forest (RF), naive bayesian model (NBM), extreme gradient boosting (XGBoost), and gradient boosting machine (GBM). A total of 906 M. azedarach locations were identified, and sixteen climate predictors were used for model building. The models’ validity was assessed using three measures (Area Under the Curves (AUC), kappa, and accuracy). Results: We found that the RF provided the most outstanding performance in prediction power and generalization capacity. The top climate factors affecting the species distribution were mean coldest month temperature (MCMT), followed by the number of frost-free days (NFFD), degree-days above 18°C (DD>18), temperature difference between MWMT and MCMT, or continentality (TD), mean annual precipitation (MAP), and degree-days below 18°C (DDConclusion: Our findings are expected to assist in better understanding the impact of climate change on the species and provide scientific basis for its planting and conservation.

The genetic efficiency of seed orchards is crucial for determining seed crops’ genetic gain and diversity. Platycladus orientalis is a conifer tree of important ecological value in China. Here, we assessed the reproductive output (fertility) variation for 166 clones in a first-generation P. orientalis seed orchard over five years and across three years for each gender (female: 2017, 2018, and 2020 and male: 2017, 2019, and 2021). Fertility variation and genetic diversity parameters were estimated for each gender-year combination. The reproductive output (fertility) variation differed among years, provinces, clones nested within provinces, and ramets within clones. We observed asymmetry in the gender reproductive output and parental imbalance and determined their profound effects on the genetic diversity of these seed crops. The maleness index revealed the existence of female-biased or male-biased clones. When seeds from multiple individuals and years were blended, we found an increase in the effective number of parents (Np) and in genetic diversity (GD), and a reduced fertility variation (Ψ) in the seed orchard. When we set the effective number of parents (Np) to 30, the GD of the seed orchard could be maintained at more than 95%. Thus, achieving genetic diversity balance in seed production can be accomplished through monitoring the fertility variation of orchards and through the utilization of the thereby generated information for the advanced generation of seed orchards.

Soil bacteria play a key role in the plant–soil system and can regulate the growth of Phoebe bournei seedlings under fertilization. However, there are few reports on how soil bacteria respond to fertilization and regulate seedling growth. This study adopted the “3414” field fertilization experiment, combined with soil microbial sequencing, nutrient contents, and biomass measurement, to explore the changes of soil chemical properties and bacterial structure under different NPK fertilization conditions and to establish the coupling relationship between soil bacteria, soil nutrients, and plant growth. The results showed that NPK fertilization decreased soil pH, increased soil N, P, and K content, reduced bacterial diversity and abundance, promoted the growth of dominant bacterial species, and enhanced Phoebe bournei seedlings’ soil N, P, and K elements. NPK fertilization promoted Proteobacteria growth, especially of three genera (Methylobacterium, Sphingobium, and Acinetobacter) and Actinobacteria, while it decreased Acidobacteria and Chloroflexi. By reducing the ratio of N to K and increasing P, NPK fertilization can slow soil acidification, promote bacterial reproduction, maintain P. bournei seedlings’ soil ecological stability, and balance the seedlings’ growth and sustainable soil utilization. AD3, Pseudomonas, and Rhodanobacter can be used as the marker species for N, P, and K fertilization, respectively, while Methylobacterium, Brevundimonas, Acinetobacter, and Sphingobium can be used as indicator species for soil pH and soil N, P, and K content changes, respectively. These results provided a theoretical basis and technical guidance for the effective fertilization and cultivation of robust P. bournei seedlings.

Natural selection on fitness-related traits can be temporally heterogeneous among populations. As climate changes, understanding population-level responses is of scientific and practical importance. We examined 18 phenotypic traits associated with phenology, biomass, and ecophysiology in 403 individuals of natural Populus trichocarpa populations, growing in a common garden.Compared with tree origin settings, propagules likely underwent drought exposures in the common garden due to significantly low rainfall during the years of measurement. All study traits showed population differentiation reflecting adaptive responses due to local genetic adaptation. Phenology and biomass traits were strongly under selection and showed plastic responses between years, co-varying with latitude. While phenological events (e.g., bud set and growth period) and biomass were under positive directional selection, post-bud set period, particularly from final bud set to the onset of leaf drop, was selected against. With one exception to water-use efficiency, ecophysiology traits were under negative directional selection. Moreover, extended phenological events jointly evolved with source niches under increased temperature and decreased rainfall exposures. High biomass coevolved with climatic niches of high temperature; low rainfall promoted high photosynthetic rates evolution.This work underpins that P. trichocarpa is likely to experience increased fitness (height gain) by evolving toward extended bud set and growth period, abbreviated post-bud set period, and increased drought resistance, potentially constituting a powerful mechanism for long-lived tree species in surviving unpredictably environmental extremes (e.g., drought).

LTR retrotransposons (LTR-RTs) are ubiquitous and represent the dominant repeat element in plant genomes, playing important roles in functional variation, genome plasticity and evolution. With the advent of new sequencing technologies, a growing number of whole-genome sequences have been made publicly available, making it possible to carry out systematic analyses of LTR-RTs. However, a comprehensive and unified annotation of LTR-RTs in plant groups is still lacking. Here, we constructed a plant intact LTR-RTs dataset, which is designed to classify and annotate intact LTR-RTs with a standardized procedure. The dataset currently comprises a total of 2,593,685 intact LTR-RTs from genomes of 300 plant species representing 93 families of 46 orders. The dataset is accompanied by sequence, diverse structural and functional annotation, age determination and classification information associated with the LTR-RTs. This dataset will contribute valuable resources for investigating the evolutionary dynamics and functional implications of LTR-RTs in plant genomes., Measurement(s) LTR_retrotransposon • genome Technology Type(s) bioinformatics method • digital curation Factor Type(s) Species Sample Characteristic - Organism Rhodophyta • Chlorophyta • Bryophyta • Tracheophyta Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14684877

Increasing evidence indicates that DNA methylation is heritable and serves as an essential marker contributing to phenotypic variation. Linkage-linkage disequilibrium mapping was used to decipher the epigenetic architecture underlying nine growth and wood property traits in a linkage population (550 F1 progeny) and a natural population (435 unrelated individuals) of Populus using methylation-sensitive amplification polymorphism (MSAP)-based analysis. The interactions between genetic and epigenetic variants in the causative genes was further unveiled using expression quantitative trait methylation (eQTM) and nucleotide (eQTN) mapping strategies. A total of 163 epigenetic quantitative trait loci (epiQTLs; LOD ≥ 3.0), explaining 1.7-44.5% of phenotypic variations, were mapped to a high-resolution epigenetic map with 19 linkage groups, which was supported by the significant MSAP associations (P < 0.001) in the two populations. There were 23 causal genes involved in growth regulation and wood formation, whose markers were located in epiQTLs and associated with the same traits in both populations. Further eQTN and eQTM mapping showed that causal genetic and epigenetic variants within the 23 candidate genes may interact more in trans in gene expression and phenotype. The present study provides strategies for investigating epigenetic architecture and the interaction between genetic and epigenetic variants modulating complex traits in forest trees.

Platycladus orientalis is an ecologically important native conifer in Northern China and exotic species in many parts of the world; however, knowledge about the species’ genetics and genome are very limited. The availability of well-developed battery of genetic markers, with large genome coverage, is a prerequisite for the species genetic dissection of adaptive attributes and efficient selective breeding. Here, we present a genome-wide genotyping method with double-digestion restriction site associated DNA sequencing (ddRAD-seq) that is effective in generating large number of Mendelian markers for genome mapping and other genetic applications. Using 139 megagametophytes collected from a single mother tree, we assembled 397,226 loci, of which 108,683 (27.4%) were polymorphic. After stringent filtering for 1:1 segregation ratio and missing rate of

Genetic structure and major climate factors may contribute to the distribution of genetic diversity of a highly valued oil tree species Xanthoceras sorbifolium (yellowhorn). Long‐term over utilization along with climate change is affecting the viability of yellowhorn wild populations. To preserve the species known and unknown valuable gene pools, the identification of genetic diversity “hotspots” is a prerequisite for their consideration as in situ conservation high priority. Chloroplast DNA (cpDNA) diversity was high among 38 natural populations (H d = 0.717, K = 4.616, Tajmas’ D = −0.22) and characterized by high genetic divergence (F ST = 0.765) and relatively low gene flow (N m = 0.03), indicating populations isolation reflecting the species’ habitat fragmentation and inbreeding depression. Six out of the studied 38 populations are defined as genetic diversity “hotspots.” The number and geographic direction of cpDNA mutation steps supported the species southwest to northeast migration history. Climatic factors such as extreme minimum temperature over 30 years indicated that the identified genetic “hotspots” are expected to experience 5°C temperature increase in next following 50 years. The results identified vulnerable genetic diversity “hotspots” and provided fundamental information for the species’ future conservation and breeding activities under the anticipated climate change. More specifically, the role of breeding as a component of a gene resource management strategy aimed at fulfilling both utilization and conservation goals.

Here, we perform cross-generational GS analysis on coastal Douglas-fir (Pseudotsuga menziesii), reflecting trans-generational selective breeding application. A total of 1321 trees, representing 37 full-sib F1 families from 3 environments in British Columbia, Canada, were used as the training population for (1) EBVs (estimated breeding values) of juvenile height (HTJ) in the F1 generation predicting genomic EBVs of HTJ of 136 individuals in the F2 generation, (2) deregressed EBVs of F1 HTJ predicting deregressed genomic EBVs of F2 HTJ, (3) F1 mature height (HT35) predicting HTJ EBVs in F2, and (4) deregressed F1 HT35 predicting genomic deregressed HTJ EBVs in F2. Ridge regression best linear unbiased predictor (RR-BLUP), generalized ridge regression (GRR), and Bayes-B GS methods were used and compared to pedigree-based (ABLUP) predictions. GS accuracies for scenarios 1 (0.92, 0.91, and 0.91) and 3 (0.57, 0.56, and 0.58) were similar to their ABLUP counterparts (0.92 and 0.60, respectively) (using RR-BLUP, GRR, and Bayes-B). Results using deregressed values fell dramatically for both scenarios 2 and 4 which approached zero in many cases. Cross-generational GS validation of juvenile height in Douglas-fir produced predictive accuracies almost as high as that of ABLUP. Without capturing LD, GS cannot surpass the prediction of ABLUP. Here we tracked pedigree relatedness between training and validation sets. More markers or improved distribution of markers are required to capture LD in Douglas-fir. This is essential for accurate forward selection among siblings as markers that track pedigree are of little use for forward selection of individuals within controlled pollinated families.

A molecular markers-based conservation strategy framework for the endemic endangered Chinese yellowhorn, Xanthoceras sorbifolium, is developed. The method utilizes pairwise kinship coefficients, a measure of genetic similarity/dissimilarity, between individuals and contrasts two simulated mating schemes that either promote “no co-ancestry” or “co-ancestry” through multiple matings of particular individuals as well as a “hybrid” scenario involving both “no co-ancestry” and “co-ancestry” mating schemes. Selection of mating individuals is focused on maximizing genetic dissimilarity, hence capturing maximum genetic diversity. These approaches were compared on a population of 79 ancient yellowhorn trees scattered across 13 populations. Simulated matings were evaluated based on their average pairwise kinship, effective population size, and average number of alleles/locus. In this regard, no co-ancestry has been proven to be superior to co-ancestry mating scheme; however, co-ancestry offered opportunities for increasing effective population size, a desirable attribute for combating the detrimental effect of genetic drift. The hybrid approach, with reduced number of crosses, produced acceptable condition with maximum genetic diversity and high effective population size and genetic dissimilarity among the produced offspring. The advantages and disadvantages of marker-based gene conservation are highlighted and discussed. Subject area: Conservation genetics and biodiversity.

Ginkgo (Ginkgo biloba L.) is a deciduous tree species with high timber, medicinal, ecological, ornamental, and scientific values, and is widely cultivated worldwide. However, for such an important tree species, the regulatory mechanisms involved in the photosynthesis of developing leaves remain largely unknown. Here, we observed variations in light response curves (LRCs) and photosynthetic parameters (photosynthetic capacity (Pnmax) and dark respiration rate (Rd)) of leaves across different developmental stages. We found the divergence in the abundance of compounds (such as 3-phospho-d-glyceroyl phosphate, sedoheptulose-1,7-bisphosphate, and malate) involved in photosynthetic carbon metabolism. Additionally, a co-expression network was constructed to reveal 242 correlations between transcription factors (TFs) and photosynthesis-related genes (p &lt, 0.05, |r| &gt, 0.8). We found that the genes involved in the photosynthetic light reaction pathway were regulated by multiple TFs, such as bHLH, WRKY, ARF, IDD, and TFIIIA. Our analysis allowed the identification of candidate genes that most likely regulate photosynthesis, primary carbon metabolism, and plant development and as such, provide a theoretical basis for improving the photosynthetic capacity and yield of ginkgo trees.

Pinus densiflora var. zhangwuensis S.J.Zhang, C.X.Li & X.Y.Yuan, a tree species with promising afforestation potential in northern China. Here, we assembled and annotated the complete chloroplast (cp) genome of P. densiflora var. zhangwuensis using the Oxford Nanopore sequencing technology. The cp genome was 119,725 bp in length, exhibiting a typical quadripartite structure with a large single-copy (LSC: 65,552 bp) and a small single-copy (SSC: 53,183 bp) separated by a pair of inverted repeats regions (IRA and IRB: each of 495 bp) region. The overall GC content is 37.3%. The genome was predicted to encode 112 distinct genes, including 72 protein-coding, 36 tRNA, and four rRNA genes. Maximum-likelihood (ML) phylogenetic for cp genome sequences of 18 Pinaceae species revealed that P. densiflora var. zhangwuensis was closely related to Pinus sylvestris.

Quercus acrodonta Seemen is an East Asian evergreen oak tree species belonging to the Quercus section Ilex. Here, we assembled and annotated the complete chloroplast (cp) genome of the species. The...

Cytokinins are important for in vitro shoot regeneration in plants. Cytokinin N-glucosides are produced via an irreversible glycosylation pathway, which regulates the endogenous cytokinin content. Although cytokinin N-glucoside pathways have been uncovered in higher plants, no regulator has been identified to date. We performed a metabolome genome-wide association study (mGWAS), weighted gene co-expression network analysis (WGCNA), and expression quantitative trait nucleotide (eQTN) mappings to build a core triple genetic network (mGWAS-gene expression-phenotype) for the trans-zeatin N-glucoside (ZNG) metabolite using data from 435 unrelated Populus tomentosa individuals. Variation of the ZNG level in poplar is attributed to the differential transcription of PtoWRKY42, a member of WRKY multigene family group IIb. Functional analysis revealed that PtoWRKY42 negatively regulated ZNG accumulation by binding directly to the W-box of the UDP-glycosyltransferase 76C 1-1 (PtoUGT761-1) promoter. Also, PtoWRKY42 was strongly induced by leaf senescence, 6-BA, wounding, and salt stress, resulting in a reduced ZNG level. We identified PtoWRKY42, a negative regulator of cytokinin N-glucosides, which contributes to the natural variation in ZNG level and mediates ZNG accumulation by directly modulating the key glycosyltransferase gene PtoUGT76C1-1.

In-depth genome characterization is still lacking for most of biofuel crops, especially for centromeres, which play a fundamental role during nuclear division and in the maintenance of genome stability. This study applied long-read sequencing technologies to assemble a highly contiguous genome for yellowhorn (Xanthoceras sorbifolium), an oil-producing tree, and conducted extensive comparative analyses to understand centromere structure and evolution, and fatty acid biosynthesis. We produced a reference-level genome of yellowhorn, ∼470 Mb in length with ∼95% of contigs anchored onto 15 chromosomes. Genome annotation identified 22,049 protein-coding genes and 65.7% of the genome sequence as repetitive elements. Long terminal repeat retrotransposons (LTR-RTs) account for ∼30% of the yellowhorn genome, which is maintained by a moderate birth rate and a low removal rate. We identified the centromeric regions on each chromosome and found enrichment of centromere-specific retrotransposons of LINE1 and Gypsy in these regions, which have evolved recently (∼0.7 MYA). We compared the genomes of three cultivars and found frequent inversions. We analyzed the transcriptomes from different tissues and identified the candidate genes involved in very-long-chain fatty acid biosynthesis and their expression profiles. Collinear block analysis showed that yellowhorn shared the gamma (γ) hexaploidy event with Vitis vinifera but did not undergo any further whole-genome duplication. This study provides excellent genomic resources for understanding centromere structure and evolution and for functional studies in this important oil-producing plant.

Polyploidization plays a key role in plant evolution, but the forces driving the fate of homoeologs in polyploid genomes, i.e., paralogs resulting from a whole-genome duplication (WGD) event, remain to be elucidated. Here, we present a chromosome-scale genome assembly of tetraploid scarlet sage (Salvia splendens), one of the most diverse ornamental plants. We found evidence for three WGD events following an older WGD event shared by most eudicots (the γ event). A comprehensive, spatiotemporal, genome-wide analysis of homoeologs from the most recent WGD unveiled expression asymmetries, which could be associated with genomic rearrangements, transposable element proximity discrepancies, coding sequence variation, selection pressure, and transcription factor binding site differences. The observed differences between homoeologs may reflect the first step toward sub- and/or neofunctionalization. This assembly provides a powerful tool for understanding WGD and gene and genome evolution and is useful in developing functional genomics and genetic engineering strategies for scarlet sage and other Lamiaceae species.

Background Hybridization and introgression are vital sources of novel genetic variation driving diversification during reticulated evolution. Quercus is an important model clade, having extraordinary diverse and abundant members in the Northern hemisphere, that are used to studying the introgression of species boundaries and adaptive processes. China is the second-largest distribution center of Quercus, but there are limited studies on introgressive hybridization. Results Here, we screened 17 co-dominant nuclear microsatellite markers to investigate the hybridization and introgression of four oaks (Quercus acutissima, Quercus variabilis, Quercus fabri, and Quercus serrata) in 10 populations. We identified 361 alleles in the four-oak species across 17 loci, and all loci were characterized by high genetic variability (HE = 0.844–0.944) and moderate differentiation (FST = 0.037–0.156) levels. A population differentiation analysis revealed the following: allopatric homologous (FST = 0.064) FST = 0.071) FST = 0.084). A Bayesian admixture analysis determined four types of hybrids (Q. acutissima × Q. variabilis, Q. fabri × Q. serrata, Q. acutissima × Q. fabri, and Q. acutissima × Q. variabilis × Q. fabri) and their asymmetric introgression. Our results revealed that interspecific hybridization is commonly observed within the section Quercus, with members having tendency to hybridize. Conclusions Our study determined the basic hybridization and introgression states among the studied four oak species and extended our understanding of the evolutionary role of hybridization. The results provide useful theoretical data for formulating conservation strategies.

Ginger (Zingiber officinale) is one of the most valued spice plants worldwide; it is prized for its culinary and folk medicinal applications and is therefore of high economic and cultural importance. Here, we present a haplotype-resolved, chromosome-scale assembly for diploid ginger anchored to 11 pseudochromosome pairs with a total length of 3.1 Gb. Remarkable structural variation was identified between haplotypes, and two inversions larger than 15 Mb on chromosome 4 may be associated with ginger infertility. We performed a comprehensive, spatiotemporal, genome-wide analysis of allelic expression patterns, revealing that most alleles are coordinately expressed. The alleles that exhibited the largest differences in expression showed closer proximity to transposable elements, greater coding sequence divergence, more relaxed selection pressure, and more transcription factor binding site differences. We also predicted the transcription factors potentially regulating 6-gingerol biosynthesis. Our allele-aware assembly provides a powerful platform for future functional genomics, molecular breeding, and genome editing in ginger.

Patterns of crop production in mast species do not track crop-year climate, but instead are regulated by climate cues in prior-years. Whether the pattern of year-to-year seed mass variation is coupled in time with mast seeding, maintaining seed mass-number trade-offs, and coherently driven by similar climate cues as other seed traits (e.g. seed germination) remains unknown. Using ca. 6,000 long-term seed inventory data over the years 1955-2015 in conifers, this retrospective study revealed the temporal patterns of mast species’ seed mass and its associated trait, seed germination. To pinpoint their ecological drivers, pairwise correlation analysis was performed between each trait and seasonal climates in crop year and four prior-years. Using climate variables key to each trait, regression models were constructed to project trait values. Findings showed minor seed mass variation among years, which rejects the generality of seed mass-number trade-offs in many plant species. This result reasonably arises as the economies of scale (compensating benefits) theory are often used to account for mast seeding but not for seed mass. Moreover, final germination fraction also varied little over time, but exhibited an increasing tendency. In addition, we found that temperature-based climate variables drive seed mass, number, and germination variation, but these variables in different seasons of crop year or prior-years did not have equal influences on trait variability. Finally, regression models showed that the number of frost-free days and evapotranspiration are crucial to the three traits and climate in autumn is a critical season, followed by summer and winter. This study holds considerable promise for explaining reproductive strategies of taxonomic groups with mast seeding characteristics in allocating reproductive resources to different life-history traits using ecological signals.

Heat stress damages plant tissues and induces multiple adaptive responses. Complex and spatiotemporally specific interactions among transcription factors (TFs), microRNAs (miRNAs), and their targets play crucial roles in regulating stress responses. To explore these interactions and to identify regulatory networks in perennial woody plants subjected to heat stress, we integrated time-course RNA-seq, small RNA-seq, degradome sequencing, weighted gene correlation network analysis, and multi-gene association approaches in poplar. Results from Populus trichocarpa enabled us to construct a three-layer, highly interwoven regulatory network involving 15 TFs, 45 miRNAs, and 77 photosynthetic genes. Candidate gene association studies in a population of P. tomentosa identified 114 significant associations and 696 epistatic SNP–SNP pairs that were linked to 29 photosynthetic and growth traits (P

Ginkgo (Ginkgo biloba L.) is a high-value medicinal tree species characterized by its flavonoids beneficial effects that are abundant in leaves. We performed a temporospatial comprehensive transcriptome and metabolome dynamics analyses of clonally propagated Ginkgo plants at four developmental stages (time: May to August) across three different environments (space) to unravel leaves flavonoids biosynthesis variation. Principal component analysis revealed clear gene expression separation across samples from different environments and leaf-developmental stages. We found that flavonoid-related metabolism was more active in the early stage of leaf development, and the content of total flavonoid glycosides and the expression of some genes in flavonoid biosynthesis pathway peaked in May. We also constructed a co-expression regulation network and identified eight GbMYBs and combining with other TF genes (3 GbERFs, 1 GbbHLH, and 1 GbTrihelix) positively regulated the expression of multiple structural genes in the flavonoid biosynthesis pathway. We found that part of these GbTFs (Gb_11316, Gb_32143, and Gb_00128) expressions was negatively correlated with mean minimum temperature and mean relative humidity, while positively correlated with sunshine duration. This study increased our understanding of the molecular mechanisms of flavonoids biosynthesis in Ginkgo leaves and provided insight into the proper production and management of Ginkgo commercial plantations.

Functional traits are organismal attributes that can respond to environmental cues, thereby providing important ecological functions. In addition, an organism’s potential for adaptation is defined by the patterns of covariation among groups of functionally related traits. Whether an organism is evolutionarily constrained or has the potential for adaptation is based on the phenotypic integration or modularity of these traits. Here, we revisited leaf morphology in two European sympatric white oaks (Quercus petraea (Matt.) Liebl. and Quercus robur L.), sampling 2098 individuals, across much of their geographical distribution ranges. At the phenotypic level, leaf morphology traditionally encompasses discriminant attributes among different oak species. Here, we estimated in situ heritability, genetic correlation, and integration across such attributes. Also, we performed Selection Response Decomposition to test these traits for potential differences in oak species’ evolutionary responses. Based on the uncovered functional units of traits (modules) in our study, the morphological module “leaf size gradient” was highlighted among functionally integrated traits. Equally, this module was defined in both oaks as being under “global regulation” in vegetative bud establishment and development. Lamina basal shape and intercalary veins’ number were not, or, less integrated within the initially defined leaf functional unit, suggesting more than one module within the leaf traits’ ensemble. Since these traits generally show the greatest species discriminatory power, they potentially underwent effective differential response to selection among oaks. Indeed, the selection of these traits could have driven the ecological preferences between the two sympatric oaks growing under different microclimates.

During their lifetimes, plants are exposed to different abiotic stress factors eliciting various physiological responses and triggering important defense processes. For UV-B radiation responses in forest trees, the genetics and molecular regulation remain to be elucidated. Here, we exposed Pinus tabuliformis Carr., a major conifer from northern China, to short-term high-intensity UV-B and employed a systems biology approach to characterize the early physiological processes and the hierarchical gene regulation, which revealed a temporal transition from primary to secondary metabolism, the buildup of enhanced antioxidant capacity and stress-signaling activation. Our findings showed that photosynthesis and biosynthesis of photosynthetic pigments were inhibited, while flavonoids and their related derivates biosynthesis, as well as glutathione and glutathione S-transferase mediated antioxidant processes, were enhanced. Likewise, stress-related phytohormones (jasmonic acid, salicylic acid and ethylene), kinase and reactive oxygen species signal transduction pathways were activated. Biological processes regulated by auxin and karrikin were, for the first time, found to be involved in plant defense against UV-B by promoting the biosynthesis of flavonoids and the improvement of antioxidant capacity in our research system. Our work evaluated the physiological and transcriptome perturbations in a conifer’s response to UV-B, and generally, highlighted the necessity of a systems biology approach in addressing plant stress biology.

Background Gender and fertility variation have an impact on mating dynamics in a population because they affect the gene exchange among parental members and the genetic composition of the resultant seed crops. Fertility is the proportional gametic contribution of parents to their progeny. An effective number of parents, derivative of effective population size, is the probability that two alleles randomly chosen from the gamete gene pool originated from the same parent. The effective number of parents is directly related to the fertility variation among parents, which should be monitored for manipulating gene diversity of seed crops. We formulated a fundamental equation of estimating the effective number of parents and applied it to a seed production population. Results Effective number of parents (Np) was derived from fertility variation (Ψ) considering covariance (correlation coefficient, r) between maternal and paternal fertility. The Ψ was calculated from the coefficient of variation in reproductive outputs and divided into female (ψf) and male (ψm) fertility variation in the population under study. The Np was estimated from the parental Ψ estimated by the fertility variation of maternal (ψf) and paternal (ψm) parents. The gene diversity of seed crops was monitored by Ψ and Np. in a 1.5 generation Pinus koraiensis seed orchard as a case of monoecious species. A large variation of female and male strobili production was observed among the studied 52 parents over four consecutive years, showing statistically significant differences across all studied years. Parental balance curve showed greater distortion in paternal than maternal parents. The Ψ ranged from 1.879 to 4.035 with greater ψm than ψf, and the Np varied from 14.8 to 36.8. When pooled, the relative effective number of parents was improved as 80.0% of the census number. Conclusions We recommend the use of fertility variation (i.e., CV, Ψ), Person’s product-moment correlation (r), and effective number of parents (Np) as tools for gauging gene diversity of seed crops in production populations. For increasing Np and gene diversity, additional management options such as mixing seed-lots, equal cone harvest and application of supplemental-mass-pollination are recommended.

Background: The significance of stumps and other coarse woody debris (CWD) in maintaining biodiversity has been widely recognized. However, there is a paucity of research on the role of stumps in tree regeneration. We studied vascular plant structure, diversity, and tree regeneration on stumps in temperate rainforests across three sites in British Columbia, Canada (Malcom Knapp Research Forest, MKRF; Pacific Spirit Regional Park, PSRP; and Stanley Park, SP).Results: 1) There were 19 vascular plant species found on stumps, including eight tree species, 2) Overall seedling abundance was higher on stumps than the nearby ground, 3) The number of established plants showed a positive linear (MKRF, SP) or Gaussian (PSRP) relationship with stump basal diameter, 4) Vegetation abundance varied with site (MKRF > SP > PSRP), 5) The overall species established on stumps were positively associated in MKRF (Variance ratio = 2.74) and SP (Variance ratio = 1.37), but negatively associated in PSRP (Variance ratio = 0.57), and 6) Tree species appeared to compete with each other on stumps and were likely to co-occur with understorey species.Conclusion: Our results highlight community and species associations on stumps. We found that stump diameter is a major factor affecting tree regeneration in these second-growth temperate rainforests. To aid future research on stump-vegetation relationships, we synthesize our results in a schematic of vascular plant biodiversity and tree regeneration on stumps. Our work and this schematic can be used to stimulate ideas for new hypothesis generation and for studies relevant for conservation, management and basic science research.

Phoebe bournei is a rare and endangered woody species and the success of its plantation development is dependent upon proper seedling cultivation. This study explored the regulation of N, P and K fertilizer and the interaction of these macronutrients on the growth of Phoebe bournei seedlings. To determine the optimum rate and ratio of N&ndash, P&ndash, K fertilizer in seedling cultivation, we used the unique &ldquo, 3414&rdquo, incomplete orthogonal regression design to evaluate the effects of N&ndash, K fertilization on seedling morphological development. One-year-old Phoebe bournei bareroot seedlings were grown for one growing season under the defined fertilization regime, with their morphological development determined by measuring seedling attributes&mdash, root, stem, leaves and total biomass, root collar diameter and seedling height. These attributes were then combined to calculate the following indices: height-diameter ratio, shoot-root ratio and the seedling quality index (QI). Results showed that the N&ndash, K fertilizer had significant and beneficial effect on seedling cultivation. N effect was highest, followed by K and P. The three-way N&times, P&times, K interaction effect was strong, and the two-way interactions effect was highest for N&times, P, followed by P&times, K and N&times, K. At the &ldquo, 2&rdquo, level of N (0.532 g&middot, plant&minus, 1), P (P2O5, 0.133 g&middot, 1), and K fertilizer (K2O, 0.356 g&middot, 1), seedling growth and biomass accumulation were at their maximum. Unary, binary, and ternary quadratic fertilizer effect function equations of QI were established. Through comparative analysis, the ternary quadratic model was the optimal model and through a simulation&ndash, optimization, the optimal N&ndash, K fertilizer rates were 0.373~0.420 g&middot, 1 (N), 0.086~0.106 g&middot, 1 (P2O5), 0.243~0.280 g&middot, 1 (K2O), with a N&ndash, K ratio of 1:0.20:0.43~1:0.65:0.75.

Floral symmetry (corolla symmetry) has important biological significance in plant genetics and evolution. However, it is often multi-dimensional and difficult to quantify. Here, we constructed a multi-dimensional data matrix [X Y Z] by extracting three qualitative variables with binary properties (X: corolla regularity of interval and coplanarity; Y: petal regularity of shape and size; Z: petal local regularity of curling and wrinkle) from different dimensions of petals (overall to individual, and then to the local): all petals (corolla), individual petals, and local areas of petals. To quantitatively express the degree of Malus corolla symmetry, these variables were then combined with weight assignments (X: 22 > Y: 21 > Z: 20) based on their contributions to the corolla symmetry and the algorithm rule of converting binary to decimal values, which facilitated the unification of qualitative and quantitative analyses. Our results revealed significant reduction in degrees of Malus corolla symmetry along the direction of local to overall. Species showed higher degree of corolla symmetry than cultivars; however, taxa with stronger corolla symmetry might not necessarily be species. These findings provide new insights into the circumscription of Malus controversial species. The matrix model should be reference for future evaluation of angiosperm flower symmetry (lack of corolla fusion).

miRNAs are important regulatory components involving in many biological processes, including plant development, vegetative and reproductive growth, and stress response. However, identification and characterization of miRNAs still remain limited for conifer species. In this study, with deep sequencing, we obtained 1,314,450 unique reads with 18-30 nt length from a stress-tolerant conifer, Sabina chinensis. We identified 37 conserved and 103 novel miRNAs, their unique characteristics were further analyzed, and 10 randomly selected were validated by qRT-PCR. Through miRNA target predictions and annotations, we found miRNA may have several targets as well a target could be regulated by several miRNAs, and a total of 2,397 mRNAs were predicted to be targets of the 140 miRNAs. These targets included not only important transcription factors such as auxin response factors, but also indispensable non-transcriptional factor proteins. Pathway-based analysis showed that S. chinensis miRNAs are involved in 172 metabolic pathways, of which 3 were discovered in adaptation-related pathways, indicating their possible relevance to the species' stress-tolerance characteristics. This study is expected to lay the foundation for exploring the regulative roles of miRNAs in development, growth, and response to environmental stresses of S. chinensis.

Local adaptation, adaptation to specialized niches and environmental clines have been extensively reported for forest trees. Investigation of the adaptive genetic variation is crucial for forest resource management and breeding, especially in the context of global climate change. Here, we utilized a Pinus yunnanensis common garden experiments established at high and low elevation sites to assess the differences in growth and survival among populations and between the two common garden sites. The studied traits showed significant variation between the two test sites and among populations, suggesting adaptive divergence. To detect genetic variation related to environment, we captured 103,608 high quality SNPs based on RNA sequencing, and used them to assess the genetic diversity and population structure. We identified 321 outlier SNPs from 131 genes showing significant divergence in allelic frequency between survival populations of two sites. Functional categories associated with adaptation to high elevation were found to be related to flavonoid biosynthesis, response to UV, DNA repair, response to reactive oxygen species, and membrane lipid metabolic process. Further investigation of the outlier genes showed overrepresentation of the flavonoid biosynthesis pathway, suggesting that this pathway may play a key role in P. yunnanensis adaptation to high elevation environments. The outlier genes identified, and their variants, provide a basic reference for advanced investigations.

Cytokinins play important roles in the growth and development of plants. Physiological and photosynthetic characteristics are common indicators to measure the growth and development in plants. However, few reports have described the molecular mechanisms of physiological and photosynthetic changes in response to cytokinin, particularly in woody plants. DNA methylation is an essential epigenetic modification that dynamically regulates gene expression in response to the external environment. In this study, we examined genome-wide DNA methylation variation and transcriptional variation in poplar (Populus tomentosa) after short-term treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA). We identified 460 significantly differentially methylated regions (DMRs) in response to 6-BA treatment. Transcriptome analysis showed that 339 protein-coding genes, 262 long non-coding RNAs (lncRNAs), and 15,793 24-nt small interfering RNAs (siRNAs) were differentially expressed under 6-BA treatment. Among these, 79% were differentially expressed between alleles in P. tomentosa, and 102,819 allele-specific expression (ASE) loci in 19,200 genes were detected showing differences in ASE levels after 6-BA treatment. Combined DNA methylation and gene expression analysis demonstrated that DNA methylation plays an important role in regulating allele-specific gene expression. To further investigate the relationship between these 6-BA-responsive genes and phenotypic variation, we performed SNP analysis of 460 6-BA-responsive DMRs via re-sequencing using a natural population of P. tomentosa, and we identified 206 SNPs that were significantly associated with growth and wood properties. Association analysis indicated that 53% of loci with allele-specific expression had primarily dominant effects on poplar traits. Our comprehensive analyses of P. tomentosa DNA methylation and the regulation of allele-specific gene expression suggest that DNA methylation is an important regulator of imbalanced expression between allelic loci.

Sustainable and efficient forestry in a rapidly changing climate is a daunting task. The sessile nature of trees makes adaptation to climate change challenging; thereby, ecological services and economic potential are under risk. Current long-term and costly gene resources management practices have been primarily directed at a few economically important species and are confined to defined ecological boundaries. Here, we present a novel in situ gene-resource management approach that conserves forest biodiversity and improves productivity and adaptation through utilizing basic forest regeneration installations located across a wide range of environments without reliance on structured tree breeding/conservation methods. We utilized 4,267 25- to 35-year-old European larch trees growing in 21 reforestation installations across four distinct climatic regions in Austria. With the aid of marker-based pedigree reconstruction, we applied multi-trait, multi-site quantitative genetic analyses that enabled the identification of broadly adapted and productive individuals. Height and wood density, proxies to fitness and productivity, yielded in situ heritability estimates of 0.23 ± 0.07 and 0.30 ± 0.07, values similar to those from traditional "structured" pedigrees methods. In addition, individual trees selected with this approach are expected to yield genetic response of 1.1 and 0.7 standard deviations for fitness and productivity attributes, respectively, and be broadly adapted to a range of climatic conditions. Genetic evaluation across broad climatic gradients permitted the delineation of suitable reforestation areas under current and future climates. This simple and resource-efficient management of gene resources is applicable to most tree species.

Genome-wide association studies are powerful tools to elucidate the genome-to-phenome relationship. In order to explain most of the observed heritability of a phenotypic trait, a sufficient number of individuals and a large set of genetic variants must be examined. The development of high-throughput technologies and cost-efficient resequencing of complete genomes have enabled the genome-wide identification of genetic variation at large scale. As such, almost all existing genetic variation becomes available, and it is now possible to identify rare genetic variants in a population sample. Rare genetic variants that were usually filtered out in most genetic association studies are the most numerous genetic variations across genomes and hold great potential to explain a significant part of the missing heritability observed in association studies. Rare genetic variants must be identified with high confidence, as they can easily be confounded with sequencing errors. In this study, we used a pre-filtered data set of 1,014 pure Populus trichocarpa entire genomes to identify rare and common small genetic variants across individual genomes. We compared variant calls between Platypus and HaplotypeCaller pipelines, and we further applied strict quality filters for improved genetic variant identification. Finally, we only retained genetic variants that were identified by both variant callers increasing calling confidence. Based on these shared variants and after stringent quality filtering, we found high genomic diversity in P. trichocarpa germplasm, with 7.4 million small genetic variants. Importantly, 377k non-synonymous variants (5% of the total) were uncovered. We highlight the importance of genomic diversity and the potential of rare defective genetic variants in explaining a significant portion of P. trichocarpa's phenotypic variability in association genetics. The ultimate goal is to associate both rare and common alleles with poplar's wood quality traits to support selective breeding for an improved bioenergy feedstock.

Comparing gene expression patterns and genetic polymorphisms between populations is of central importance for understanding the origin and maintenance of biodiversity. Based on population-specific gene expression levels and allele frequency differences, we sought to identify population divergence (PD) genes across the introgression-resistant genomic regions of Populus trichocarpa. Genes containing highly diverged loci [i.e., genetic divergence (GD)] or showing expression divergence (ED) between populations were widely distributed in the genome and substantially enriched in functional categories related to stress responses, disease resistance, timing of flowering, cell cycle regulation, plant growth, and development. Nine genomic regions showing evidence of strong positive selection were overlapped with GD genes, which had significant differences between Oregon (a southernmost peripheral deme) and the other demes. However, we did not find evidence that genes under positive selection show an enrichment for ED. PD genes and genes under selection pertained to the same gene classes, such as SERINE/CYSTEINE PROTEASE, ABC TRANSPORTER, GLYCOSYLTRANSFERASE and other transferases. Our analysis also revealed that GD genes were polymorphic within the species (41.9 ± 3.66 biallelic variants per gene), as previously reported in herbaceous plants. By contrast, ED genes contained less genetic variants (10.73 ± 1.14) and were likely highly expressed. In addition, we found that trans- rather than cis-acting variants considerably contribute to the evolution of >90% PD genes. Overall, this study elucidates that cohorts of PD genes agree with the general attributes of known speciation genes and GD genes will provide substrates for positive selection to operate on.

Azaleas (Ericaceae) comprise one of the most diverse ornamental plants, renowned for their cultural and economic importance. We present a chromosome-scale genome assembly for Rhododendron simsii, the primary ancestor of azalea cultivars. Genome analyses unveil the remnants of an ancient whole-genome duplication preceding the radiation of most Ericaceae, likely contributing to the genomic architecture of flowering time. Small-scale gene duplications contribute to the expansion of gene families involved in azalea pigment biosynthesis. We reconstruct entire metabolic pathways for anthocyanins and carotenoids and their potential regulatory networks by detailed analysis of time-ordered gene co-expression networks. MYB, bHLH, and WD40 transcription factors may collectively regulate anthocyanin accumulation in R. simsii, particularly at the initial stages of flower coloration, and with WRKY transcription factors controlling progressive flower coloring at later stages. This work provides a cornerstone for understanding the underlying genetics governing flower timing and coloration and could accelerate selective breeding in azalea., Azaleas are one of the most diverse ornamental plants and have cultural and economic importance. Here, the authors report a chromosome-scale genome assembly for the primary ancestor of the azalea cultivar Rhododendro simsi and identify transcription factors that may function in flower coloration at different stages.

Cytokinins play important roles in the growth and development of plants. Physiological and photosynthetic characteristics are common indicators to measure the growth and development in plants. However, few reports have described the molecular mechanisms of physiological and photosynthetic changes in response to cytokinin, particularly in woody plants. DNA methylation is an essential epigenetic modification that dynamically regulates gene expression in response to the external environment. In this study, we examined genome-wide DNA methylation variation and transcriptional variation in poplar (Populus tomentosa) after short-term treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA). We identified 460 significantly differentially methylated regions (DMRs) in response to 6-BA treatment. Transcriptome analysis showed that 339 protein-coding genes, 262 long non-coding RNAs (lncRNAs), and 15,793 24-nt small interfering RNAs (siRNAs) were differentially expressed under 6-BA treatment. Among these, 79% were differentially expressed between alleles in P. tomentosa, and 102,819 allele-specific expression (ASE) loci in 19,200 genes were detected showing differences in ASE levels after 6-BA treatment. Combined DNA methylation and gene expression analysis demonstrated that DNA methylation plays an important role in regulating allele-specific gene expression. To further investigate the relationship between these 6-BA-responsive genes and phenotypic variation, we performed SNP analysis of 460 6-BA-responsive DMRs via re-sequencing using a natural population of P. tomentosa, and we identified 206 SNPs that were significantly associated with growth and wood properties. Association analysis indicated that 53% of loci with allele-specific expression had primarily dominant effects on poplar traits. Our comprehensive analyses of P. tomentosa DNA methylation and the regulation of allele-specific gene expression suggest that DNA methylation is an important regulator of imbalanced expression between allelic loci.

The genetic gain of spruce (Picea spp.) breeding programs is impeded by long recurrent selection cycles stemming from biological constraints such as late expression of traits, weak juvenile mature correlations, and late onset of sexual maturity. Genomic selection (GS) is capable of addressing these barriers to improving the rate of genetic gain via early prediction of phenotypes using dense genetic marker arrays. Results from GS studies focused on spruce species in Canada thus far have produced encouraging results to capture additional genetic gain for wood quality, growth, and insect resistance traits either through the re-analysis of existing progeny trials with genomic information or via prediction of phenotypes for untested candidate trees. With the continual improvement of phenotyping technologies and spruce genomic resources, we expect the capability of GS to capture genetic gain to greatly exceed that of traditional pedigree-based selection methods in the future.

In recent years, the scientific community has become aware that epigenetic mechanisms play a more important role in gene regulatory networks (GRNs) than was hitherto thought, as accumulating evidence has shown that changes in epigenetics without genetic variation can affect complex traits over multiple generations. Within the epigenetic machinery, small non-coding RNAs (sRNAs, 18-24 nucleotides in length) are evolutionarily conserved RNA molecules that target mRNAs for deregulation or translational repression. They commonly have high-level regulatory functions in GRNs by mediating DNA and/or histone methylation and gene silencing essential for plant developmental programs and adaptability. Local adaptation enables plants to acquire a high fitness by, for example, properly timing developmental transitions to match plant growth stages with organism's favorable seasons. In particular, the seed represents a key evolutionary adaptation of seed plants that facilitates dispersal and reinitiates the development coupled in time with suitable environmental conditions. With the advent of high-throughput sequencing for sRNAs and computational approaches for sRNA detection and categorization, it is now feasible to unravel how sRNAs contribute to the fitness of tree species that can survive hundreds of years (e.g., conifers). Of particular interest is to disentangle the roles of sRNAs from complex genomic information in tree species with intimidating genomic sizes (commonly 20-30 Gb in conifers) and abundant nongenic components (e.g., >60% transposable elements). In this chapter, we use seeds of the conifer Picea glauca as a study system to describe the methods and protocols we used or have recently updated, from high-quality RNA isolation to sRNA identification, sequence conservation, abundance comparison, and functional analysis.

The spruce reference genomes (Picea abies, P. glauca, P. glauca x engelmannii) were published eight years after the release of Populus trichocarpa’s genome, the first forest tree species ever sequenced (Science 313:1596–1604 (2006)). Although sequencing these highly complex 20 Gigabases spruce genomes represented a major achievement, the current assemblies present many limitations and a chromosome-level assembly reference in this ecologically and economically important genus is still far from reach (Chaps. 2, 7, 8). Nevertheless, improvements in next-generation sequencing technologies and bioinformatic methods have led to important advancements as outlined in this book (Chap. 3). Increasingly complete and more accurate spruce genome sequences will serve the research community in many aspects. Foremost, they will enable improved annotations in the gene space, new discoveries in plant secondary metabolism, and support new breeding strategies for physiologically active compounds and beyond (Chaps. 5, 9, 12, and 13). An even more uncharted territory is the elucidation of epigenetic memory (Chaps. 4 and 6). In the context of adaptation to future climates, spruces are prime study subjects, as important representatives of boreal ecosystems (Chaps. 10 and 11). Following the example of plant species with less complex genomes, whole-genome resequencing will become state-of-the-art in population genomics of spruces and thus invaluable for finding the most important genetic/genomic variants underlying traits of interest. This volume details the progress of the spruce genome sequencing efforts as a cornerstone for exciting discoveries in gymnosperm genomics.

Background Natural selection on fitness-related traits can be temporally heterogeneous among populations. As climate changes, understanding population-level responses is of scientific and practical importance. We examined 18 phenotypic traits associated with phenology, biomass, and ecophysiology in 403 individuals of natural Populus trichocarpa populations, growing in a common garden. Results Compared with tree origin settings, propagules likely underwent drought exposures in the common garden due to significantly low rainfall during the years of measurement. All study traits showed population differentiation reflecting adaptive responses due to local genetic adaptation. Phenology and biomass traits were strongly under selection and showed plastic responses between years, co-varying with latitude. While phenological events (e.g., bud set and growth period) and biomass were under positive directional selection, post-bud set period, particularly from final bud set to the onset of leaf drop, was selected against. With one exception to water-use efficiency, ecophysiology traits were under negative directional selection. Moreover, extended phenological events jointly evolved with source niches under increased temperature and decreased rainfall exposures. High biomass coevolved with climatic niches of high temperature; low rainfall promoted high photosynthetic rates evolution. Conclusions This work underpins that P. trichocarpa is likely to experience increased fitness (height gain) by evolving toward extended bud set and growth period, abbreviated post-bud set period, and increased drought resistance, potentially constituting a powerful mechanism for long-lived tree species in surviving unpredictably environmental extremes (e.g., drought).

Pollination dynamics was studied in a first-generation Platycladus orientalis (L.) Franco seed orchard with pedigree reconstruction using eight nuclear and four chloroplast SSRs. The pedigree reconstruction assigned 371 of 448 studied seeds to one of the orchard’s 192 candidate male parents and showed a high level of outcrossing and pollen contamination in the orchard’s seed crop. While the orchard’s seed population showed greater allelic richness compared with the parental population, a few alleles present in the parental population were missing in the seed crop. Additionally, we detected no significant correlation between male reproductive energy (pollen yield) and male reproductive success; however, uneven parental contribution was also observed. Pollen management practices were recommended to ensure the maintenance of genetic diversity in the seed crops and increase in genetic gain.