PPGR: a comprehensive perennial plant genomes and regulation database.

Perennial woody plants hold vital ecological significance, distinguished by their unique traits. While significant progress has been made in their genomic and functional studies, a major challenge persists: the absence of a comprehensive reference platform for collection, integration and in-depth analysis of the vast amount of data. Here, we present PPGR (Resource for Perennial Plant Genomes and Regulation; https://ngdc.cncb.ac.cn/ppgr/) to address this critical gap, by collecting, integrating, analyzing and visualizing genomic, gene regulation and functional data of perennial plants. PPGR currently includes 60 species, 847 million protein-protein/TF (transcription factor)-target interactions, 9016 transcriptome samples under various environmental conditions and genetic backgrounds. Noteworthy is the focus on genes that regulate wood production, seasonal dormancy, terpene biosynthesis and leaf senescence representing a wealth of information derived from experimental data, literature mining, public databases and genomic predictions. Furthermore, PPGR incorporates a range of multi-omics search and analysis tools to facilitate browsing and application of these extensive datasets. PPGR represents a comprehensive and high-quality resource for perennial plants, substantiated by an illustrative case study that demonstrates its capacity in unraveling gene functions and shedding light on potential regulatory processes.

Growth-regulating factor 15-mediated gene regulatory network enhances salt tolerance in poplar.

Soil salinity is an important determinant of crop productivity and triggers salt stress response pathways in plants. The salt stress response is controlled by transcriptional regulatory networks that maintain regulatory homeostasis through combinations of transcription factor (TF)-DNA and TF-TF interactions. We investigated the transcriptome of poplar 84 K (Populus alba × Populus glandulosa) under salt stress using samples collected at 4- or 6-h intervals within 2 days of salt stress treatment. We detected 24,973 differentially expressed genes, including 2,231 TFs that might be responsive to salt stress. To explore these interactions and targets of TFs in perennial woody plants, we combined gene regulatory networks, DNA affinity purification sequencing, yeast two-hybrid-sequencing, and multi-gene association approaches. Growth-regulating factor 15 (PagGRF15) and its target, high-affinity K+ transporter 6 (PagHAK6), were identified as an important regulatory module in the salt stress response. Overexpression of PagGRF15 and PagHAK6 in transgenic lines improved salt tolerance by enhancing Na+ transport and modulating H2O2 accumulation in poplar. Yeast two-hybrid assays identified more than 420 PagGRF15-interacting proteins, including ETHYLENE RESPONSE FACTOR TFs and a zinc finger protein (C2H2) that are produced in response to a variety of phytohormones and environmental signals and are likely involved in abiotic stress. Therefore, our findings demonstrate that PagGRF15 is a multifunctional TF involved in growth, development, and salt stress tolerance, highlighting the capability of a multifaceted approach in identifying regulatory nodes in plants.

The transcriptional landscape of Populus pattern/effector-triggered immunity and how PagWRKY18 involved in it.

Plants trigger a robust immune response by activating massive transcriptome reprogramming through crosstalk between PTI and ETI. However, how PTI and ETI contribute to the quantitative or/and qualitative output of immunity and how they work together when both are being activated were unclear. In this study, we performed a comprehensive overview of pathogen-triggered transcriptomic reprogramming by analyzing temporal changes in the transcriptome up to 144 h after Colletotrichum gloeosporioides inoculated in Populus. Moreover, we constructed a hierarchical gene regulatory network of PagWRKY18 and its potential target genes to explore the underlying regulatory mechanisms of PagWRKY18 that are not yet clear. Interestingly, we confirmed that PagWRKY18 protein can directly bind the W-box elements in the promoter of a transmembrane leucine-rich repeat receptor-like kinase, PagSOBIR1 gene, to trigger PTI. At the same time, PagWRKY18 functions in disease tolerance by modulation of ROS homeostasis and induction of cell death via directly targeting PagGSTU7 and PagPR4 respectively. Furthermore, PagPR4 can interact with PagWRKY18 to inhibit the expression of PagPR4 genes, forming a negative feedback loop. Taken together, these results suggest that PagWRKY18 may be involved in regulating crosstalk between PTI and ETI to activate a robust immune response and maintain intracellular homeostasis.

Genomic insights into selection for heterozygous alleles and woody traits in Populus tomentosa.

Heterozygous alleles are widespread in outcrossing and clonally propagated woody plants. The variation in heterozygosity that underlies population adaptive evolution and phenotypic variation, however, remains largely unknown. Here, we describe a de novo chromosome-level genome assembly of Populus tomentosa, an economic and ecologically important native tree in northern China. By resequencing 302 natural accessions, we determined that the South subpopulation (Pop_S) encompasses the ancestral strains of P. tomentosa, while the Northwest subpopulation (Pop_NW) and Northeast subpopulation (Pop_NE) experienced different selection pressures during population evolution, resulting in significant population differentiation and a decrease in the extent of heterozygosity. Analysis of heterozygous selective sweep regions (HSSR) suggested that selection for lower heterozygosity contributed to the local adaptation of P. tomentosa by dwindling gene expression and genetic load in the Pop_NW and Pop_NE subpopulations. Genome-wide association studies (GWAS) revealed that 88 single nucleotide polymorphisms (SNPs) within 63 genes are associated with nine wood composition traits. Among them, the selection for the homozygous AA allele in PtoARF8 is associated with reductions in cellulose and hemicellulose contents by attenuating PtoARF8 expression, and the increase in lignin content is attributable to the selection for decreases in exon heterozygosity in PtoLOX3 during adaptive evolution of natural populations. This study provides novel insights into allelic variations in heterozygosity associated with adaptive evolution of P. tomentosa in response to the local environment and identifies a series of key genes for wood component traits, thereby facilitating genomic-based breeding of important traits in perennial woody plants.

Multifeature analysis of age-related microbiome structures reveals defense mechanisms of Populus tomentosa trees.

Root microbiota composition shifts during the development of most annual plants. Although some perennial plants can live for centuries, the host-microbiome partnerships and interaction mechanisms underlying their longevity remain unclear. To address this gap, we investigated age-related changes in the root metabolites, transcriptomes, and microbiome compositions of 1- to 35-yr-old Populus tomentosa trees. Ten co-response clusters were obtained according to their accumulation patterns, and members of each cluster displayed a uniform and clear pattern of abundance. Multi-omics network analysis demonstrated that the increased abundance of Actinobacteria with tree age was strongly associated with the flavonoid biosynthesis. Using genetic approaches, we demonstrate that the flavonoid biosynthesis regulator gene Transparent Testa 8 is associated with the recruitment of flavonoid-associated Actinobacteria. Further inoculation experiments of Actinobacteria isolates indicated that their colonization could significantly improve the host's phenotype. Site-directed mutagenesis revealed that the hyBl gene cluster, involved in biosynthesis of an aminocyclitol hygromycin B analog in Streptomyces isolate bj1, is associated with disease suppression. We hypothesize that interactions between perennial plants and soil microorganisms lead to gradual enrichment of a subset of microorganisms that may harbor a wealth of currently unknown functional traits.

Occurrence, Characteristics, and qPCR-Based Identification of Pectobacterium versatile Causing Soft Rot of Chinese Cabbage in China.

Pectobacterium is one of the most important genera of phytopathogenic bacteria. It can cause soft-rot diseases on a wide range of plant species across the world. In this study, three Pectobacterium strains (KC01, KC02, and KC03) were isolated from soft-rotted Chinese cabbage in Beijing, China. These three strains were identified as Pectobacterium versatile based on phylogenetic analysis of Pectobacterium 16S ribosomal RNA, pmrA, and 504 Pectobacterium core genes, as well as a genomic average nucleotide identity analysis. Their biochemical characteristics were found to be similar to the P. versatile type strain ICMP9168T but differed in response to citric acid, stachyose, D-glucuronic acid, dextrin, and N-acetyl-ß-D-mannosamine. All of the tested P. versatile strains showed different carbohydrate utilization abilities compared with P. carotovorum and P. odoriferum, particularly in their ability to utilize D-arabitol, L-rhamnose, and L-serine. Under laboratory conditions, the maceration ability of P. versatile on Chinese cabbage was the highest at 28°C, compared with those at 13, 28, 23, and 33°C. Additionally, P. versatile could infect all of the 17 known Pectobacterium host plants, except for Welsh onion (Allium fistulosum). A SYBR Green quantitative PCR (qPCR) detection system was developed to distinguish P. versatile from other soft-rot bacteria based on the combined performance of melting curve (with a single melting peak at around 85°C) and fluorescence curve (with cycle threshold <30) when the bacterial genomic DNA concentration was in the range of 10 pg/µl to 10 ng/µl. This study is the first to report the presence of P. versatile on Chinese cabbage in China, as well as a specific and sensitive qPCR assay that can be used to quickly identify P. versatile. The work contributes to a better understanding of P. versatile and will facilitate the effective diagnosis of soft-rot disease, ultimately benefitting commercial crop production.

Evolutionary Origins of Pseudogenes and Their Association with Regulatory Sequences in Plants.

Pseudogenes (Ψs), nonfunctional relatives of functional genes, form by duplication or retrotransposition, and loss of gene function by disabling mutations. Evolutionary analysis provides clues to Ψ origins and effects on gene regulation. However, few systematic studies of plant Ψs have been conducted, hampering comparative analyses. Here, we examined the origin, evolution, and expression patterns of Ψs and their relationships with noncoding sequences in seven angiosperm plants. We identified ∼250,000 Ψs, most of which are more lineage specific than protein-coding genes. The distribution of Ψs on the chromosome indicates that genome recombination may contribute to Ψ elimination. Most Ψs evolve rapidly in terms of sequence and expression levels, showing tissue- or stage-specific expression patterns. We found that a surprisingly large fraction of nontransposable element regulatory noncoding RNAs (microRNAs and long noncoding RNAs) originate from transcription of Ψ proximal upstream regions. We also found that transcription factor binding sites preferentially occur in putative Ψ proximal upstream regions compared with random intergenic regions, suggesting that Ψs have conditioned genome evolution by providing transcription factor binding sites that serve as promoters and enhancers. We therefore propose that rapid rewiring of Ψ transcriptional regulatory regions is a major mechanism driving the origin of novel regulatory modules.

Revisiting the Mg/TMSCl/Dipolar Solvent System for Dearomatic Silylation of Aryl Carbonyl Compounds: Substrate Scope, Transformations, and Mechanistic Studies.

Dearomatic silylation of arene derivatives is an intriguing synthetic target, which represents an elegant extension of Birch reduction and produces silylated cyclohexene derivatives with great potential of further transformation. Herein, we report a systematic study on dearomatic silylation of aryl carbonyl compounds with Mg and the TMSCl/NMP adduct. The protocol displays a wide range of substrate scope, including alkyl aryl ketones, aromatic amides, benzonitriles, tert-butyl benzoates, and even 2,2'-bipyridines. Synthetic utility is demonstrated using the products as versatile substrate in various transformations. The detailed mechanism is presented with both control experimental analyses and theoretical calculations. An unusual five-coordinated silicon dianion intermediate is first proposed and described here. The selectivity is influenced by the relative rates of single electron reductions (the TMSCl/NMP adduct versus the substrate) and the steric effects.

Enantio- and diastereoselective boron conjugate addition to α-alkyl α,ß-unsaturated esters.

We developed a copper-catalyzed enantio- and diastereoselective boron conjugate addition to α-alkyl α,ß-unsaturated esters under base-free conditions. The approach showed excellent enantioselectivities (87-99% ee) and moderate to good conversions (51-99%), albeit with moderate diastereoselectivities (1 : 1-17 : 1 dr). The synthetic utility of this protocol was demonstrated.

Assessment of carbon emission and primary energy input of a gasoline combustion-powered 2.0 T SUV in China.

With the use of vehicles, large amounts of carbon dioxide are emitted by combustion of gasoline and energy consumes in their lifecycle. Therefore, the objective of this study is to evaluate the lifecycle carbon emission and primary energy input of a widely used sport utility vehicle (SUV) in China with the lifecycle assessment method. The results show that total petrol consumption of an SUV in lifetime is 21,300 kg; the CO2 emissions and primary energy input in the manufacturing, assembly, operation, and decommissioning phase are respectively 8857, 443, 54,925, and 443 kg and 123,413, 6171, 12,341, and 6171 MJ. The average CO2 emission intensity and energy input intensity of materials are respectively 2.74 kg/kg and 64.9 MJ/kg. The primary energy input of materials in manufacturing phase occupies 83.3%, and CO2 emission in use phase is 64,267.3 kg (occupied 92.62%), mainly attributed to the combustion of petrol.

Secondary Mutation-Induced Alternative Splicing Suppresses RNA Splicing Defect of the jhs1 Mutant.

jing he sheng1 (jhs1) is a mutant of the DNA2 homolog in Arabidopsis (Arabidopsis thaliana), which was previously identified as being involved in DNA damage repair, cell cycle regulation, and meristem maintenance. A mutation at the 3' intron splice site of the 11th intron causes alternative splicing of this intron at two other sites, which results in frame shifts and premature stop codons. Here, we screened suppressors of jhs1 to further study the function and regulatory networks of JHS1 Three suppressors with wild-type-like phenotypes were obtained. Sequencing analysis results showed that each of the suppressors has a second mutation in jhs1 that causes further alternative splicing of the intron and corrects the shifted reading frame with small insertions. Precursor mRNA sequence analysis and intron splice site evaluation results suggested that intron splicing was disturbed in the suppressors, and this switched the splice site, resulting in small insertions in the coding regions of JHS1. Structural analysis of JHS1 suggested that the insertions are in a disordered loop region of the DNA2 domain and do not seem to have much deleterious effect on the function of the protein. This work not only has implications for the evolution of protein sequences at exon junctions but also provides a strategy to study the mechanism of precursor mRNA splicing.

Synonymous mutation in Growth Regulating Factor 15 of miR396a target sites enhances photosynthetic efficiency and heat tolerance in poplar.

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<0.0001, q<0.05). We also identified miR396a and its target, Growth-Regulating Factor 15 (GRF15) as an important regulatory module in the heat-stress response. Transgenic plants of hybrid poplar (P. alba × P. glandulosa) overexpressing a GRF15 mRNA lacking the miR396a target sites exhibited enhanced heat tolerance and photosynthetic efficiency compared to wild-type plants. Together, our observations demonstrate that GRF15 plays a crucial role in responding to heat stress, and they highlight the power of this new, multifaceted approach for identifying regulatory nodes in plants.

A Re-evaluation of the Taxonomy and Classification of the Type III Secretion System in a Pathogenic Bacterium Causing Soft Rot Disease of Pleurotus eryngii.

Pantoea beijingensis, a gram-negative pathogenic bacterium, causes soft rot disease in the fungus Pleurotus eryngii in China. However, the taxonomic classification of this pathogen is controversial due to close relationships between bacteria of the genera Pantoea and Erwinia. This study aimed to resolve the identity of P. beijingensis using phylogenomic and systematic analyses of Pantoea and Erwinia by whole-genome sequencing. Single-copy orthologs identified from the Erwinia/Pantoea core genomes were used to delineate Erwinia/Pantoea phylogeny. P. beijingensis LMG27579T clustered within a single Erwinia clade. A whole-genome-based phylogenetic tree and average nucleotide and amino-acid identity values indicate that P. beijingensis LMG27579T should be renamed Erwinia beijingensis. The hrp/hrc genes encoding type III secretion system (T3SS) proteins in Erwinia and Pantoea were divided into five groups according to gene contents and organization. Neighbor-joining-inferred phylogenetic trees based on concatenated HrcU, HrcN, and HrcR in the main hrp/hrc cluster showed that E. beijingensis T3SS proteins are closely related to those in Ewingella americana, implying that E. beijingensis and E. americana have a recent common hrp/hrc gene ancestor. Furthermore, T3SS proteins of Erwinia and Pantoea were clustered in different clades separated by other bacterial T3SS proteins. Thus, T3SS genes in Pantoea and Erwinia strains might have been acquired by horizontal gene transfer. Overall, our findings clarify the taxonomy of the bacterium causing soft rot in P. eryngii, as well as the genetic structure and classification of the hrp/hrc T3SS virulence factor. We propose that T3SS acquisition is important for E. beijingensis emergence and pathogenesis.

Distinct and Overlapping Functions of Miscanthus sinensis MYB Transcription Factors SCM1 and MYB103 in Lignin Biosynthesis.

Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.

Key Genes and Genetic Interactions of Plant-Pathogen Functional Modules in Poplar Infected by Marssonina brunnea.

Marssonina brunnea, the causative pathogen of Marssonina leaf spot of poplars (MLSP), devastates poplar plantations by forming black spots on leaves and defoliating trees. Although MLSP has been studied for over 30 years, the key genes that function during M. brunnea infection and their effects on plant growth are poorly understood. Here, we used multigene association studies to investigate the effects of key genes in the plant-pathogen interaction pathway, as revealed by transcriptome analysis, on photosynthesis and growth in a natural population of 435 Populus tomentosa individuals. By analyzing transcriptomic changes during three stages of infection, we detected 628 transcription factor genes among the 7,611 differentially expressed genes that might be associated with basal defense responses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that transcriptomic changes across different stages of infection lead to the reprogramming of metabolic processes possibly related to defense activation. We identified 29,399 common single-nucleotide polymorphisms (SNPs) within 221 full-length genes in plant-pathogen interaction pathways that were significantly associated with photosynthetic and growth traits. We also detected 4,460 significant epistatic pairs associated with stomatal conductance, tree diameter, and tree height. Epistasis analysis uncovered significant interactions between 2,561 SNP-SNP pairs from different functional modules in the plant-pathogen interaction pathway, revealing possible genetic interactions. This analysis revealed many key genes that function during M. brunnea infection and their potential roles in mediating photosynthesis and plant growth, shedding light on genetic interactions between functional modules in the plant-pathogen interaction pathway.

Genetic interactions among Pto-miR319 family members and their targets influence growth and wood properties in Populus tomentosa.

MicroRNAs (miRNAs) play crucial roles in all aspects of plant growth and development, but the genetic interactions of miRNAs and their target genes in woody plants are largely unknown. Here, we integrated association genetics and expression profiling to decipher the allelic variations and interactions of the Pto-MIR319 family of miRNAs and 12 putative Pto-miR319 target genes related to wood formation in 435 unrelated individuals of Populus tomentosa Carrière (Chinese white poplar). Expression pattern analysis showed that among all pairings between expressions of pre-miRNA of Pto-MIR319 members and targets, 70.0% showed negative correlation of expression levels (r = - 0.944 to 0.674, P < 0.01) in eight tissues and organs of poplar, suggesting that Pto-miR319 may participate in the regulatory network of wood formation. Single SNP-based association studies identified 137 significant associations (P < 0.01, Q < 0.1), representing 126 unique SNPs from Pto-MIR319 members and their targets, with 10 tree growth traits, revealing that these genetic factors have common roles related to wood formation. Epistasis analysis uncovered 105 significant SNP-SNP associations (P < 0.01) influencing the 10 traits, demonstrating the close genetic interactions between Pto-MIR319 family members and the 12 Pto-miR319 target genes. Notably, one common SNP, in the precursor region of Pto-MIR319e, affected the stability of Pto-MIR319e's secondary structure by altering the stem-loop structure and minimum free energy, contributing to variations in the expression of Pto-MIR319e and Pto-miR319e target genes. This study enriches the understanding of the functions of miR319 family miRNAs in poplar and exemplifies a feasible approach to exploring the genetic effects underlying miRNA-mRNA interactions related to complex traits in trees.

Genetic architecture underlying the lignin biosynthesis pathway involves noncoding RNAs and transcription factors for growth and wood properties in Populus.

Lignin provides structural support in perennial woody plants and is a complex phenolic polymer derived from phenylpropanoid pathway. Lignin biosynthesis is regulated by coordinated networks involving transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). However, the genetic networks underlying the lignin biosynthesis pathway for tree growth and wood properties remain unknown. Here, we used association genetics (additive, dominant and epistasis) and expression quantitative trait nucleotide (eQTN) mapping to decipher the genetic networks for tree growth and wood properties in 435 unrelated individuals of Populus tomentosa. We detected 124 significant associations (P ≤ 6.89E-05) for 10 growth and wood property traits using 30 265 single nucleotide polymorphisms from 203 lignin biosynthetic genes, 81 TF genes, 36 miRNA genes and 71 lncRNA loci, implying their common roles in wood formation. Epistasis analysis uncovered 745 significant pairwise interactions, which helped to construct proposed genetic networks of lignin biosynthesis pathway and found that these regulators might affect phenotypes by linking two lignin biosynthetic genes. eQTNs were used to interpret how causal genes contributed to phenotypes. Lastly, we investigated the possible functions of the genes encoding 4-coumarate: CoA ligase and cinnamate-4-hydroxylase in wood traits using epistasis, eQTN mapping and enzymatic activity assays. Our study provides new insights into the lignin biosynthesis pathway in poplar and enables the novel genetic factors as biomarkers for facilitating genetic improvement of trees.

Time-specific and pleiotropic quantitative trait loci coordinately modulate stem growth in Populus.

In perennial woody plants, the coordinated increase of stem height and diameter during juvenile growth improves competitiveness (i.e. access to light); however, the factors underlying variation in stem growth remain unknown in trees. Here, we used linkage-linkage disequilibrium (linkage-LD) mapping to decipher the genetic architecture underlying three growth traits during juvenile stem growth. We used two Populus populations: a linkage mapping population comprising a full-sib family of 1,200 progeny and an association mapping panel comprising 435 unrelated individuals from nearly the entire natural range of Populus tomentosa. We mapped 311 quantitative trait loci (QTL) for three growth traits at 12 timepoints to 42 regions in 17 linkage groups. Of these, 28 regions encompassing 233 QTL were annotated as 27 segmental homology regions (SHRs). Using SNPs identified by whole-genome re-sequencing of the 435-member association mapping panel, we identified significant SNPs (P ≤ 9.4 × 10-7 ) within 27 SHRs that affect stem growth at nine timepoints with diverse additive and dominance patterns, and these SNPs exhibited complex allelic epistasis over the juvenile growth period. Nineteen genes linked to potential causative alleles that have time-specific or pleiotropic effects, and mostly overlapped with significant signatures of selection within SHRs between climatic regions represented by the association mapping panel. Five genes with potential time-specific effects showed species-specific temporal expression profiles during the juvenile stages of stem growth in five representative Populus species. Our observations revealed the importance of considering temporal genetic basis of complex traits, which will facilitate the molecular design of tree ideotypes.

Genetic variants in microRNA biogenesis genes as novel indicators for secondary growth in Populus.

MicroRNAs (miRNAs) function as key regulators of complex traits, but how genetic alterations in miRNA biogenesis genes (miRBGs) affect quantitative variation has not been elucidated. We conducted transcript analyses and association genetics to investigate how miRBGs, miRNA genes (MIRNAs) and their respective targets contribute to secondary growth in a natural population of 435 Populus tomentosa individuals. This analysis identified 29 843 common single-nucleotide polymorphisms (SNPs; frequency > 0.10) within 682 genes (80 miRBGs, 152 MIRNAs, and 457 miRNA targets). Single-SNP association analysis found SNPs in 234 candidate genes exhibited significant additive/dominant effects on phenotypes. Among these, specific candidates that associated with the same traits produced 791 miRBG-MIRNA-target combinations, suggesting possible genetic miRBG-MIRNA and MIRNA-target interactions, providing an important clue for the regulatory mechanisms of miRBGs. Multi-SNP association found 4672 epistatic pairs involving 578 genes that showed significant associations with traits and identified 106 miRBG-MIRNA-target combinations. Two multi-hierarchical networks were constructed based on correlations of miRBG-miRNA and miRNA-target expression to further probe the mechanisms of trait diversity underlying changes in miRBGs. Our study opens avenues for the investigation of miRNA function in perennial plants and underscored miRBGs as potentially modulating quantitative variation in traits.

Conserved noncoding sequences conserve biological networks and influence genome evolution.

Comparative genomics approaches have identified numerous conserved cis-regulatory sequences near genes in plant genomes. Despite the identification of these conserved noncoding sequences (CNSs), our knowledge of their functional importance and selection remains limited. Here, we used a combination of DNA methylome analysis, microarray expression analyses, and functional annotation to study these sequences in the model tree Populus trichocarpa. Methylation in CG contexts and non-CG contexts was lower in CNSs, particularly CNSs in the 5'-upstream regions of genes, compared with other sites in the genome. We observed that CNSs are enriched in genes with transcription and binding functions, and this also associated with syntenic genes and those from whole-genome duplications, suggesting that cis-regulatory sequences play a key role in genome evolution. We detected a significant positive correlation between CNS number and protein interactions, suggesting that CNSs may have roles in the evolution and maintenance of biological networks. The divergence of CNSs indicates that duplication-degeneration-complementation drives the subfunctionalization of a proportion of duplicated genes from whole-genome duplication. Furthermore, population genomics confirmed that most CNSs are under strong purifying selection and only a small subset of CNSs shows evidence of adaptive evolution. These findings provide a foundation for future studies exploring these key genomic features in the maintenance of biological networks, local adaptation, and transcription.