Rare variations within the serine/arginine-rich splicing factor PtoRSZ21 modulate stomatal size to determine drought tolerance in Populus.

Rare variants contribute significantly to the 'missing heritability' of quantitative traits. The genome-wide characteristics of rare variants and their roles in environmental adaptation of woody plants remain unexplored. Utilizing genome-wide rare variant association study (RVAS), expression quantitative trait loci (eQTL) mapping, genetic transformation, and molecular experiments, we explored the impact of rare variants on stomatal morphology and drought adaptation in Populus. Through comparative analysis of five world-wide Populus species, we observed the influence of mutational bias and adaptive selection on the distribution of rare variants. RVAS identified 75 candidate genes correlated with stomatal size (SS)/stomatal density (SD), and a rare haplotype in the promoter of serine/arginine-rich splicing factor PtoRSZ21 emerged as the foremost association signal governing SS. As a positive regulator of drought tolerance, PtoRSZ21 can recruit the core splicing factor PtoU1-70K to regulate alternative splicing (AS) of PtoATG2b (autophagy-related 2). The rare haplotype PtoRSZ21hap2 weakens binding affinity to PtoMYB61, consequently affecting PtoRSZ21 expression and SS, ultimately resulting in differential distribution of Populus accessions in arid and humid climates. This study enhances the understanding of regulatory mechanisms that underlie AS induced by rare variants and might provide targets for drought-tolerant varieties breeding in Populus.

Temporal dynamics of genetic architecture governing leaf development in Populus.

Leaf development is a multifaceted and dynamic process orchestrated by a myriad of genes to shape the proper size and morphology. The dynamic genetic network underlying leaf development remains largely unknown. Utilizing a synergistic genetic approach encompassing dynamic genome-wide association study (GWAS), time-ordered gene co-expression network (TO-GCN) analyses and gene manipulation, we explored the temporal genetic architecture and regulatory network governing leaf development in Populus. We identified 42 time-specific and 18 consecutive genes that displayed different patterns of expression at various time points. We then constructed eight TO-GCNs that covered the cell proliferation, transition, and cell expansion stages of leaf development. Integrating GWAS and TO-GCN, we postulated the functions of 27 causative genes for GWAS and identified PtoGRF9 as a key player in leaf development. Genetic manipulation via overexpression and suppression of PtoGRF9 revealed its primary influence on leaf development by modulating cell proliferation. Furthermore, we elucidated that PtoGRF9 governs leaf development by activating PtoHB21 during the cell proliferation stage and attenuating PtoLD during the transition stage. Our study provides insights into the dynamic genetic underpinnings of leaf development and understanding the regulatory mechanism of PtoGRF9 in this dynamic process.

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.

Local diversity of drought resistance and resilience in Populus tomentosa correlates with the variation of DNA methylation.

Little information is known about DNA methylation variation in shaping environment-specific drought resistance and resilience for tree adaptation. In this study, we leveraged RNA sequencing and whole-genome bisulfite sequencing data to dissect the distinction of epigenetic regulation under drought stress and rewater condition of Populus tomentosa accessions from three geographical regions. We demonstrated low resistance and high resilience for accessions from South. Non-CG methylation levels in promoter regions of Southern accessions were lower than accessions from higher latitudes and negatively regulated gene expression. CHH context methylation was more sensitive to drought stress, and the geographical-specific differentially methylated regions were scarcely changed by environmental fluctuation. We identified 60 conserved hub genes within the co-expression networks that correlate with photosynthetic and stomatal morphological traits. Epigenome-wide association studies and genome-wide association studies of these 60 hub genes revealed the interdependency between genetic and epigenetic variation in GATA9 and LECRK-VIII.2, which was associated with stomatal morphology and chlorophyll content. The natural epigenetic variation in GATA9 was also faithfully transmitted to progenies in two family-based F1 populations. This study indicates a functional relationship of DNA methylation diversity with drought resistance and resilience which offers new insights into plants' local adaptation to a stressful environment.

Leaf physiology variations are modulated by natural variations that underlie stomatal morphology in Populus.

Stomata are essential for photosynthesis and abiotic stress tolerance. Here, we used multiomics approaches to dissect the genetic architecture and adaptive mechanisms that underlie stomatal morphology in Populus tomentosa juvenile natural population (303 accessions). We detected 46 candidate genes and 15 epistatic gene-pairs, associated with 5 stomatal morphologies and 18 leaf development and photosynthesis traits, through genome-wide association studies. Expression quantitative trait locus mapping revealed that stomata-associated gene loci were significantly associated with the expression of leaf-related genes; selective sweep analysis uncovered significant differentiation in the allele frequencies of genes that underlie stomatal variations. An allelic regulatory network operating under drought stress and adequate precipitation conditions, with three key regulators (DUF538, TRA2 and AbFH2) and eight interacting genes, was identified that might regulate leaf physiology via modulation of stomatal shape and density. Validation of candidate gene variations in drought-tolerant and F1 hybrid populations of P. tomentosa showed that the DUF538, TRA2 and AbFH2 loci cause functional stabilisation of spatiotemporal regulatory, whose favourable alleles can be faithfully transmitted to offspring. This study provides insights concerning leaf physiology and stress tolerance via the regulation of stomatal determination in perennial plants.

Genetic Architecture and Genome-Wide Adaptive Signatures Underlying Stem Lenticel Traits in Populus tomentosa.

The stem lenticel is a highly specialized tissue of woody plants that has evolved to balance stem water retention and gas exchange as an adaptation to local environments. In this study, we applied genome-wide association studies and selective sweeping analysis to characterize the genetic architecture and genome-wide adaptive signatures underlying stem lenticel traits among 303 unrelated accessions of P. tomentosa, which has significant phenotypic and genetic variations according to climate region across its natural distribution. In total, we detected 108 significant single-nucleotide polymorphisms, annotated to 88 candidate genes for lenticel, of which 9 causative genes showed significantly different selection signatures among climate regions. Furthermore, PtoNAC083 and PtoMYB46 showed significant association signals and abiotic stress response, so we overexpressed these two genes in Arabidopsis thaliana and found that the number of stem cells in all three overexpression lines was significantly reduced by PtoNAC083 overexpression but slightly increased by PtoMYB46 overexpression, suggesting that both genes are involved in cell division and expansion during lenticel formation. The findings of this study demonstrate the successful application of an integrated strategy for dissecting the genetic basis and landscape genetics of complex adaptive traits, which will facilitate the molecular design of tree ideotypes that may adapt to future climate and environmental changes.

Genetic Architecture Underlying the Metabolites of Chlorogenic Acid Biosynthesis in Populus tomentosa.

Chlorogenic acid (CGA) plays a crucial role in defense response, immune regulation, and the response to abiotic stress in plants. However, the genetic regulatory network of CGA biosynthesis pathways in perennial plants remains unclear. Here, we investigated the genetic architecture for CGA biosynthesis using a metabolite-based genome-wide association study (mGWAS) and expression quantitative trait nucleotide (eQTN) mapping in a population of 300 accessions of Populus tomentosa. In total, we investigated 204 SNPs which were significantly associated with 11 metabolic traits, corresponding to 206 genes, and were mainly involved in metabolism and cell growth processes of P. tomentosa. We identified 874 eQTNs representing 1066 genes, in which the expression and interaction of causal genes affected phenotypic variation. Of these, 102 genes showed significant signatures of selection in three geographical populations, which provided insights into the adaptation of CGA biosynthesis to the local environment. Finally, we constructed a genetic network of six causal genes that coordinately regulate CGA biosynthesis, revealing the multiple regulatory patterns affecting CGA accumulation in P. tomentosa. Our study provides a multiomics strategy for understanding the genetic basis underlying the natural variation in the CGA biosynthetic metabolites of Populus, which will enhance the genetic development of abiotic-resistance varieties in forest trees.