SummaryDeciphering the genetic architecture underlying polygenic traits in perennial species can inform molecular marker-assisted breeding. Recent advances in high-throughput sequencing have enabled strategies that integrate linkage-linkage disequilibrium (LD) mapping in Populus.We used an integrated method of quantitative trait locus (QTL) dissection with a high-resolution linkage map and multi-gene association mapping to decipher the nature of genetic architecture (additive, dominant, and epistatic effects) of potential QTLs for growth traits in a Populus linkage population (1200 progeny) and a natural population (435 individuals).Seventeen QTLs for tree height, diameter at breast height, and stem volume mapped to 11 linkage groups (logarithm of odds (LOD) ≥ 2.5), and explained 2.7-18.5% of the phenotypic variance. After comparative mapping and transcriptome analysis, 187 expressed genes (10 046 common single nucleotide polymorphisms (SNPs)) were selected from the segmental homology regions (SHRs) of 13 QTLs. Using multi-gene association models, we observed 202 significant SNPs in 63 promising genes from 10 QTLs (P ≤ 0.0001; FDR ≤ 0.10) that exhibited reproducible associations with additive/dominant effects, and further determined 11 topranked genes tightly linked to the QTLs. Epistasis analysis uncovered a uniquely interconnected gene-gene network for each trait.This study opens up opportunities to uncover the causal networks of interacting genes in plants using an integrated linkage-LD mapping approach.

Silicon is essential for bone formation. A low-silicon diet leads to bone defects, and numerous animal models have demonstrated that silicon supplementation increases bone mineral density (BMD) and reduces bone fragility. However, the exact mechanism of this action has not been characterized. In this study, we aimed to determine the role of biological silicon in the induction of osteoblast differentiation and the possible underlying mechanism. We examined whether orthosilicic acid promotes collagen type 1 (COL-1) and osteocalcin synthesis through the bone morphogenetic protein-2 (BMP-2)/Smad1/5/runt-related transcription factor 2 (RUNX2) signaling pathway by investigating its effect in vitro at several concentrations on COL-1 and osteocalcin synthesis in human osteosarcoma cell lines (MG-63 and U2-OS). The expression of relevant proteins was detected by Western blotting following exposure to noggin, an inhibitor of BMP-2. In MG-63 cells, immunofluorescence methods were applied to detect changes in the expression of BMP-2, phosphorylated Smad1/5 (P-Smad1/5), and RUNX2. Furthermore, rat bone mesenchymal stem cells (BMSCs) were used to determine the effect of orthosilicic acid on osteogenic differentiation. Exposure to 10 μM orthosilicic acid markedly increased the expression of BMP-2, P-Smad1/5, RUNX2, COL-1, and osteocalcin in osteosarcoma cell lines. Enhanced ALP activity and the formation of mineralized nodules were also observed under these conditions. Furthermore, preconditioning with noggin inhibited the silicon-induced upregulation of P-Smad1/5, RUNX2, and COL-1 expression. In conclusion, the BMP-2/Smad1/5/RUNX2 signaling pathway participates in the silicon-mediated induction of COL-1 and osteocalcin synthesis, and orthosilicic acid promotes the osteogenic differentiation of rat BMSCs.

Long non-coding RNAs (lncRNAs) function in various biological processes. However, their roles in secondary growth of plants remain poorly understood. Here, 15,691 lncRNAs were identified from vascular cambium, developing xylem, and mature xylem of Populus tomentosa with high and low biomass using RNA-seq, including 1,994 lncRNAs that were differentially expressed (DE) among the six libraries. 3,569 cis-regulated and 3,297 trans-regulated protein-coding genes were predicted as potential target genes (PTGs) of the DE lncRNAs to participate in biological regulation. Then, 476 and 28 lncRNAs were identified as putative targets and endogenous target mimics (eTMs) of Populus known microRNAs (miRNAs), respectively. Genome re-sequencing of 435 individuals from a natural population of P. tomentosa found 34,015 single nucleotide polymorphisms (SNPs) within 178 lncRNA loci and 522 PTGs. Single-SNP associations analysis detected 2,993 associations with 10 growth and wood-property traits under additive and dominance model. Epistasis analysis identified 17,656 epistatic SNP pairs, providing evidence for potential regulatory interactions between lncRNAs and their PTGs. Furthermore, a reconstructed epistatic network, representing interactions of 8 lncRNAs and 15 PTGs, might enrich regulation roles of genes in the phenylpropanoid pathway. These findings may enhance our understanding of non-coding genes in plants.

Lignocellulosic biomass from trees provides a renewable feedstock for biofuels, lumber, pulp, paper, and other uses. Dissecting the mechanism underlying natural variation of the complex traits controlling growth and lignocellulose biosynthesis in trees can enable marker-assisted breeding to improve wood quality and yield. Here, we combined linkage disequilibrium (LD)-based association analysis with traditional linkage analysis to detect the genetic effect of a Populus tomentosa cellulose synthase gene, PtoCesA4. PtoCesA4 is strongly expressed in developing xylem and leaves. Nucleotide diversity and LD in PtoCesA4, sampled from the P. tomentosa natural distribution, revealed that PtoCesA4 harbors high single nucleotide polymorphism (SNP) diversity (πT = 0.0080 and θw = 0.0098) and low LD (r2 ≥ 0.1, within 1400 bp), demonstrating that the potential of a candidate-gene-based LD approach in understanding the molecular basis underlying quantitative variation in this species. By combining single SNP, multi-SNP, and haplotype-based associations in an association population of 460 individuals with single SNP linkage analysis in a family-based linkage populations (1200 individuals), we identified three strong associations (false discovery rate Q < 0.05) in both populations. These include two nonsynonymous markers (SNP49 associated with α-cellulose content and SNP59 associated with fiber width) and a noncoding marker (SNP18 associated with α-cellulose content). Variation in RNA transcript abundance among genotypic classes of SNP49 was confirmed in these two populations. Therefore, combining different methods allowed us to examine functional PtoCesA4 allelic variation underlying natural variation in complex quantitative traits related to growth and lignocellulosic biosynthesis.

In trees, xylem tissues play a key role in the formation of woody tissues, which have important uses for pulp and timber production; also DNA methylation plays an important part in gene regulation during xylogenesis in trees. In our study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to analyze the role cytosine methylation plays in wood formation in the commercially important tree species Populus tomentosa. This analysis compared the methylation patterns between xylem tissues (developing xylem and mature xylem) and non-xylem tissues (cambium, shoot apex, young leaf, mature leaf, phloem, root, male catkin, and female catkin) and found 10,316 polymorphic methylation sites. MSAP identified 132 candidate genes with the same methylation patterns in xylem tissues, including seven wood-related genes. The expression of these genes differed significantly between xylem and non-xylem tissue types (P < 0.01). This indicated that the difference of expression of specific genes with unique methylation patterns, rather than relative methylation levels between the two tissue types plays a critical role in wood biosynthesis. However, 46.2% of candidate genes with the same methylation pattern in vascular tissues (cambium, phloem, and developing xylem) did not have distinct expression patterns in xylem and non-xylem tissue. Also, bisulfite sequencing and transcriptome sequencing of MYB, NAC and FASCICLIN-LIKE AGP 13 revealed that the location of cytosine methylation in the gene might affect the expression of different transcripts from the corresponding gene. The expression of different transcripts that produce distinct proteins from a single gene might play an important role in the regulation of xylogenesis.

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.

MicroRNAs (miRNAs) regulate gene expression in many biological processes, but the significance of the interaction between a miRNA and its targets in perennial trees remains largely unknown. Here, we employed transcript profiling and association studies in Populus tomentosa (Pto) to decipher the effect of genetic variation and interactions between Pto-miR156c and its potential targets (Pto-SPL15, Pto-SPL20, and Pto-SPL25) in 435 unrelated individuals from a natural population of P. tomentosa. Single-SNP (single-nucleotide polymorphism) based association studies with analysis of the underlying additive and dominant effects identified 69 significant associations (P < 0.01), representing 51 common SNPs (minor allele frequency > 0.05) from Pto-MIR156c and its three potential targets, with six wood and growth traits, revealing their common roles in wood formation. Epistasis analysis uncovered 129 significant SNP-SNP associations with ten traits, indicating the potential genetic interactions of Pto-MIR156c and its three putative targets. Interestingly, expression analysis in stem (phloem, cambium, and xylem) revealed that Pto-miR156c expression showed strong negative correlations with Pto-SPL20 (r = −0.90, P < 0.01) and Pto-SPL25 (r = −0.65, P < 0.01), and a positive correlation with Pto-SPL15 (r = 0.40, P < 0.01), which also indicated the putative interactions of Pto-miR156c and its potential targets and their common roles in wood formation. Thus, our study provided an alternative approach to decipher the interaction between miRNAs and their targets and to dissect the genetic architecture of complex traits in trees.

In the burgeoning microbiome field, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. The proper preservation of samples before being processed is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that microbial consortia in human fecal samples were substantially preserved within a temporary storage of 4 h, independent of the storage temperature. We also verified a previous reported self-made preservation buffer (PB buffer) could not only preserve fecal microbiota at room temperature up to 4 weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize samples if neither freezing facilities nor liquid nitrogen is available.