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.

Genetic architecture of growth traits in Populus revealed by integrated quantitative trait locus (QTL) analysis and association studies.

Deciphering 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 top-ranked 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.

Allelic variation within the S-adenosyl-L-homocysteine hydrolase gene family is associated with wood properties in Chinese white poplar (Populus tomentosa).

BACKGROUND: S-adenosyl-l-homocysteine hydrolase (SAHH) is the only eukaryotic enzyme capable of S-adenosyl-l-homocysteine (SAH) catabolism for the maintenance of cellular transmethylation potential. Recently, biochemical and genetic studies in herbaceous species have obtained important discoveries in the function of SAHH, and an extensive characterization of SAHH family in even one tree species is essential, but currently lacking. RESULTS: Here, we first identified the SAHH family from Populus tomentosa using molecular cloning method. Phylogenetic analyses of 28 SAHH proteins from dicotyledons, monocotyledons, and lower plants revealed that the sequences formed two monophyletic groups: the PtrSAHHA with PtoSAHHA and PtrSAHHB with PtoSAHHB. Examination of tissue-specific expression profiles of the PtoSAHH family revealed similar expression patterns; high levels of expression in xylem were found. Nucleotide diversity and linkage disequilibrium (LD) in the PtoSAHH family, sampled from P. tomentosa natural distribution, revealed that PtoSAHH harbors high single-nucleotide polymorphism (SNP) diversity (π = 0.01059 ± 0.00122 and 0.00930 ± 0.00079,respectively) and low LD (r2 > 0.1, within 800 bp and 2,200 bp, respectively). Using an LD-linkage analysis approach, two noncoding SNPs (PtoSAHHB_1065 and PtoSAHHA_2203) and the corresponding haplotypes were found to significantly associate with α-cellulose content, and a nonsynonymous SNP (PtoSAHHB_410) within the SAHH signature motifs showed significant association with fiber length, with an average of 3.14% of the phenotypic variance explained. CONCLUSIONS: The present study demonstrates that PtoSAHHs were split off prior to the divergence of interspecies in Populus, and SAHHs may play a key role promoting transmethylation reactions in the secondary cell walls biosynthesis in trees. Hence, our findings provide insights into SAHH function and evolution in woody species and also offer a theoretical basis for marker-aided selection breeding to improve the wood quality of Populus.

Genetic variation in transcription factors and photosynthesis light-reaction genes regulates photosynthetic traits.

Transcription factors (TFs) play crucial roles in regulating the production of the components required for photosynthesis; elucidating the mechanisms by which underlying genetic variation in TFs affects complex photosynthesis-related traits may improve our understanding of photosynthesis and identify ways to improve photosynthetic efficiency. Promoter analysis of 96 nuclear-encoded Populus tomentosa Carr. genes within this pathway revealed 47 motifs responsive to light, stress, hormones and organ-specific regulation, as well as 86 TFs that might bind these motifs. Using phenotype-genotype associations, we identified 244 single-nucleotide polymorphisms (SNPs) within 105 genes associated with 12 photosynthesis-related traits. Most (30.33%) of these SNPs were located in intronic regions and these SNPs explained 18.66% of the mean phenotypic variation in the photosynthesis-related traits. Additionally, expression quantitative trait loci (eQTL) mapping identified 216 eQTLs associated with 110 eGenes (genes regulated by eQTLs), explaining 14.12% of the variability of gene expression. The lead SNPs of 12.04% of the eQTLs also contributed to phenotypic variation. Among these, a SNP in zf-Dof 5.6 (G120_9287) affected photosynthesis by modulating the expression of a sub-regulatory network of eight other TFs, which in turn regulate 55 photosynthesis-related genes. Furthermore, epistasis analysis identified a large interacting network representing 732 SNP-SNP pairs, of which 354 were photosynthesis gene-TF pairs, emphasizing the important roles of TFs in affecting photosynthesis-related traits. We combined eQTL and epistasis analysis and found 32 TFs harboring eQTLs being epistatic to their targets (identified by eQTL analysis), of which 15 TFs were also associated with photosynthesis traits. We therefore constructed a schematic model of TFs involved in regulating the photosynthetic light reaction pathway. Taken together, our results provide insight into the genetic regulation of photosynthesis, and may drive progress in the marker-assisted selection of desirable P. tomentosa genotypes with more efficient photosynthesis.

Genome-Wide Association Studies to Improve Wood Properties: Challenges and Prospects.

Wood formation is an excellent model system for quantitative trait analysis due to the strong associations between the transcriptional and metabolic traits that contribute to this complex process. Investigating the genetic architecture and regulatory mechanisms underlying wood formation will enhance our understanding of the quantitative genetics and genomics of complex phenotypic variation. Genome-wide association studies (GWASs) represent an ideal statistical strategy for dissecting the genetic basis of complex quantitative traits. However, elucidating the molecular mechanisms underlying many favorable loci that contribute to wood formation and optimizing GWAS design remain challenging in this omics era. In this review, we summarize the recent progress in GWAS-based functional genomics of wood property traits in major timber species such as Eucalyptus, Populus, and various coniferous species. We discuss several appropriate experimental designs for extensive GWAS in a given undomesticated tree population, such as omics-wide association studies and high-throughput phenotyping technologies. We also explain why more attention should be paid to rare allelic and major structural variation. Finally, we explore the potential use of GWAS for the molecular breeding of trees. Such studies will help provide an integrated understanding of complex quantitative traits and should enable the molecular design of new cultivars.

Identification and allelic dissection uncover roles of lncRNAs in secondary growth of Populus tomentosa.

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.

The Role of DNA Methylation in Xylogenesis in Different Tissues of Poplar.

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.