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DNA methylation is a pervasive feature of eukaryotic genomes, and an important epigenetic modification. Much attention has been focused on the regulatory role of DNA methylation underlying the growth and developmental processes, and the field garnered increased interests through the recent studies of functional potential in heritable epigenetic variation. In this review, we briefly summarized the relevant concepts and research methods for analyzing DNA methylation in perennial trees. Furthermore, we reviewed current research progress of DNA methylation in perennial woody plants, and provided integrated strategy of epigenome-wide association study (EWAS), CRISPR/dCas9 (deactivated CRISPR-associated protein 9) and epigenome sequencing technologies to systematically characterize the putative function of DNA methylation on development process. These system biological methods provide important insights to dissect the genetic regulatory mechanisms of DNA methylation underlying complex traits and decipher the function of DNA methylation in molecular breeding of forest trees.

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.

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.

Flavonoids are widely distributed secondary metabolites as well as a major class of chemical defenses in response to light intensity, temperature, and other external factors. However, systematic investigation on the genetic basis of flavonoids in perennial trees is poorly documented. In this study, we married metabolite-based genome wide association analysis (mGWAS), expression quantitative trait loci (eQTL) mapping and weighted co-expression network analysis (WGCNA) analysis to systematically construct the regulatory network underlying flavonoids decoration in Populus. We firstly characterized 172 flavonoids features in 300 natural accessions of Populus tomentosa collected in three widespread geographical regions. Then, we conducted systematical genetic strategy to identify casual candidate genes, and constructed the regulatory network of flavonoids biosynthesis of Populus. Selection signature revealed 83 metabolites exhibited significant divergence among three geographical regions of Populus, we thus determined candidate genes contributing to flavonoids divergence based on the selection sweep analysis. Furthermore, we focused on PtoLDOX, a key gene responsible for the levels of anthocyanin and dihydrokaempferol, and dissected it through allelic frequency and haplotype analysis. Collectively, our study provides new insights into the genetic basis of flavonoids biosynthesis, and sheds light on the adaptive selection to different geographical regions that potentially dominated by the flavonoids of Populus, facilitating marker-based breeding in poplar varieties.