DNA Methylation Analysis of Growth Differences between Upright and Inverted Cuttings of Populus yunnanensis

DNA methylation is an important mechanism for epigenetic modifications that have been shown to be associated with responses to plant development. Previous studies found that inverted Populus yunnanensis cuttings were still viable and could develop into complete plants. However, the growth status of inverted cuttings was weaker than that of upright cuttings, and the sprouting time of inverted cuttings was later than that of upright cuttings. There is currently no research on DNA methylation patterns in inverted cuttings of Populus yunnanensis. In this study, we detected genome-wide methylation patterns of stem tips of Populus yunnanensis at the early growth stage and the rapid growth stage by Oxford Nanopore Technologies (ONT) methylation sequencing. We found that the methylation levels of CpG, CHG, CHH, and 6mA were 41.34%, 33.79%, 17.27%, and 12.90%, respectively, in the genome of inverted poplar cuttings, while the methylation levels of the four methylation types were higher in the genome of upright poplar cuttings than in inverted cuttings, 41.90%, 34.57%, 18.09%, and 14.11%, suggesting important roles for DNA methylation in poplar cells. In all comparison groups, CpG-type methylation genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were annotated to pathways associated with carbon metabolism, ribosome biogenesis in eukaryotes, glycolysis/gluconeogenesis, pyruvate metabolism, and mRNA detection pathways, suggesting that different biological processes are activated in upright and inverted cuttings. The results show that methylation genes are commonly present in the poplar genome, but only a few of them are involved in the regulation of expression in the growth and development of inverted cuttings. From this, we screened the DET2 gene for significant differences in methylation levels in upright or inverted cuttings. The DET2 gene is a key gene in the Brassinolide (BRs) synthesis pathway, and BRs have an important influence on the growth and development process of plants. These results provide important clues for studying DNA methylation patterns in P. yunnanensis.