Fig. 5: Lineage-specific differences in subgenome expression and regulatory patterns of retained duplicated gene pairs within the Populus-Salix and two sister lineages.

a Cluster results of expression divergence of duplicated gene pairs between the A and B subgenomes during different developmental stages of female and male flowers and fruits in I. orientalis (Ior), I. polycarpa (Ipo) and P. deltoides (Pde). The subgenome expression divergence was represented by the TPM ratio (A - B)/(A + B), where a ratio > 0 indicates expression dominance in the A subgenome, while a ratio < 0 indicates expression dominance in the B subgenome. The heatmap results are shown on the left, and the mean and standard deviation of the TPM ratio for each sample are displayed on the right. Each sample has three biological replicates. b The expression levels of duplicated gene pairs related to the development of traits in different tissues and developmental stages. These genes are distributed in four clusters, including C1, which is more highly expressed in the A subgenome across all species, and clusters (C4-C6) with lineage-specific expression divergence in Populus. The circle size and color are positively correlated with the expression levels. c Expression of FT1 and FT2 in leaves and buds/shoot apex of I. orientalis (Ior), I. polycarpa (Ipo) and P. deltoides (Pde) from late autumn to early spring. Relative fold change in expression levels of FT1 and FT2 relative to the lowest expression (TPM) within a tissue are shown. The results for P. deltoides (Pde) are adapted from previous studies^74,79. d Overall rosette leaf phenotype of A. thaliana WT (wild-type) plant, 35S:ZFP6, and 35S:GIS3 transgenic plants. Arrows indicate the 4th rosette leaf. Bar = 1 mm. e Phenotypes and trichome distribution on the 4th rosette leaves of A. thaliana. Bar = 1 mm. f Statistical analysis of trichome density on the 4th rosette leaves of A. thaliana. Each line included five biological replicates. Data are presented as mean ± SD, and p-values from two-tailed t-tests are shown. g VISTA sequence conservation plot of the Populus-Salix specific CNE around ZFP6 and GIS3, using S. rehderiana as a reference. The A and B subgenomes of Clade I species are represented in blue and green, respectively. All species names are the same as abbreviations in Fig. 4. h The Dual-luciferase assay revealed four CNEs could significantly improve the expression level of luciferases and have potential enhancer activities. CNE3 and CNE4 were tested with 3 and 6 replicates, respectively, while the others were tested with 4 replicates. Data are presented as mean ± SD, and p-values from two-tailed t-tests are shown. Source data are provided as a Source Data file.