Samples for Differential Expression Analysis
To comprehend the transcriptional control mechanism responsible for the concomitant formation of the self-supporting and lianescent xylem in different portions of the same plant of B. magnifica , we sampled the cambium and differentiating xylem of entire internodes samples (approximately 30 cm long) collected from 60 cm from the shoot apex to the base of the stem (from around the 10th to the 20th internodes), along different stems of a single plant. After two years of cultivation, the sampling was conducted in April 2018 during the period of active cambium by peeling the bark and gently scraping the exposed xylem (Allona et al. , 1998; Sterky et al. , 1998; Perry et al. , 2021). The cambial activity was assessed by anatomical analysis (Supporting Information Fig. S1) and the ease with which the bark was peeled (Wilcox, 1962; Melder et al. , 2015). We chose this sampling strategy to avoid that the observed differences could be a product of, or be masked by, allelic variations, as B. magnifica is a wild, highly heterozygous species with no available sequence data. Given our focus on cambium and differentiating xylem, we also avoided sampling tissues below the peeled bark to prevent collecting other tissues, such as phloem and cortex, as the bark in the species was fragile and wrapped immediately after removal. This also prevented sampling the cambium and phloem from the phloem wedges (Supporting Information Fig. S2), what would add complexity to our analysis and was out of the scope of the present work. The innermost position of the variant cambium in the stem facilitated the collection of only regular portions for differential expression analysis, which were the only ones analyzed for this purpose. Tissues were immediately frozen in liquid nitrogen and stored at -80 °C. Before tissue harvest, each stem segment had its base cut and stored in 50 % alcohol to determine the xylem phase, self-supporting or lianescent, and any segment showing dubious or transitional anatomy, and the segment next to it were discarded. Six pools of self-supporting and six pools of lianescent cambium and differentiating xylem, composed of 8-19 segments each, were collected for sequencing and differential expression analysis.