RNA editing validation by Sanger sequencing

For individual WM and a secondary female individual (WF_2), RNA and DNA were extracted from the same inflorescence sample, at different developmental stages using Spectrum Plant Total RNA Kit (Sigma-Aldrich) and DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), respectively, following manufacturer’s instructions. This approach allows to exclude putative somatic mutations that may occur along the development of the new branches. Total RNA was additionally treated with 30 units of DNase I (Qiagen). Nucleic acid concentration was measured using a microplate reader Synergy HT (Biotek, Germany), with the software Gen5 (Biotek, Germany). The cDNA was synthesized from 100 ng of total RNA, using oligo(dT)₂₀ and RevertAid Reverse Transcriptase (Thermo Fisher Scientific) in a 20 μL reaction volume, following the manufacturer’s instructions. cDNA concentration was measured as described above.

Two genes in chromosome 2, VviYABBY (VIT_202s0154g00070) and VIT_202s0033g01400 were manually inspected using IGV. Primers were designed for exons of VviYABBY and VIT_202s0033g01400 genes using Primer Premier5 (Supplementary Table ^S2). For VviYABBY, a 336 bp fragment (DNA and cDNA) was amplified and for VIT_202s0033g01400 a 829 bp fragment was obtain in DNA and 632 bp in cDNA amplification.

The DNA and the cDNA amplifications were performed through PCR in 25 μL total volume composed by 0.2 µg of DNA (or cDNA), PCR buffer (20 mM Tris–HCl [pH 8.4], 50 mM KCl), 1.5 mM of Mg₂, 0.2 mM of dNTP mix, 0.4 μM of each forward and reverse primers, 10 U of Taq DNA polymerase and autoclaved MiliQ water. The applied program had 3 min for initial denaturation at 95 °C followed by 34 cycles of 45 s at 95 °C (denaturation), 45 s at 50 °C (annealing) for VviYABBY and 55 °C for VIT_202s0033g01400, and 90 s at 72 °C (extension), followed by a final extension for 5 min at 72 °C. PCR amplifications (1 μL) were inspected on an agarose gel (1%). The remaining product was purified using PureLink Quick PCR Purification kit (Thermo Fisher Scientific), according to the kit protocol. For each gene DNA and cDNA were Sanger sequenced in three technical replicates without cloning. The provided electropherograms were manually inspected.