All primer sequences used in this study are available in Supplementary Data Sheet 1 . Reverse transcription was performed on 500 ng of total pokeweed RNA in a 20 μL reaction volume containing 5 mM DTT, 1 μM reverse primer, 1X First Strand Buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.5 mM dNTPs, 20 units Murine RNase Inhibitor (NEB) and 25 units Superscript III reverse transcriptase (Thermo Fisher). The reaction was incubated at 42°C for 1 h and heat inactivated at 70°C for 20 min.
Following cDNA synthesis, a PCR reaction was conducted, containing 1X Q5 buffer (NEB), 0.5 μM forward primer, 0.5 μM reverse primer, 200 mM dNTPs, 2 μL cDNA and 1 unit Q5 DNA polymerase (NEB) in a total volume of 50 μL. The PCR program included an initial denaturation of 95°C for 30 s, 30 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 120 s and finished with an extension at 72°C for 180 s. PCR products were separated on low-melt agarose and the correct size band excised and purified with EZ-10 Spin columns (Biobasic). The purified product was digested with BamHI and SalI, ligated into pBS-KSII and sequenced.
Following cDNA synthesis, a PCR reaction was conducted, containing 1X Q5 buffer (NEB), 0.5 μM forward primer, 0.5 μM reverse primer, 200 mM dNTPs, 2 μL cDNA and 1 unit Q5 DNA polymerase (NEB) in a total volume of 50 μL. The PCR program included an initial denaturation of 95°C for 30 s, 30 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 120 s and finished with an extension at 72°C for 180 s. PCR products were separated on low-melt agarose and the correct size band excised and purified with EZ-10 Spin columns (Biobasic). The purified product was digested with BamHI and SalI, ligated into pBS-KSII and sequenced.