Sp6 or t7 polymerase

Specific primers were designed for genes of interest retrieved from the transcriptome resources of M. fuliginosus, and fragments were amplified from either mixed stage or stage-specific cDNA. The PCR fragments were inserted into pGEM-T-easy vector (Promega) and cloned in Top10 E. coli cells (Invitrogen). DIG/FITC labeled RNA probes were generated by DIG RNA labeling mix (Roche) or using transcription reagents along with DIG-UTP/FITC-UTP (Roche) by SP6 or T7 polymerases (Roche).

cDNA sequences were amplified using primers for target genes. Amplified fragments were cloned into the pZErO-2 vector. Templates for probe synthesis were amplified by PCR using M13 forward (5′-GTAAAACGACGGCCAGT-3′) and M13 reverse (5′-CAGGAAACAGCTATGAC-3′) primers. Using the templates, we prepared DIG-labelled antisense and sense probes with SP6 or T7 polymerases (Roche, Basel, Switzerland) using DIG RNA Labeling Mix (Roche). Whole-mount in situ hybridisation (WISH) was performed as described previously^{27 (link),36 (link)}. For the WISH, we used embryos at the heart stage and plants at the anisocotyledonous stage in which GM and BM were evident and reproductive stage in which an inflorescence was visible. These were immersed in the fixative solution (4% (w/v) paraformaldehyde with 15% (v/v) dimethyl sulfoxide in phosphate-buffered saline with 0.1% (v/v) Tween-20) for 1 h at room temperature. DIG was detected using a DIG Detection Kit (Roche) with anti-digoxigenin antibody (Roche). Samples were visualised using a MZ16F stereomicroscope (Leica Microsystems, Wetzlar, Germany) or an OM-D E-M10 digital camera (Olympus, Tokyo, Japan) with a SZ61 dissecting microscope (Olympus).