Droplet Digital PCR (ddPCR)

Genotyping of single seeds: individual seeds were removed from the fruit, deposited in a 1.5ml Eppendorf tube, and grinded with a blue plastic pestle. DNA was extracted with the Mag-Bind Plant DS DNA kit (OMEGA Bio-Tek) following the manufacturer’s instructions. Elution was done with water into a 1.5ml low-binding Eppendorf tube. The DNA samples were then concentrated in a Speedvac to 20μl final volume. Total DNA was digested for 30min at 37°C with EcoRV, and then pre-amplified with specific primers for GFP (GFP-fw + GFP-rev), RFP (RFP-fw + RFP-rev), and internal control (Control-fw + Control-rev) genes together in the same reaction tube, using the ExTaq linear polymerase (Takara, RR001A) as follows: DNA 3μl, 40nM of each primer, 0.6 units Extaq in 1x buffer containing 1.5mM MgCl₂ with the following PCR protocol: 98°C x 3min, [98°C x 10sec; 59°C x 20sec; 72°C x 20sec]x15 cycles. For ddPCR, 5μl of pre-amplified DNA were used in duplex assays GFP/Control and RFP/Control. Assay conditions: 500nM primers, 200nM probes, and QX200 ddPCR Probe no dUTPS Supermix (BioRad). The PCR protocol was the manufacturer’s recommendation for Probe assays (95°C: 10min, 94°C 30s, Ramp 2.5°C/s, 60°C 1min, Ramp 2.5°C/s[40 cycles], 98°C 10min, 4°C until further process). Fluorescence was detected with the QX200 droplet digital reader (Bio-Rad), and analyzed with the provided Quanta Soft version 1.7 software. Presence of each gene was calculated relative to the endogenous control. The sum was given as 100% and the ratio of each gene was calculated as % relative to the total.

Expression analysis of CYCD1;1 on isolated embryos: seeds were removed from siliques, placed in a 1.5ml Eppendorf tube containing PBS1X, and gently pressed with a blue plastic pestle with up-and-down movements to release the embryos. Collection time did not exceed 15min. The sample was then passed through a 100μm pore-size cell strainer (CellTrics) to remove excess of debris. The flow-through, containing the embryos, was collected in a small plastic rectangular box with low walls (we used the lid of the 8-well Tissue Culture Chambers REF94.6190.802, SARSTEDT). Embryos were collected with a 50μm diameter size capillary (ES-blastocyst injection pipettes, BioMedical Instruments, BM100T-10P) and an oil micromanipulator (CellTram Vario, Eppendorf), mounted on a Leica SP2 inverted microscope. Embryos were collected in maximum 1h shifts and washed thoroughly in fresh PBS1X. The drop of PBS1X buffer containing the embryos was then ejected directly from the capillary onto a piece of parafilm to create a round-shaped droplet. The parafilm was then placed for 2min at -70°C to let the droplet freeze. Frozen droplets were collected in a 1.5ml low-binding Eppendorf tube and stored at -70°C until the extraction. For our experiment, a total of 1000 embryos around the early globular stage (with and without suspensor) were collected per replicate from wild-type and mea plants, and ddPCR was performed on biological triplicates (total of 3000 embryos per genotype). For RNA extraction, 4-6 glass beads (1.7-2.1mm diameter, ROTH A556-1) were added to each tube containing the frozen droplets with the embryos, frozen in liquid nitrogen, and grinded 3-4 times with a single-tube tissue grinder (Silamat S6). The RNA extraction was done with the Qiagen RNeasy Plant Mini extraction kit, and subsequently treated with Turbo-Dnase (Ambion) following the manufacturer’s protocol. cDNA synthesis was performed using Maxima Reverse Transcriptase (Invitrogen) and OligodT (Invitrogen) following the manufacturer’s protocol. 5μl of a 1:2 dilution of cDNA were then used for ddPCR assays of CYCD1;1/UBI21, with 100nM final concentration of each primer, in a total reaction volume of 25μl, 20 of which were used to generate droplets in 1X Master mix EVAGREEN (BIORAD). PCR conditions were according to manufacturer’s recommendation for EVAGREEN.

Primers are listed in the Table S2.