Lightcycler 480 real time sybr green pcr system

Expression analysis of DA1 was checked by qRT-PCR. Total RNA was extracted from each repeat using Trizol (Life Technologies, Invitrogen) and DNA was removed by RQ1 DNase (Promega) treatment. Preparation of cDNA was performed using the iScript cDNA synthesis kit (BioRad) according to the manufacturer's recommendations starting with 1µg of RNA. qRT-PCR was performed with the LightCycler 480 Real-Time SYBR Green PCR System (Roche), used primers are listed in Table S2. For qPCR on maize samples, we used 18S RNA as the housekeeping gene.

RNA was isolated from the respective tissues with the RNeasy isolation kit (Qiagen).
DNase treatment with the RQ1 RNase-Free DNase (Promega) was performed before cDNA synthesis with the iScript cDNA Synthesis Kit (Bio-Rad). Relative expression levels were determined by qRT-PCR with the LightCycler 480 Real-Time SYBR Green PCR System (Roche). The primers used are described in Supplementary Table 3. The RPS26C and EMB2386 reference genes were used for normalization. In three biological repetitions, total RNA was isolated by means of the RNeasy Plant mini kit (Qiagen). For the root tips, seedlings were sown and grown for 5 days on nylon meshes (Prosep) and subsequently harvested using a scalpel. Quantitative PCR data were analyzed using the 2(-ΔΔCt) method.

Plant material was harvested at the indicated time points for DNA or RNA extraction with the CTAB method (Clarke, 2009) or an RNeasy plant mini kit (Qiagen), respectively. qRT-PCR experiments were performed in a Light-Cycler480 Real-Time SYBR green PCR system (Roche). For gene expression studies, 500 ng or 1 mg of RNA was reverse-transcribed with the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's specifications. qRT-PCR results were normalized against three reference genes (AT1G13320, AT2G32170, and AT2G28390), except for the mitochondrial gene expression analysis for which five nuclear genes (RPL5B, YSL8, UBQ, TUBULIN, and ACTIN) were used as a reference (Delannoy et al., 2015) . For quantification of the mitochondrial copy number and the accumulation of recombination products, published primer pairs and reference genes were used (Miller-Messmer et al., 2012; Wallet et al., 2015) . For each experiment, at least two technical replicates were performed on the same biological replicate; the number of biological replicates is indicated for each experiment in the figure legends. Each biological replicate consists of multiple plants grown under the same conditions; harvesting and further sample handling were done in separate tubes for the indicated tissues and/or time points. All primers used in this study are listed in Supplemental Data Set 2.

RNA was isolated from 2-week-old Arabidopsis T3 seedlings (consisting of four pools of five seedlings for the AtNUDX7 overexpression lines and three pools of five seedlings for the Atnudx7-1 mutant line) and from 10-to 12-day-old division zone tissue of the 4th leaf of T1 maize (consisting of five pools of three transgenic (+) and the same for the azygous (-) maize seedlings) with the RNeasy Plant Mini Kit (Qiagen) and the cDNA prepared with the SuperScript III First-Strand Synthesis System for reverse-transcription PCR (Invitrogen), according to the manufacturers' protocols. qPCR experiments were carried out in a LightCycler480 Real-Time SYBR Green PCR System (Roche) and all reactions were done in three technical replicates. For the Arabidopsis samples, the expression levels were normalized to the reference genes SAND (AT2G28390), PP2A (AT1G13320), and YLS8 (AT5G08290), whereas for the maize samples, the expression levels were normalized to the reference genes 18SrRNA and EF1a (GenBank accession X00794.1 and NM_001112117.1, respectively).