Gene Expression Analysis Using Real-Time qPCR.
RNA extraction, cDNA synthesis, and qPCR were performed as previously described (Wakao et al., 2014). All primer pairs were confirmed as having 90–105% amplification efficiency and linear amplification within their dynamic range in experimental samples using serial dilutions of cDNA prior to experiments. Relative transcript levels were calculated by the ΔΔCt method (Livak & Schmittgen, 2001) usingPEX4 (AT5G25760) as the internal reference. PEX4 , a peroxisomal ubiquitin-conjugating enzyme, is an established RT-qPCR internal reference (Dekkers et al., 2012) and was confirmed in the RNAseq dataset to have constant expression levels in all conditions and ecotypes. Primers were designed using Primer3 (Untergasser et al., 2012) against the 3´-UTR of each gene to avoid binding to off-target paralogous genes. A single peak in melt-curve analysis with a unique melting temperature was observed for each amplicon, verifying that off-target amplification of paralogous genes was negligible.