7300 thermocycler

Sixteen primers were designed using Primer 5 to amplify 100 to 400 bp regions within four genes related to the flower color pathway (Supplementary Table 1). Dihydroflavonol 4-Reductase (DFR) and chalcone isomerase (CHI) were both key enzymes of the flavonoid biosynthesis pathway (Nishihara et al., 2005 (link)). They play impartment roles in pigment accumulation of the seed coat and flower. The over-expression of DFR and CHI make flowers color darker (Aida et al., 2000 (link); Nishihara et al., 2005 (link)). Genomic DNA was extracted from fresh leaves of mutants and wild type plants (85-1) according to the procedure described by Beek et al. (1992) (link). For the PCR analysis, 10-μL reaction volumes were used: 2 μL of 5X PCR buffer, 0.8 μL of 2.5 mM of dNTP, 0.5 μL of 2.5 mM of both forward and reverse primers, 1 μL of 1X LC green Plus, 0.1 μL of 5U of Taq enzyme, 1 μL of 50 ng of genomic DNA, and 4.5 μL of double-distilled water. The PCR reactions were performed using a 7300 Thermocycler (Bio-Rad, USA) with the following conditions: 98°C for 30 s, 35 cycles at 98°C for 10 s, annealing at 60°C for 10 s, and an extension at 72°C for 30 s, followed by an additional denaturation step at 94°C for 30 s and a cool down to 25°C for 30 s to facilitate heteroduplex formation.

Total RNA was isolated from N. benthamiana leaves by a NucleoSpin RNA plant mini-kit (Clontech). cDNA synthesis was performed on 1 µg of total plant RNA using an oligo(dT) primer and M-MLV reverse transcriptase (Invitrogen). Quantitative reverse transcription–PCR (qRT-PCR) was performed using SYBR Green master mix (Promega), gene-specific primers (Supplementary Table S3), and 2 µl of 10-fold diluted cDNA using a Bio-Rad 7300 thermocycler. Gene expression levels were normalized to Actin expression (Gabriëls et al., 2007 (link)).