RT-qPCR Protocol for Seed Shattering Genes

The expression of the genes of interest was performed using reverse transcription quantitative PCR (RT–qPCR) with a Rotor-Gene Q real-time PCR instrument (Qiagen, Germany). Specific PCR primers were designed for reference genes and target genes, using Primer Premier 6.20 [seeSupporting Information—Table S3]. A reaction volume of 15 μL was used for all qPCRs containing 1 μL of 10-fold diluted cDNA, the relevant primers and home-made SYBR Green master mix (Song et al. 2012 (link)). PCR products were either directly Sanger-sequenced or cloning-sequenced to confirm their identity. At least three technical replicates for each of the three biological replicates were carried out for each sample set. The relative expression of putative seed shattering genes was corrected using two reference genes, LpElongation Factor (LpEF) and LpGAPDH (LpGAP), and calculated using modified 2^−∆∆Ct method as described in previous studies (Pfaffl 2001 (link); Song et al. 2012 (link)).