Transcriptome Analysis of Plant Tissues

For expression analyses, stigmas, corms, roots, leaves, stamens and tepals were dissected from ten plants and the same kind of tissues were pooled. Three independent pools of these tissues were used as three biological replicas. TRIzol reagent (Invitrogen) was utilized for RNA extraction. 1 µg of total RNA was used for first-strand cDNAs by reverse transcription (RT) using an oligo dT primer and a First-strand cDNA Synthesis Kit (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer’s instructions. The cDNAs were used as templates using gene-specific primers (Table S1). The cycling parameters of qPCR consisted of an initial denaturation at 94 °C for 5 min, 40 cycles at 94 °C for 20 s, 58 °C for 20 s, 72 °C for 20 s, and a final extension at 72 °C for 5 min. The assays were conducted in a StepOne™ Thermal Cycler (Applied Biosystems, Foster City, CA, USA) and analyzed using StepOne software v2.0 (Applied Biosystems, Foster City, CA, USA). DNA melt curves were created for each primer combination in order to confirm the presence of a single product. Gene expression was calculated using the 2^−ΔΔCT method. The expression data were normalized based on CsRP18S [8 (link),59 (link)].