2.10 RNA extraction, purification and qRT-PCR analysis
Eight candidate housekeeping genes (Kumar et al., 2013) were screened to select an appropriate reference gene for SA and SV. These eight genes have been reported on Setaria italica (Foxtail Millet), representing different functional classes and gene families (Kumar et al., 2013). These genes are: viz., 18S rRNA (18S), elongation factor-1a (EF-1a), actin2 (Act2), alpha tubulin (Tub α), beta tubulin (Tub β), translation factor (TLF), RNA polymerase II (RNA POL II), adenine phosphoribosyl transferase (APRT; Kumar et al., 2013). In a recent study on Spartina alterniflora , tubulin was used as a housekeeping gene (Karan and Subudhi, 2012a). Based on the similarity index between the sequences of each housekeeping gene in SV andSA , we obtained the highest similarity index in Tubulin alpha (Tub α), which is around 85%. In this study, we therefore selected Tub  as reference gene for qRT-PCR.
Total RNA was extracted from mature leaves using Purelink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Concentration of each RNA sample was measured using NanoDrop 2000 spectrophotometer (NanoDrop Technologies). Leaves were sampled from both species and total RNA was extracted using TRIzol Plus RNA Purification kit (Invitrogen Life Technologies, http:// www.invitrogen.com). One microgram (1 μg) of total RNA was used to synthesize first strand cDNA with SuperScript VILO cDNA Synthesis Kit (Invitrogen Life Technologies, http://www. invitrogen.com). Quantitative real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, USA) with the fist strand cDNA as a template on a Real-Time PCR System (ABI StepOnePlus, Applied Biosystems lco., USA), with the following cycling parameters: 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s. Primers for qRT-PCR were designed using Primer-Blast of the National Center for Biotechnology Information website (NCBI; https://www.ncbi.nlm.nih.gov). The primers for PTOX and Tubulin-alpha used for qPCR analysis were listed in Table S2. Relative expression of gene against housekeeping gene tubulin-alpha was calculated as: 2−ΔΔCT (ΔCT = CT, gene of interest−CT, Tubulin-alpha), as described by Livak & Schmittgen (2001). Six complete biological and technical replicates were determined for the analysis.