Quantifying C4 and Salinity Genes

Transcripts of the following C₄ pathway-related genes were quantified using total RNA extracted from the first fully expanded leaf samples collected at 42 days after sowing: NADPME, (PEPC, PPDK, RBCL and RBCS and salinity tolerance related genes NHX1, VHA-C, VPPase, HKT1;4, SOS1, SOS2, SOS3, -ATPaseAHA1, HAK1and HAK5 (Liu et al. 2014 (link); Wang et al. 2016b (link)). Reverse transcription was performed as per the manufacturer’s instructions (Bioline, Australia). Quantitative real-time PCR (qPCR) was performed using a Quantinova SYBR Green Kit (QIAGEN, USA) in a Rotor-Gene 3000 quantitative PCR thermoscycler (QIAGEN, USA). Relative gene expression was calculated using the comparative threshold cycle (C_t) 2^-∆∆Ct method (Livak et al. 2001 (link)); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and elongation factor 1-alpha (EF1A) were used as the internal reference genes. The experiments were conducted with three biological replicates and three technical replicates. The primer pairs are listed in Supplementary Table S1.