Semi-quantitative PCR and quantitative RT-PCR analysis
In the present study, a total of 34 genes were analysed for their salinity induced expression profiles. Direct primers were used for 22 genes based on available transcriptome given by Sreeharsha et al., (2016) and Huang et al., (2012), while indirect primers were designed for 12 genes based on the available gene sequences of Glycine maxfrom SoyKB (www.soykb.org) database since the transcriptome studies showed that P. pinnata is closely related to G. max (list of primers given in Supplementary Table 7). All genes were amplified by PCR using P. pinnata cDNA. The amplicons obtained based onG. max indirect primers as well as P. pinnata direct primers were sequenced for confirming the identity of target gene. Quantitative PCR analysis was performed on Eppendorf Realplex Master Cycler (Eppendorf, Germany) using KAPASYBRFAST [Mastermix (2×) Universal; KAPA Biosystems] real-time PCR kit following manufacturer’s instructions. For relative quantification analysis, 0.25 µ g of RNA template was used, which was extracted from the pool of six biological replicates of both control and salt treated plants by using Sigma spectrumTM Total RNA kit (Sigma, USA) and cDNA synthesis was performed by using the PrimeScriptTM 1st strand cDNA synthesis kit (TAKARA, Japan). Target genes were amplified with the following cycling programme: 1 cycle at 95°C for 2 min, followed by 40 cycles of 30 s at 95°C, 30 s at 60°C and 20 s at 72°C, followed by the dissociation (melting) curve. The intensity of fluorescence was measured by using the Realplex software (Eppendorf, Germany). The limit and efficiency of each primer pair of both target and reference gene was allowed to measure accurate comparison of gene expression using the 2-∆∆CT method for relative quantification (the Applied Biosystems User Bulletin No. 2 (P/N 4303859)) (Livak & Schmittgen, 2001). The 18s rRNA was used as reference gene after confirming its consistency expression under salt stress.