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.