Quantitative analysis by qRT-PCR
We firstly analyzed the tissue specificity of the expression of genes
related to SLs signaling-mediated tillering in S. alterniflora .
The genes including D10 and D17 which involved in SLs
biosynthesis, and D14 and D53 which involved in SLs
signaling, were selected. Six tissues including stem, basal node of the
main stem, root, rhizome, bud and leaf were used to perform the gene
expression analysis. Based on the results of above experiment, we
further analyzed the expression level of D10 , D17 in roots
and D14 , D53 in nodes under different salinities. A 20 μL
of qRT-PCR mixture containing 7.4 μL ddH2O, 10 μL
Fast-start universal SYBR Green Master (ROX, Mannheim, Germany), 1 μL
cDNA and 0.8 μL forward and reverse primers was used. The condition of
qRT-PCR was performed as followed: initially denatured at
95oC for 10 min, 45 cycles of 95 oC
for 15 sec, the appropriate annealing temperature for 15 sec
(Supplementary Table S4) and 72 oC for 15 sec and the
protocol of thermal dissociation was run from 60 to 90oC. At least three biological replicates were
conducted for each treatment. Relative expression levels of genes were
calculated by 2-ΔΔCT method and SaTublin was
used as the reference gene.