Fig 1.
Illustrative figure of each S. viridis tissue and developmental stage that was sampled.
A. Seedling stage (3–5 DAS); B. Young S. viridis, with third leaf fully expanded; C. Mature S. viridis; D. The third leaf 0.5 cm sections that comprised the leaf gradient analysis. Bar corresponds to 1.0 cm.
Table 1.
Candidate genes and their primer sequences that were selected for evaluation of expression stability using qPCR analysis on S. viridis tissues, and the sequences of two genes of interest.
Fig 2.
The assessment of the best methodology for RNA extraction for each representative tissue: seedling (A); shoot (B); root (C), inflorescence (D) and axis (E). Electropherograms were obtained using an Agilent 2100 Bioanalyzer. RNA quality is expressed as the RNA integrity number (RIN).
Fig 3.
Quantification cycle (Cq) boxplot.
Graphical representation of Cq distribution for each gene analyzed in the developmental (A) and leaf gradient (B) datasets. The line shows the distribution of the maximum and minimum Cq values, while the grey box outlines the first and third quartiles.
Table 2.
Candidate genes ranked by geNorm algorithm according to their average pairwise variation compared with all other genes.
Fig 4.
geNorm results for expression stability values (M) and ranking of the candidate reference genes.
Average expression stability values (M) of the reference genes measured during geNorm stepwise exclusion of the least stable reference genes. Both the developmental (A) and leaf gradient (B) datasets are shown; lower values of average expression stability, M, indicate more stable expression.
Fig 5.
Optimal number of control genes for an accurate normalization.
The geNorm pairwise variation Vn(n+1) was analyzed between the normalization factors (NF) for both developmental (A) and leaf gradient (B) datasets. All values are below the cutoff of 0.15.
Table 3.
S. viridis ranked reference genes and the best combination pair of genes with their stability values calculated by the NormFinder software.
Table 4.
Summary of the best normalization pair of genes for each developmental set based on geNorm and NormFinder software programs.
Fig 6.
Expression profile of the target genes SvAP3/PI and SvPEPC.
The plot corresponding to the expression profile of the MADS-box gene SvAP3/PI (magenta bars) and the enzyme SvPEPC (green bars) responsible for the initial carbon fixation on C4 photosynthesis is shown in (A). The developmental dataset was normalized using the Si034613 and Si035045 genes. It can be observed in (B) that the expression profile of SvPEPC on the leaf gradient was normalized using the gene pair that included Si021373 and Si034613. This result is compared with the transcriptome data (light blue line) for the same gene (personal communication with Todd Mockler). The small plots correspond to the same described target genes normalized with the poorly ranked reference genes: Si018607 and Si025395 for the developmental set and Si000245 and Si018607 for leaf gradient. The reference samples are indicated with an asterix.