Table 1.
Description of the 10 candidate reference genes and primer sequences for RT-qPCR.
Table 2.
PCR amplification efficiency of each primer pair.
Figure 1.
Range of Cq values of the candidate reference genes obtained for all cDNA samples.
Each box corresponding to Act, CACs, EF-1α, GAPDH, His3, Ubq, PsaH, Sand, PP2A and ß-Tub indicates the 25% and 75% percentiles. Whiskers represent the maximum and minimum values. The median is depicted by the line across the box.
Figure 2.
Stability values of candidate reference genes (RG) calculated by different statistical methods using all cDNA samples.
Ranking of each RG (Act, CACs, EF-1α, GAPDH, His3, Ubq, PP2A, PsaH, Sand, β-Tub), calculated by geNorm, NormFinder and CV method, for all tested samples [leaves, branches (1st, 2nd, 3rd active, 3rd dormancy) and cork].
Figure 3.
Venn diagram showing the most stable genes identified by the geNorm, NormFinder and CV method.
The most stable genes were identified using data from the developmental stage and seasonal growth sample sets.
Table 3.
Stability values for the candidate RG in individual sample types.
Figure 4.
Determination of the optimal number of reference genes for normalization according to geNorm software.
Pairwaise variation (Vn/n+1) analysis between the normalization factors NFn and NFn+1, carried out for all the samples (Total), individual samples [leaves, periderm from 1-year-old (1stB), 2-year-old (2ndB) and 3-year-old branches during active growth (3rdB) or dormancy (3rdDB) and cork], developmental stage sample set (DS) and seasonal growth sample set (SG).
Figure 5.
Validation of the reference genes (RG).
Relative expression levels of GPAT5 in periderm from 3-year-old branches collected in spring (April) and summer (July). Normalization factors were calculated with RG obtained in the analysis of data from all samples (Total) by geNorm/NormFinder (A) and CV method (C) or data from periderm of 3-year-old branches (3rdB), also in geNorm/NormFinder (B) and CV method (D). The normalization factors were based on the geometric means of the two most stable genes [NF2(S)] and the two most unstable [NF2(U)].
Figure 6.
Average GPAT5 gene specific variation.
Determination of the coefficient of variation in percentage, for the two most stable, NF(2S), and the two most unstable genes, NF(2U). This analysis was performed for the RG selected from all tested samples (total) and from periderm from 3-year-old branches (3rdB), using three statistical methods (geNorm, NormFinder and CV method).