Figure 1.
Gene specificity and amplicon size.
(a) Agarose gel (2%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene. (b) Melting curves of 11 reference genes and 2 target genes showing single peaks. M1 and M2 represent 2000 bp and 100 bp DNA ladder marker respectively.
Table 1.
Candidate reference genes, primers and different parameters derived from RT-qPCR analysis.
Figure 2.
RT-qPCR Ct values for reference genes.
Expression data displayed as Ct values for each reference gene in all tung samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles, whisker caps represent the maximum and minimum values, dots represent outliers.
Figure 3.
Average expression stability values (M) calculated by geNorm.
(a) all 31 samples, (b) different tissue/organs, (c) the cultivar “Jinhua”, (d) the cultivar “Jiangchengxu 79-9”, (e) the cultivar “Henglu 20”, (f) the cultivar “Chengjiaxu 9–24”, (g) four cultivars from 9 September. Lower average expression stability (M value) indicates more stable expression.
Figure 4.
Pairwise variation (V) calculated by geNorm to determine the optimal number of reference genes.
The average pairwise variations Vn/Vn+1 was analyzed between the normalization factors NFn and NFn+1 to indicate the optimal number of reference genes required for RT-qPCR data normalization in different samples.
Table 2.
Ranking of candidate reference genes in order of their expression stability as calculated by NormFinder.
Table 3.
Statistics results by BestKeeper software for ten selected genes based on Ct values.
Figure 5.
Expression levels of FAD2 and FADX in different tissues/organs and seeds development of the cultivar “Jiangchengxu 79-9”.
(a1 and a2) Expression levels of FAD2 in different tissues/organs and seeds development, (b1 and b2) Expression levels of FADX in different tissues/organs and seeds development. Genes were normalized to individual and/or combined reference genes. Error bars show mean standard error calculated from two biological replicates.