Table 1.
Expression level of the selected candidate reference genes tested in pearl millet.
Table 2.
Expression levels of candidate reference genes across four experimental sets of pearl millet.
Table 3.
Stability ranking of the reference genes in all tissue and experimental sets studied in pearl millet.
Table 4.
Stability ranking of the reference genes in developmental tissues of pearl millet.
Table 5.
Stability ranking of the reference genes in hormone treatments of pearl millet.
Table 6.
Stability ranking of the reference genes in abiotic stress conditions of pearl millet.
Table 7.
Stability ranking of the reference genes among three genotypes of pearl millet.
Table 8.
Summary of the best combination of reference genes for accurate normalization across five experimental sets of pearl millet using geNorm and RefFinder programs.
Figure 1.
Estimation of pairwise variation to determine the optimal number of control reference genes required for accurate normalization using geNorm.
Pairwise variation (V, Vn/Vn+1) was calculated between successively ranked normalization factors NFn and NFn+1. Arrowheads on the bar graph indicate the minimum number of genes required at the cut-off value 0.15 [31]. The V between the normalization factors of the two first-ranked and the three first-ranked is represented by V2/3 and so on, respectively.
Figure 2.
Validation of PEPC gene expression after normalization using optimal number of control reference genes in developmental tissue samples from genotypes (A) ICMR01004, (B) IPCI1466 and (C) IP300088.
Results are presented as mean relative expression with SD from three biological replicates after normalization using the best combination of reference genes recommended by geNorm and RefFinder (see Table 8) for developmental tissue samples. Leaf- 7D, 15D and 30D represent 7DPS, 15DPS and 30DPS leaf samples while flag leaf, sheath, node and internode are from 60DPS plants. Different letters on the bars indicate significant differences at the P≤0.05 level as tested by Tukey's Range Test.
Figure 3.
Validation of ERF gene expression after normalization using optimal number of control reference genes in hormone treated samples from genotypes (A) ICMR01004, (B) IPCI1466 and (C) IP300088.
Data are presented as mean relative expression with SD from three biological replicates after normalization using the best combination of reference genes recommended by geNorm and RefFinder (see Table 8) for hormone treatments. ABA (abscisic acid), Bra (brassinolide), GA (gibberellic acid), IAA (indole-3-acetic acid), MeJa (methyl jasmonate), SA (salicylic acid) and Zea (zeatin) treatments of 15DPG plants. Different letters on the bars indicate significant differences at the P≤0.05 level as tested by Tukey's Range Test.
Figure 4.
Validation of DREB gene expression after normalization using optimal number of control reference genes in genotypes (A) ICMR01004, (B) IPCI1466 and (C) IP300088 subjected to abiotic stress conditions.
Results are presented as mean relative expression with SD from three biological replicates after normalization using the best combination of reference genes recommended by geNorm and RefFinder (see Table 8) for abiotic stress conditions. Dehydration (mannitol), drought (no water), heat (42°C) and cold (4°C) stresses are presented. Different letters on the bars indicate significant differences at the P≤0.05 level as tested by Tukey's Range Test.
Figure 5.
Validation of expression of gus, gfp and hpt transgenes using optimal number of control reference genes in hygromycin resistant calli from genotypes (A) ICMR01004, (B) IPCI1466 and (C) IP300088 after 30 days post particle bombardment-mediated transformation using pCAMBIA1201 and pCAMBIA1302, respectively.
Results are presented as mean relative expression with SD from three biological replicates after normalization using the best combination of reference genes recommended by geNorm and RefFinder (see Table 8) for developmental tissue samples. Different letters on the bars indicate significant differences at the P≤0.05 level as tested by Tukey's Range Test.