Table 1.
The ten candidate reference genes and six target genes used in this study.
Table 2.
Amplification characteristics of the ten candidate reference genes and six target genes in qRT-PCR.
Fig 1.
Distribution of quantification cycle (Cq) values for ten candidate reference genes in all samples.
Expression levels of the ten candidate reference genes were examined in all the samples in this study and shown by the boxplots. The solid line within each box represents the 50th percentile. The lower boundary and upper boundary of each box represents the 25th and 75th percentile, respectively. The line above and below the vertical dashed lines represents the maximum and minimum values, respectively. The circles represents potential outliers.
Fig 2.
The expression stability of the ten soybean reference genes across all treatments in this study.
Y-axis represents the average expression stability (M) values analyzed by geNorm. (A) Leaf samples, (B) Root samples, (C) Leaf and root samples together.
Table 3.
Rankings of the expression stability of ten candidate reference genes by RefFinder.
Fig 3.
Relative expression levels of GmALMT1 (A) and GmARI1 (B) in soybean roots under 25 μM AlCl3 (pH4.3) treatment normalized by the most (TUA4) and two least (CYP2 and ABC) stable reference genes.
The relative expression levels of two target genes relative to the control samples at corresponding time points were calculated using the 2-ΔΔCT method. Bars represent the mean ± SE (standard error) from three replications. Different letters represent significant difference (P < 0.05) at each time point based on Duncan’s multiple range test.
Fig 4.
Relative expression levels of GmHMA13 and GmHMA19 in soybean leaves and roots under 100 μM CdCl2 treatment normalized by the most (UKN2) and two least (ELF1A and Fbox) stable reference genes in leaves and the most (Fbox) and two least (ELF1A and TUB4) stable reference genes in roots.
(A) GmHMA13 in soybean leaves, (B) GmHMA19 in soybean leaves, (C) GmHMA13 in soybean roots, and (D) GmHMA19 in soybean roots. The relative expression levels of two target genes relative to the control samples at corresponding time points were calculated using the 2-ΔΔCT method. Bars represent the mean ± SE (standard error) from three replications. Different letters represent significant difference (P < 0.05) at each time point based on Duncan’s multiple range test.
Fig 5.
Relative expression levels of GmGBP1 and GmHsfA1 in soybean leaves and roots under 42°C heat stress normalized by the most (UKN2) and two least (TUB4 and CYP2) stable reference genes in leaves and the most (TUA4) and two least (60S and Fbox) stable reference genes in roots.
(A) GmGBP1 in soybean leaves, (B) GmHsfA1 in soybean leaves, (C) GmGBP1 in soybean roots, and (D) GmHsfA1 in soybean roots. The relative expression levels of two target genes relative to the control samples at corresponding time points were calculated using the 2-ΔΔCT method. Bars represent the mean ± SE (standard error) from three replications. Different letters represent significant difference (P < 0.05) at each time point based on Duncan’s multiple range test.