Selection and evaluation of reference genes for expression analysis of Cassi

Figures & data

Table 1. Details of primers and parameters derived from qRT-PCR in this study.

Abbreviation	Gene name	Forward/reverse primer (5′-3′)	Amplicon length (bp)	Efficiency	R^2
ACT1	Actin 1	TAACACTCCAGCCATGTACGTC/GTGGTGAAAGAGTATCCACGCT	167	1.969	0.996
ACT2	Actin 2	TTGATTGGAATGGAAGCAGCAG/CTGTACTTTCTCTCGGGTGGTG	211	1.943	0.991
CYP1	Cyclophilin 1	TGTGTCAGGGAGGAGATTTCAC/AACTGAGAACCATTCGTGTTGG	154	1.912	0.996
CYP2	Cyclophilin 2	CACTACAAGGGCTCCACCTTC/CCGTGCAGATGAAGAACTGAGA	114	1.959	0.994
EF1α2	Elongation factor 1α2	TTGAAGAACGGTGATGCAGGTA/CACACTCTTGATGACTCCCACA	237	1.918	0.993
UBQ1	Ubiquitin 1	CCGTACTCTTGCCGATTACAAC/GATCTTCTTCTTTGGCCTCAGC	152	1.942	0.992
UBQ2	Ubiquitin 2	TTGCGACTTCGAGGTGGTATG/CACGAAGACGGAGGACAAGATG	195	1.872	0.992
α-TUB1	α-Tubulin 1	TCGCATTGACCACAAGTTTGAC/GCTCCCACTTCTTCGTAATCCT	218	1.908	0.993
α-TUB2	α-Tubulin 2	TGACATTGACCGACCAACCTAC/TATGCCTTCTCTGCCGAAATGA	225	1.919	0.992
β-TUB2	β-Tubulin 2	TCAACGTGCAGAACAAGAACTC/CTGCTCACTAACCCTCCTGAAC	223	1.968	0.996
WRKY	WRKY transcription factor	TGGAGGAAGTACGGACAGAAAC/GAGAGGGAGTGGTTATGATCGC	144	1.927	0.995

Fig. 1. Cycle threshold (Ct) values of the candidate reference genes across the experimental samples.

Notes: The box represented the 25th and 75th percentiles of data. Whiskers represented the maximum and minimum values. Horizontal line within the box represented the median Ct value.

Fig. 2. Average expression stability (M) values of candidate reference genes by geNorm.

Notes: (A) The M values calculated for all plant samples. (B) The M values calculated for 12 various tissues. (C) The M values calculated under ABA treatment. (D) The M values calculated under cold stress. (E) The M values calculated under PEG treatment. (F) The M values calculated under salt stress.

Fig. 3. Pairwise variation analysis of the candidate reference genes.

Notes: Pairwise variation (V_n/V_n+1) was analyzed between the normalization factors NF_n and NF_n+1. Arrows indicated the optimal number of reference genes required for normalization.

Table 2. Ranking of the candidate reference genes according to their stability estimated by NormFinder.

Rank	Total		Tissue		ABA		Cold		PEG		Salt
	Gene	Stability	Gene	Stability	Gene	Stability	Gene	Stability	Gene	Stability	Gene	Stability
1	ACT2	0.006	CYP1	0.014	CYP1	0.011	EF1α2	0.008	UBQ1	0.007	EF1α2	0.007
2	UBQ1	0.017	UBQ1	0.024	β-TUB2	0.011	CYP2	0.010	CYP1	0.010	CYP1	0.024
3	CYP1	0.020	ACT2	0.024	EF1α2	0.012	UBQ1	0.011	ACT2	0.021	UBQ1	0.025
4	EF1α2	0.030	EF1α2	0.025	ACT2	0.015	ACT2	0.035	EF1α2	0.051	β-TUB2	0.028
5	α-TUB2	0.089	ACT1	0.041	UBQ1	0.023	CYP1	0.036	ACT1	0.082	ACT2	0.030
6	ACT1	0.098	α-TUB2	0.043	α-TUB2	0.063	β-TUB2	0.058	α-TUB1	0.143	α-TUB2	0.071
7	α-TUB1	0.105	β-TUB2	0.046	ACT1	0.072	α-TUB2	0.066	UBQ2	0.161	ACT1	0.071
8	β-TUB2	0.107	CYP2	0.051	α-TUB1	0.079	ACT1	0.072	CYP2	0.161	α-TUB1	0.074
9	CYP2	0.122	α-TUB1	0.054	CYP2	0.093	UBQ2	0.104	α-TUB2	0.163	UBQ2	0.087
10	UBQ2	0.147	UBQ2	0.062	UBQ2	0.148	α-TUB1	0.112	β-TUB2	0.221	CYP2	0.095

Table 3. Ranking of the candidate reference genes according to their stability calculated by BestKeeper.

Rank	Total		Tissue		ABA		Cold		PEG		Salt
	Gene	CV ± SD	Gene	CV ± SD	Gene	CV ± SD	Gene	CV ± SD	Gene	CV ± SD	Gene	CV ± SD
1	UBQ1	3.162 ± 0.737	UBQ1	2.104 ± 0.468	ACT2	1.376 ± 0.307	UBQ1	2.417 ± 0.546	CYP1	2.902 ± 0.727	EF1α2	3.318 ± 0.696
2	EF1α2	3.540 ± 0.757	CYP1	2.781 ± 0.677	β-TUB2	1.550 ± 0.355	ACT2	2.501 ± 0.587	EF1α2	3.057 ± 0.748	CYP1	3.321 ± 0.772
3	CYP1	3.779 ± 0.872	ACT2	3.127 ± 0.743	EF1α2	1.818 ± 0.350	EF1α2	2.998 ± 0.663	UBQ1	3.378 ± 0.780	UBQ1	3.870 ± 0.904
4	ACT2	4.217 ± 0.827	CYP2	3.618 ± 0.848	CYP1	2.376 ± 0.528	CYP2	3.246 ± 0.725	ACT2	3.441 ± 0.793	ACT2	3.920 ± 0.785
5	α-TUB2	4.708 ± 1.389	EF1α2	3.689 ± 0.858	UBQ1	2.849 ± 0.483	CYP1	4.075 ± 0.729	ACT1	4.202 ± 0.875	CYP2	4.838 ± 1.520
6	ACT1	5.123 ± 1.370	UBQ2	3.999 ± 0.744	α-TUB1	3.640 ± 0.984	UBQ2	4.200 ± 1.281	α-TUB2	5.771 ± 1.435	α-TUB1	5.209 ± 1.088
7	CYP2	6.940 ± 1.446	β-TUB2	4.085 ± 0.930	CYP2	3.782 ± 0.669	β-TUB2	4.237 ± 0.752	α-TUB1	7.592 ± 1.535	β-TUB2	5.595 ± 1.057
8	α-TUB1	7.365 ± 1.710	α-TUB1	4.341 ± 1.162	α-TUB2	3.823 ± 1.151	α-TUB1	4.531 ± 1.217	CYP2	8.401 ± 1.842	UBQ2	5.759 ± 0.946
9	β-TUB2	7.631 ± 1.427	ACT1	4.437 ± 1.054	ACT1	5.202 ± 1.450	α-TUB2	4.642 ± 0.872	UBQ2	9.494 ± 1.684	ACT1	5.925 ± 1.666
10	UBQ2	7.804 ± 1.405	α-TUB2	5.352 ± 1.046	UBQ2	6.021 ± 0.951	ACT1	6.464 ± 1.053	β-TUB2	10.383 ± 2.134	α-TUB2	6.660 ± 1.493

Table 4. Ranking of candidate reference gene stability according to geNorm, NormFinder, and BestKeeper.

Various samples	Most stable genes	Most stable two genes combination (geNorm)	The least stable genes
Total	CYP1, EF1α2, ACT2, UBQ1	CYP1/EF1α2	UBQ2
Various tissues	EF1α2, UBQ1, CYP1, ACT2	EF1α2/UBQ1	UBQ2, α-TUB2
ABA	EF1α2, β-TUB2, CYP1	EF1α2/β-TUB2	UBQ2
Cold	UBQ1, CYP2, EF1α2	UBQ1/CYP2	UBQ2, α-TUB1, ACT1
PEG	ACT2, UBQ1, CYP1, EF1α2	ACT2/UBQ1	β-TUB2
Salt	EF1α2, UBQ1, CYP1	EF1α2/UBQ1	UBQ2, CYP2, α-TUB2

Fig. 4. The relative transcript level of WRKY gene in 12 tissues and under different stress normalized by the selected reference genes.

Notes: (A) The relative transcript level of WRKY gene in 12 tissues. (B) The relative transcript level of WRKY gene under ABA treatment. (C) The relative transcript level of WRKY gene under cold stress. (D) The relative transcript level of WRKY gene under PEG treatment. (E) The relative transcript level of WRKY gene under salt stress.