Selection and validation of reference genes for qRT-PCR in cultivated octoploid strawberry

Jianxin Mao; Jiqi Li; Yan Wang; Zhihong Zhang; Jianxin Mao; Jiqi Li; Yan Wang; Zhihong Zhang

doi:10.48130/frures-0024-0003

Figures (6) Tables (2)

Figure 1.
Expression stability value of five candidate reference genes calculated by geNorm in 'Yanli' and 'Chuliandeweidao'. (a) M value of five candidate reference genes. A lower M-value indicates more stable gene expression. (b) Pairwise variation (V) analysis of five candidate reference genes. A lower value indicates a more stable combination number of reference genes.
Figure 2.
Expression stability value of five candidate reference genes calculated by NormFinder in 'Yanli' and 'Chuliandeweidao'. A lower value indicates more stable gene expression.
Figure 3.
Expression stability value of five candidate reference genes calculated by BestKeeper in 'Yanli' and 'Chuliandeweidao'. A lower value indicates more stable gene expression.
Figure 4.
Expression stability value of five candidate reference genes calculated by delta-CT method in 'Yanli' and 'Chuliandeweidao'. A lower value indicates more stable gene expression.
Figure 5.
Relative expression levels of FaMYB10, FaUGT1, and FaCHS in five different development stages of 'Yanli' fruit. The bars of different colors represent the relative expression levels of the validation genes calculated using different reference genes. (SG = Small Green, BG = Big Green, W = White, TR = Turning Red, R = Red).
Figure 6.
Boxplot analysis of the expression profiles of FaADPrf1, FaGAPC2, and Fa26S rRNA in root, crown stem, leaf, flower, and five fruit developmental stages. Solid dots represent the expression cycle threshold (CT) values of candidate reference genes in different organs. The line across the box represents the median. The boxes represent the 25/75 percentiles.

Gene symbol	Gene name	Primer sequence (5'-3')	Amplification length (bp)	Amplification efficiency (%)	Correlation coefficiency (R²)
ADPrf1	ADP–ribosylation factor 1	F: 5'-TGCGAATTCTGATGGTCGGT-3' R: 5'-CTCCACAATGGACGGATCTT-3'	144 bp	95.43%	0.9984
GAPC2	Glyceraldehyde-3-phosphate dehydrogenase	F: 5'-GAATCAACGGATTCGGAAGA-3' R: 5'-ACAATATCGGCACCAACTGA-3'	231 bp	101.51%	0.9996
EF1-α	Elongation factor 1 - alpha	F: 5'-CACATCAACATTGTGGTCAT-3' R: 5'-GTCTCAAACTTCCACAAGGC-3'	187 bp	99.69%	0.9984
PPC1	Peptidyl-prolyl cis-trans isomerase 1	F: 5'-TACAAGGGATCGTCCTTCCA-3' R: 5'-ACCCAACCTTCTCGATGTTC-3'	258 bp	89.21%	0.9955
26S rRNA	26S ribosomal RNA	F: 5'-TAACCGCATCAGGTCTCCAA-3' R: 5'-CTCGAGCAGTTCTCCGACAG-3'	132 bp	95.69%	0.9996

Table 1.

Primers sequence and amplification characteristics of five candidate reference genes.

Rank Yanli Chuliandeweidao
Gene name Ranking value Gene name Ranking value

1 GAPC2 1.32 GAPC2 1.00
2 ADPrf1 1.86 ADPrf1 1.68
3 EF1-α 2.59 26S 3.22
4 26S 3.98 EF1-α 3.72
5 PPC1 4.00 PPC1 5.00

Table 2.
The comprehensive ranking of five candidate reference genes in 'Yanli' and 'Chuliandeweidao' analyzed by RefFinder.

Rank	Yanli		Chuliandeweidao
Rank	Gene name	Ranking value	Gene name	Ranking value
1	GAPC2	1.32	GAPC2	1.00
2	ADPrf1	1.86	ADPrf1	1.68
3	EF1-α	2.59	26S	3.22
4	26S	3.98	EF1-α	3.72
5	PPC1	4.00	PPC1	5.00