Selection and Validation of Reference Genes for RT-qPCR of Flower Development in Calanthe triplicata

doi:10.16420/j.issn.0513-353x.2023-0954

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (9): 2063-2074.doi: 10.16420/j.issn.0513-353x.2023-0954

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Selection and Validation of Reference Genes for RT-qPCR of Flower Development in Calanthe triplicata

WU You¹, JI Hongyu¹, DENG Shuwen¹, LIANG Cuiyi¹, WAN Zhiting², WU Shasha¹, ZHAI Junwen¹^,^*()

¹ Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization，College of Landscape Architecture and Art，Fujian Agriculture and Forestry University，Fuzhou 350002，China
² College of Landscape Architecture，Beijing Forestry University，Beijing 100083，China

Received:2024-01-24 Revised:2024-05-17 Online:2024-09-25 Published:2024-09-19
Contact: ZHAI Junwen

Abstract

Abstract:

To facilitate studies on gene expression in Calanthe triplicata，nine candidate reference genes were identified and evaluated across three different groups（the whole flower group，the floral structures group and the mixed group）using geNorm，NormFinder，BestKeeper and RefFinder. The results showed although there were slight differences in the ranking of RGs between groups，UBQ1 and UBQ2 were the most stable. To valid the accuracy of the evaluation results，four target genes related to flower development were amplified by RT-qPCR，the results further supported that UBQ1 and UBQ2 were the most optimal for reference genes.

Key words: Calanthe triplicata, RT-qPCR, reference gene, stability evaluation

WU You, JI Hongyu, DENG Shuwen, LIANG Cuiyi, WAN Zhiting, WU Shasha, ZHAI Junwen. Selection and Validation of Reference Genes for RT-qPCR of Flower Development in Calanthe triplicata[J]. Acta Horticulturae Sinica, 2024, 51(9): 2063-2074.

Figures/Tables 11

References 44

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基因 Gene	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature	扩增长度/bp Amplicon length	扩增效率/% PCR efficiency	R²	序列号 Accession
ACTB	GCTTTCCCTTTATGCGAGT CATCAGTCAAATCCCTACCAG	57.0 57.1	145	98.72	0.9996	MZ005277
ADF	GGAGAAAACTGGAGGTGCT TGCGTCACAAAATCAAAATC	56.9 57.2	101	93.41	0.9964	MZ005280
EF1-α	TCATCATCGACTCCACCAC TTGCATCCATCTTATTGCAG	57.8 57.3	131	99.97	0.9996	MZ005274
GAPDH	GCGAGTCAGAACATCATCC GGCAAGTCAAGTCCACAAC	56.6 56.6	138	98.04	0.9992	MZ005278
PP2A	TCCGGTTGTTTGAATATGG GATAGGAGGAGGCAGATGG	56.8 57.2	108	101.76	0.9988	MZ005275
TIP41	TGACTACACATTCACGACACC TCACAATCCTCCCAGTTTG	57.0 57.0	107	92.42	0.9982	MZ005279
TUB	GCCTCACCCAGACTTTTATG TGTATGCCTTGCTTCCTTG	57.3 57.4	128	90.41	0.9901	MZ005276
UBQ1	CACCTTGTTCTCCGTCTCA GATGCCCTCCTTATCTTGG	57.2 57.1	97	103.44	0.9950	MZ005281
UBQ2	CCACTCTTCATCTGGTGCT GAATGCCCTCCTTATCCTG	57.7 57.4	143	98.16	0.9973	MZ005282

基因 Gene	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature	扩增长度/bp Amplicon length	扩增效率/% PCR efficiency	R²	序列号 Accession
ACTB	GCTTTCCCTTTATGCGAGT CATCAGTCAAATCCCTACCAG	57.0 57.1	145	98.72	0.9996	MZ005277
ADF	GGAGAAAACTGGAGGTGCT TGCGTCACAAAATCAAAATC	56.9 57.2	101	93.41	0.9964	MZ005280
EF1-α	TCATCATCGACTCCACCAC TTGCATCCATCTTATTGCAG	57.8 57.3	131	99.97	0.9996	MZ005274
GAPDH	GCGAGTCAGAACATCATCC GGCAAGTCAAGTCCACAAC	56.6 56.6	138	98.04	0.9992	MZ005278
PP2A	TCCGGTTGTTTGAATATGG GATAGGAGGAGGCAGATGG	56.8 57.2	108	101.76	0.9988	MZ005275
TIP41	TGACTACACATTCACGACACC TCACAATCCTCCCAGTTTG	57.0 57.0	107	92.42	0.9982	MZ005279
TUB	GCCTCACCCAGACTTTTATG TGTATGCCTTGCTTCCTTG	57.3 57.4	128	90.41	0.9901	MZ005276
UBQ1	CACCTTGTTCTCCGTCTCA GATGCCCTCCTTATCTTGG	57.2 57.1	97	103.44	0.9950	MZ005281
UBQ2	CCACTCTTCATCTGGTGCT GAATGCCCTCCTTATCCTG	57.7 57.4	143	98.16	0.9973	MZ005282

基因 Gene	引物序列（5′-3′） Primer sequence
CtARF3	F：TGTATCCTTCGTTGTTCCCT；R：ATCTTCCCTGAGCCACATT
CtARF16	F：AAAGCTCAAGTGGTCGGAT；R：AAAGCCTCTCTAAGCTCCTCA
CtARF17	F：GAAGACACCGAACTAAGGCT；R：CCTCTCGAACTCAGAATCATC
CtARF22	F：GTGATTTGTCGGGTCGTAG；R：TCCACTTCTTCCCCTTCTC

基因 Gene	引物序列（5′-3′） Primer sequence
CtARF3	F：TGTATCCTTCGTTGTTCCCT；R：ATCTTCCCTGAGCCACATT
CtARF16	F：AAAGCTCAAGTGGTCGGAT；R：AAAGCCTCTCTAAGCTCCTCA
CtARF17	F：GAAGACACCGAACTAAGGCT；R：CCTCTCGAACTCAGAATCATC
CtARF22	F：GTGATTTGTCGGGTCGTAG；R：TCCACTTCTTCCCCTTCTC

排名 Rank	整花组 Flower group		花部结构组 Floral structure group		混合组 Mixed group
排名 Rank	基因 Gene	M	基因 Gene	M	基因 Gene	M
1	UBQ1	0.004	UBQ1	0.024	UBQ1	0.022
2	UBQ2	0.004	UBQ2	0.024	UBQ2	0.022
3	ADF	0.013	ADF	0.069	PP2A	0.073
4	ACTB	0.029	PP2A	0.083	EF1-α	0.084
5	TUB	0.048	EF1-α	0.089	TUB	0.087
6	PP2A	0.055	TUB	0.092	GAPDH	0.094
7	EF1-α	0.059	GAPDH	0.098	ACTB	0.098
8	GAPDH	0.063	ACTB	0.102	TIP41	0.113
9	TIP41	0.085	TIP41	0.114	ADF	0.129

Selection and Validation of Reference Genes for RT-qPCR of Flower Development in Calanthe triplicata

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排名 Rank	整花组 Flower group				花部结构组 Floral structure group				混合组 Mixed group
排名 Rank	基因 Gene	SD	Cov	r	基因 Gene	SD	Cov	r	基因 Gene	SD	Cov	r
1	UBQ1	0.86	4.30	0.984	UBQ1	0.98	4.89	0.642	UBQ1	0.95	4.75	0.663
2	UBQ2	0.88	4.35	0.984	UBQ2	1.00	4.94	0.500	UBQ2	0.97	4.81	0.529
3	PP2A	0.50	1.38	0.411	PP2A	1.04	2.99	0.389	PP2A	1.04	2.96	0.252
4	EF1-α	0.40	1.09	0.176	EF1-α	1.12	3.17	0.567	EF1-α	1.12	3.14	0.367
5	TUB	0.21	0.58	0.137	TUB	1.20	3.41	0.219	TUB	1.15	3.24	0.085
6	ACTB	1.61	5.04	0.812	ADF	1.24	4.00	0.662	ACTB	1.63	5.06	0.667
7	GAPDH	0.98	2.84	0.017	ACTB	1.62	5.04	0.697	GAPDH	1.77	5.09	0.641
8	ADF	1.28	5.25	0.964	GAPDH	1.98	5.70	0.677	ADF	2.40	8.11	0.530
9	TIP41	1.98	6.56	0.029	TIP41	2.47	7.84	0.786	TIP41	2.46	7.88	0.710

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