Selection and Validation of Reference Genes for RT-qPCR in Phalaenopsis- type Dendrobium Hybrid

doi:10.16420/j.issn.0513-353x.2021-0763

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (11): 2489-2501.doi: 10.16420/j.issn.0513-353x.2021-0763

• New Technology and New Methods • Previous Articles Next Articles

Selection and Validation of Reference Genes for RT-qPCR in Phalaenopsis- type Dendrobium Hybrid

HOU Tianze¹, YI Shuangshuang²^,³, ZHANG Zhiqun²^,³, WANG Jian¹^,^*(), LI Chonghui²^,³^,^*

¹College of Horticulture，Hainan University，Haikou 570208，China
²Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China，Ministry of Agriculture and Rural affairs，Tropical Crops Genetic Resources Institute，Chinese Academy of Tropical Agricultural Sciences，Haikou 571101，China
³The Engineering Technology Research Center of Tropical Ornamental Plant Germplasm Innovation and Utilization，Hainan Province，Danzhou，Hainan 571737，China

Received:2021-10-15 Revised:2022-04-30 Online:2022-11-25 Published:2022-11-25
Contact: WANG Jian,LI Chonghui E-mail:blchh@sina.com

Abstract

Abstract:

In order to screen reference genes stably expressing in Phalaenopsis-type Dendrobium hybrid for analysis of real-time fluorescent quantitative PCR，the whole floral buds，including the sepals，petals and labellums，from various cultivars and the floral bud at different development stages of dendrobium were used as materials. The expression stability of seven candidate gene was calculated by three commonly used software（geNorm，NormFinder and BestKeeper），comprehensive stability rankings were merged by RefFinder and the selected reference genes were verified. The results suggested that the expression stability of each reference gene in dendrobium differed greatly. In floral buds of different cultivars at the same developmental stage，β-actin，TUA，and GADPH were the most stable reference genes. The expression of β-actin，TUA，and CYP were the most stable during the development of the floral buds. For different tissues of floral organ，β-actin，TUA，and GADPH were the most stable reference genes in the sepals，β-actin，CYP，and PGK were in the petals and CYP，TUA，and PGK were in the labellum.

Key words: Phalaenopsis-type Dendrobium hybrid, floral bud, developmental stage, tissues, RT-qPCR, reference gene, gene expression

CLC Number:

S682.31

HOU Tianze, YI Shuangshuang, ZHANG Zhiqun, WANG Jian, LI Chonghui. Selection and Validation of Reference Genes for RT-qPCR in Phalaenopsis- type Dendrobium Hybrid[J]. Acta Horticulturae Sinica, 2022, 49(11): 2489-2501.

Figures/Tables 11

References 46

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基因 Gene	引物序列（5′-3′） Primer sequences	产物长度/bp Product length	T_m/℃ （F/R）	E/% Amplifcation efficiency	r² Correlation coefficient
PGK	F：ATCGGTGAGGAAGTTGAGAAAAC;R：GCCAATTTCTTAGCAAACTCTGG	165	60.6/60.5	100	0.997
TUA	F：CAAAGAAGATGCAGCCAACAAC;R：AAGACCAGTGCAGTTGTCAGCTA	111	60.1/59.4	104	0.992
TUB	F：TCACGGTGAGACGGACCTG;R：TATCCGGGCGAAAAATCTGA	157	59.4/60.6	104	0.998
EF1α	F：GATGGATGCGACCACACCC;R：TCGAGAAGAGTTGGTCCCTTG	191	61.1/59.2	105	0.996
CYP	F：TCTACGCCGACACGACTCCT;R：GGTGAAAGGTAGAGCCCTTGAA	113	60.4/60.3	102	0.998
GADPH	F：AGCTGCACAACCAACTGTTTG;R：GCTCTTCCACCCCTCCAGTC	151	58.7/60.7	100	0.993
β-actin	F：GTCAGGGACATCAAGGAGAAG;R：TGGGCACCTAAATCTCTCAGC	153	60.5/60.6	95	0.993

基因 Gene	引物序列（5′-3′） Primer sequences	产物长度/bp Product length	T_m/℃ （F/R）	E/% Amplifcation efficiency	r² Correlation coefficient
PGK	F：ATCGGTGAGGAAGTTGAGAAAAC;R：GCCAATTTCTTAGCAAACTCTGG	165	60.6/60.5	100	0.997
TUA	F：CAAAGAAGATGCAGCCAACAAC;R：AAGACCAGTGCAGTTGTCAGCTA	111	60.1/59.4	104	0.992
TUB	F：TCACGGTGAGACGGACCTG;R：TATCCGGGCGAAAAATCTGA	157	59.4/60.6	104	0.998
EF1α	F：GATGGATGCGACCACACCC;R：TCGAGAAGAGTTGGTCCCTTG	191	61.1/59.2	105	0.996
CYP	F：TCTACGCCGACACGACTCCT;R：GGTGAAAGGTAGAGCCCTTGAA	113	60.4/60.3	102	0.998
GADPH	F：AGCTGCACAACCAACTGTTTG;R：GCTCTTCCACCCCTCCAGTC	151	58.7/60.7	100	0.993
β-actin	F：GTCAGGGACATCAAGGAGAAG;R：TGGGCACCTAAATCTCTCAGC	153	60.5/60.6	95	0.993

排名 Rank	萼片 Sepal			花瓣 Petal		唇瓣 Labellum		不同品种花芽 Floral bud of different cultivars		发育不同阶段花芽 Floral bud of different developmental stages
排名 Rank	内参基因 Reference gene		M值 M	内参基因 Reference gene	M值 M	内参基因 Reference gene	M值 M	内参基因 Reference gene	M值 M	内参基因 Reference gene	M值 M
1	TUA	0.840		β-actin	0.759	CYP	0.499	β-actin	0.857	β-actin	1.334
2	GADPH	0.840		CYP	0.759	GADPH	0.499	TUA	0.857	TUA	1.334
3	β-actin	0.954		TUA	0.862	β-actin	0.587	GADPH	0.879	CYP	1.434
4	PGK	1.225		GADPH	0.912	TUA	0.628	CYP	1.001	GADPH	1.461
5	CYP	1.478		PGK	0.954	PGK	0.795	PGK	1.125	PGK	1.735
6	TUB	1.813		TUB	1.180	TUB	1.142	TUB	1.441	TUB	2.040
7	EF1α	2.254		EF1α	1.788	EF1α	1.699	EF1α	1.977	EF1α	2.234

排名 Rank	萼片 Sepal			花瓣 Petal		唇瓣 Labellum		不同品种花芽 Floral bud of different cultivars		发育不同阶段花芽 Floral bud of different developmental stages
排名 Rank	内参基因 Reference gene		M值 M	内参基因 Reference gene	M值 M	内参基因 Reference gene	M值 M	内参基因 Reference gene	M值 M	内参基因 Reference gene	M值 M
1	TUA	0.840		β-actin	0.759	CYP	0.499	β-actin	0.857	β-actin	1.334
2	GADPH	0.840		CYP	0.759	GADPH	0.499	TUA	0.857	TUA	1.334
3	β-actin	0.954		TUA	0.862	β-actin	0.587	GADPH	0.879	CYP	1.434
4	PGK	1.225		GADPH	0.912	TUA	0.628	CYP	1.001	GADPH	1.461
5	CYP	1.478		PGK	0.954	PGK	0.795	PGK	1.125	PGK	1.735
6	TUB	1.813		TUB	1.180	TUB	1.142	TUB	1.441	TUB	2.040
7	EF1α	2.254		EF1α	1.788	EF1α	1.699	EF1α	1.977	EF1α	2.234

排名 Rank	萼片 Sepal		花瓣 Petal		唇瓣 Labellum		不同品种花芽 Floral bud of different cultivars		不同发育阶段花芽 Floral bud of different developmental stages
排名 Rank	内参基因 Reference gene	稳定值 Stability value	内参基因 Reference gene	稳定值 Stability value	内参基因 Reference gene	稳定值 Stability value	内参基因 Reference gene	稳定值 Stability value	内参基因 Reference gene	稳定值 Stability value
1	β-actin	0.022	β-actin	0.017	β-actin	0.021	β-actin	0.019	β-actin	0.013
2	TUA	0.022	CYP	0.032	CYP	0.022	TUA	0.032	TUA	0.034
3	PGK	0.042	TUB	0.032	TUA	0.030	PGK	0.038	PGK	0.058
4	GADPH	0.047	PGK	0.032	GADPH	0.036	GADPH	0.045	CYP	0.063
5	TUB	0.066	TUA	0.035	TUB	0.042	TUB	0.048	GADPH	0.074
6	CYP	0.100	GADPH	0.052	PGK	0.045	CYP	0.065	TUB	0.092
7	EF1α	0.179	EF1α	0.163	EF1α	0.163	EF1α	0.173	EF1α	0.121

Selection and Validation of Reference Genes for RT-qPCR in Phalaenopsis- type Dendrobium Hybrid

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排名	萼片Sepal			花瓣Petal			唇瓣Labellum
Rank	基因	标准差	变异系数/%	基因	标准差	变异系数/%	基因	标准差	变异系数/%
	Gene	SD	CV	Gene	SD	CV	Gene	SD	CV
1	β-actin	0.45	2.17	PGK	0.99	3.88	PGK	0.63	2.41
2	GADPH	0.6	2.95	CYP	0.99	4.65	GADPH	0.73	3.44
3	TUA	0.79	3.48	β-actin	1.01	4.67	CYP	0.89	4.2
4	PGK	0.9	3.61	TUA	1.04	4.56	TUA	1.09	4.76
5	CYP	1.33	6.2	GADPH	1.31	6.36	β-actin	1.16	5.43
6	TUB	1.87	7.11	TUB	1.76	6.27	EF1α	1.83	8.86
7	EF1α	2.3	13.03	EF1α	1.88	9.33	TUB	2.23	7.98
排名	不同品种花芽			不同发育阶段花芽
Rank	Floral bud of different cultivars			Floral bud of different developmental stages
	基因	标准差	变异系数/%	基因	标准差	变异系数/%
	Gene	SD	CV	Gene	SD	CV
1	β-actin	0.89	4.2	β-actin	0.85	4.02
2	TUA	0.91	3.97	PGK	0.91	3.64
3	GADPH	0.92	4.43	GADPH	0.99	4.92
4	PGK	0.95	3.73	TUA	1.04	4.59
5	CYP	1	4.7	CYP	1.14	5.44
6	TUB	1.95	7.15	TUB	1.52	5.31
7	EF1α	2.71	14.19	EF1α	1.77	8.39

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