Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

doi:10.1016/j.rsci.2019.04.005

Abstract

Abstract:

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R² of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

Key words: endogenous reference gene, rice, genetically modified crop, phosphoenolpyruvate carboxylase gene, sucrose-phosphate synthase gene, phospholipase D gene, starch branching enzyme 4 gene, rice root-specific GOS9 gene

Xiujie Zhang, Wujun Jin, Wentao Xu, Xiaying Li, Ying Shang, Sha Li, Hongsheng Ouyang. Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice[J]. Rice Science, 2019, 26(4): 248-256.

Figures/Tables 12

References 21

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Species	GM variety	Non-GM variety
Rice	Huahui 1, II Kefeng 6, Kefeng 6, Bar 68-1, Wan 21B, Kangyou 97	II Minghui 86, Minghui 86, Minghui 63, Shanyou 10, Shanyou 63, 80-4B, Donglong, Songjing 6, Zhonghua 11, Zhongzuo 58, Sanjiang 1, Liaojing 371
Cotton	LLcotton25, Mon15985, Mon1445, Mon88913	Non-GM variety
Maize	Bt176, TC1507, Mon810, Mon863	Non-GM variety
Soybean	Mon89788, A5547-127, GTS40-3-2	Non-GM variety
Beet	H7-1	Non-GM variety
Rapeseed	GT73, ToPas19/2
Papaya	Huanong 1
Tomato		Tomato
Potato		Potato
Gramineous plant		Wheat, oats, barley, mung bean, buckwheat
Model plant		Arabidopsis, cauliflower, tobacco
GM, Genetically modified.

Species	GM variety	Non-GM variety
Rice	Huahui 1, II Kefeng 6, Kefeng 6, Bar 68-1, Wan 21B, Kangyou 97	II Minghui 86, Minghui 86, Minghui 63, Shanyou 10, Shanyou 63, 80-4B, Donglong, Songjing 6, Zhonghua 11, Zhongzuo 58, Sanjiang 1, Liaojing 371
Cotton	LLcotton25, Mon15985, Mon1445, Mon88913	Non-GM variety
Maize	Bt176, TC1507, Mon810, Mon863	Non-GM variety
Soybean	Mon89788, A5547-127, GTS40-3-2	Non-GM variety
Beet	H7-1	Non-GM variety
Rapeseed	GT73, ToPas19/2
Papaya	Huanong 1
Tomato		Tomato
Potato		Potato
Gramineous plant		Wheat, oats, barley, mung bean, buckwheat
Model plant		Arabidopsis, cauliflower, tobacco
GM, Genetically modified.

Gene	Primer	Primer sequence (5′-3′)	Product size (bp)	Annealing temperature (ºC)	Reference
PLD	PLD-F PLD-R PLD-P	TGGTGAGCGTTTTGCAGTCT CTGATCCACTAGCAGGAGGTCC FAM-GTTGTGCTGCCAATGTGGCCTG-TAMRA	68	58	JRC, 2006
	PLD-F1 PLD-R1	CGGCTTGGGATGTGCTCTA TGGATGCTCCTGTCAATGGTG	268	54
	PLD-F2 PLD-R2	CTGAGTGTCGTCTGTGGGTGTTCC CCGTGCTAAAGATGGGTGAGG	245	58
	PLD-F3 PLD-R3	GAACAACACCATTGACAGGAGCAT CCACATTGGCAGCACAACATAGA	241	56
SPS	SPS-F SPS-R SPS-P	CGGTTGATCTTTTCGGGATG TTGCGCCTGAACGGATAT FAM-GACGCACGGACGGCTCGGA-TAMRA	81	54	Ding et al, 2004
	SPS-F1 SPS-R1	TTGCGCCTGAACGGATAT GGAGAAGCACTGGACGAGG	277	54	Jiang et al, 2009
	SPS-F2 SPS-R2	GCCATGGATTACATATGGCAAGA ATCTGTTTACTCGTCAAGTGTCATCTC	287	56
RBE4	RBE4-F RBE4-R RBE4-P	GTTTTAGTTGGGTGAAAGCGGTT CCTGTTAGTTCTTCCAATGCCCTTA FAM-TCTGGTTGGGAATAGATACT-MGBNFQ	106	54	Jeong et al, 2007
	RBE4-F1 RBE4-R1	TCCAATGCCCTTAAACTTTATCA AACGATCTAATTCTGGGCTTTAT	206	54	Jeong et al, 2007
	RBE4-F2 RBE4-R2	AGGGCATTGGAAGAACTAACAG CACCACCATCTTGAACAGGAAT	264	54
	RBE4-F3 RBE4-R3	TAGTTGGGTGAAAGCGGTTAG CACCACCATCTTGAACAGGAAT	343	54
GOS9	GOS9-F GOS9-R GOS9-P	TTAGCCTCCCGCTGCAGA AGAGTCCACAAGTGCTCCCG FAM-CGGCAGTGTGGTTGGTTTCTTCGG-TAMRA	68	56	Hernández et al, 2005
	GOS9-F1 GOS9-R1	ACTTACGGGATGGTCTTTGCA GCAATCAATCCATTACCTGCTC	255	54
	GOS9-F2 GOS9-R2	TAAGTTGCTTCGACGTACAGGA TAGATGACGGTGACGAGGATAGAGT	303	56
	GOS9-F3 GOS9-R3	GCTGCCCGTTAATACATGTTCCT CTAAAAGCAGTTCCACCACCTTGT	301	56
PEPC	PEPC-F PEPC-R PEPC-P	TAGGAATCACGGATACGCA TGAACTCAGGTTGCTGGAC FAM-AGGGAGATCCTTACTTGAGGCAGAGAC-TAMRA	80	54
	PEPC-F1 PEPC-R1	GGTCCCTGATACGCTTTA TCTGCCCACTCACTGCTA	153	54
	PEPC-F2 PEPC-R2	GCCGTTGTCGCTTGTTGTGTTC CAGAGACTGCGTATCCGTGATTCCT	183	60
	PEPC-F3 PEPC-R3	TCCCTCCAGAAGGTCTTTGTGTC GCTGGCAACTGGTTGGTAATG	271	56

Gene	Primer	Primer sequence (5′-3′)	Product size (bp)	Annealing temperature (ºC)	Reference
PLD	PLD-F PLD-R PLD-P	TGGTGAGCGTTTTGCAGTCT CTGATCCACTAGCAGGAGGTCC FAM-GTTGTGCTGCCAATGTGGCCTG-TAMRA	68	58	JRC, 2006
	PLD-F1 PLD-R1	CGGCTTGGGATGTGCTCTA TGGATGCTCCTGTCAATGGTG	268	54
	PLD-F2 PLD-R2	CTGAGTGTCGTCTGTGGGTGTTCC CCGTGCTAAAGATGGGTGAGG	245	58
	PLD-F3 PLD-R3	GAACAACACCATTGACAGGAGCAT CCACATTGGCAGCACAACATAGA	241	56
SPS	SPS-F SPS-R SPS-P	CGGTTGATCTTTTCGGGATG TTGCGCCTGAACGGATAT FAM-GACGCACGGACGGCTCGGA-TAMRA	81	54	Ding et al, 2004
	SPS-F1 SPS-R1	TTGCGCCTGAACGGATAT GGAGAAGCACTGGACGAGG	277	54	Jiang et al, 2009
	SPS-F2 SPS-R2	GCCATGGATTACATATGGCAAGA ATCTGTTTACTCGTCAAGTGTCATCTC	287	56
RBE4	RBE4-F RBE4-R RBE4-P	GTTTTAGTTGGGTGAAAGCGGTT CCTGTTAGTTCTTCCAATGCCCTTA FAM-TCTGGTTGGGAATAGATACT-MGBNFQ	106	54	Jeong et al, 2007
	RBE4-F1 RBE4-R1	TCCAATGCCCTTAAACTTTATCA AACGATCTAATTCTGGGCTTTAT	206	54	Jeong et al, 2007
	RBE4-F2 RBE4-R2	AGGGCATTGGAAGAACTAACAG CACCACCATCTTGAACAGGAAT	264	54
	RBE4-F3 RBE4-R3	TAGTTGGGTGAAAGCGGTTAG CACCACCATCTTGAACAGGAAT	343	54
GOS9	GOS9-F GOS9-R GOS9-P	TTAGCCTCCCGCTGCAGA AGAGTCCACAAGTGCTCCCG FAM-CGGCAGTGTGGTTGGTTTCTTCGG-TAMRA	68	56	Hernández et al, 2005
	GOS9-F1 GOS9-R1	ACTTACGGGATGGTCTTTGCA GCAATCAATCCATTACCTGCTC	255	54
	GOS9-F2 GOS9-R2	TAAGTTGCTTCGACGTACAGGA TAGATGACGGTGACGAGGATAGAGT	303	56
	GOS9-F3 GOS9-R3	GCTGCCCGTTAATACATGTTCCT CTAAAAGCAGTTCCACCACCTTGT	301	56
PEPC	PEPC-F PEPC-R PEPC-P	TAGGAATCACGGATACGCA TGAACTCAGGTTGCTGGAC FAM-AGGGAGATCCTTACTTGAGGCAGAGAC-TAMRA	80	54
	PEPC-F1 PEPC-R1	GGTCCCTGATACGCTTTA TCTGCCCACTCACTGCTA	153	54
	PEPC-F2 PEPC-R2	GCCGTTGTCGCTTGTTGTGTTC CAGAGACTGCGTATCCGTGATTCCT	183	60
	PEPC-F3 PEPC-R3	TCCCTCCAGAAGGTCTTTGTGTC GCTGGCAACTGGTTGGTAATG	271	56

	Reaction system (µL)					Reaction program
	2×FastStart Universal Probe Master	dNTP (10 Mm)	Primer Each (10 µM)	Probe (10 µM)	DNA (50 ng/μL)	Step1	Step 2		Cycles of Step 2
SPS-F/R/P	10	0.4	0.8	0.4	1.0	95 °C 10 min	95 °C 15 s	60 °C 60 s	40
PLD-F/R/P	10	0.4	0.4	0.4	1.0	95 °C 10 min	95 °C 15 s	60 °C 60 s	45
RBE4-F/R/P	10	1.0	1.0	0.4	1.0	95 °C 10 min	95 °C 30 s	60 °C 60 s	40
GOS-F/R/P	10	1.6	0.6	0.4	1.0	95 °C 10 min	95 °C 30 s	60 °C 60 s	40
PEPC-F/R/P	10	0.4	0.8	0.4	1.0	95 °C 10 min	95 °C 15 s	60 °C 60 s	40