Screening of Stable Reference Genes for qRT-PCR Analysis in Macadamia integrifolia

doi:10.3969/j.issn.1000-2561.2020.08.001

Abstract

Abstract:

Macadamia nut is an important economics crop in subtropical areas of China. The selection of a suitable reference gene is an important prerequisite for successful gene expression analysis by real-time fluorescence quantitative PCR (RT-qPCR). In order to select the appropriate reference genes, we investigated the expression stability of 11 candidate genes (18S rRNA, Actin, CYP, EF1a, EF1b, GAPDH, MDH, TUBa, TUBb, UBQ, UBC) in RT-qPCR experiments in different tissues, including kernel, peel, roots, stem, leaf from Macadamia with geNorm, NormFinder, BestKeeper, ΔCt, RefFinder program software packages. As determined by geNorm, MDH/EF1b were the most stable reference genes, TUBa was the least stable gene. BestKeeper revealed that MDH was the most stables reference gene, and EF1b ranked the second. The rank in BestKeeper was similarly with that in geNorm. The result by NormFinder showed GAPDH was the most stable gene, CYP ranks the second, the least stable gene was TUBa. ΔCt algorithm demonstrated that18S was the most stable gene, GAPDH ranks the second, MDH and EF1b ranked the third and fourth. To obtain a consensus result of the most stable reference genes according to the RefFinder approach, the geometric mean of the four algorithms corresponding rankings for each candidate gene were calculated: MDH>18S>GADPH>EF1b>CYP>UBC>EF1a>Actin>TUBb>UBQ>TUBa. The result showed that MDH was the most suitable reference gene for macadamia in different tissues.

Key words: Macadamia integrifolia, reference genes, real-time fluorescence quantitative PCR, stability analysis

CLC Number:

S664.9

YANG Qian,YANG Ziping,ZHOU Yali,CHEN Dongquan,LIU Heng. Screening of Stable Reference Genes for qRT-PCR Analysis in Macadamia integrifolia[J]. Chinese Journal of Tropical Crops, 2020, 41(8): 1505-1512.

Figures/Tables 7

References 31

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基因名称 Genes symbol	基因 ID Gene ID	引物序列（5°?3°） Primers sequences(5°?3°)	扩增效率 Primer efficiency/%	相关系数 R²
18S	MN650750	F: CTGAGAAACGGCTACCACATC	102.51	0.963
18S	MN650750	R: CGAAGAGCCCGGTATTGTTATT	102.51	0.963
Actin	MN627205	F: GAGGAGAGGATCTGTCGTAAA	91.96	0.998
Actin	MN627205	R: GATAACAAGGAGAGGCCAAAG	91.96	0.998
CYP	MN627206	F: AACAAGTTCGCCGATGAG	95.05	0.999
CYP	MN627206	R: GTCTTCGCAGTGCAAATAAAG	95.05	0.999
EF1a	MN627207	F: CCCACTTCAGGGTGTTTAC	97.99	0.994
EF1a	MN627207	R: CGAAGGTGACAACCATACC	97.99	0.994
EF1b	MN627208	F: GGCTGCTAAAGCATCTACAA	94.41	0.998
EF1b	MN627208	R: CGAACAGCTTCCTCTAGTTTC	94.41	0.998
GADPH	MN627209	F: GTTGGTGACTGTAGGTCAAG	93.73	0.999
GADPH	MN627209	R: AGGTCCAACACTCGGTTA	93.73	0.999
MADH	MN627210	F: GCTGGTCTCATCTATTCTTTCC	98.61	0.999
MADH	MN627210	R: CGTCCAACTTCTTCCTTGAG	98.61	0.999
TUBa	MN627211	F: GGCTTGTGTCTCAGGTTATT	94.90	0.999
TUBa	MN627211	R: GTGGATATGGGACCAAGTTAG	94.90	0.999
TUBb	MN627212	F: ATATGAGGATGAGGAGGAAGG	98.35	0.999
TUBb	MN627212	R: CCCATAATCAGCCACTGTAAA	98.35	0.999
UBQ	MN627213	F: GTGGATGTTGATGGATGAAAC	98.68	0.999
UBQ	MN627213	R: GTACTTACAGAGCGTCCTTAC	98.68	0.999
UBC	MN627214	F: CCACCCAAGGTAGCATTTAG	95.95	0.982
UBC	MN627214	R: CTGGGCTCCATTGTTCTTTA	95.95	0.982

基因名称 Genes symbol	基因 ID Gene ID	引物序列（5°?3°） Primers sequences(5°?3°)	扩增效率 Primer efficiency/%	相关系数 R²
18S	MN650750	F: CTGAGAAACGGCTACCACATC	102.51	0.963
18S	MN650750	R: CGAAGAGCCCGGTATTGTTATT	102.51	0.963
Actin	MN627205	F: GAGGAGAGGATCTGTCGTAAA	91.96	0.998
Actin	MN627205	R: GATAACAAGGAGAGGCCAAAG	91.96	0.998
CYP	MN627206	F: AACAAGTTCGCCGATGAG	95.05	0.999
CYP	MN627206	R: GTCTTCGCAGTGCAAATAAAG	95.05	0.999
EF1a	MN627207	F: CCCACTTCAGGGTGTTTAC	97.99	0.994
EF1a	MN627207	R: CGAAGGTGACAACCATACC	97.99	0.994
EF1b	MN627208	F: GGCTGCTAAAGCATCTACAA	94.41	0.998
EF1b	MN627208	R: CGAACAGCTTCCTCTAGTTTC	94.41	0.998
GADPH	MN627209	F: GTTGGTGACTGTAGGTCAAG	93.73	0.999
GADPH	MN627209	R: AGGTCCAACACTCGGTTA	93.73	0.999
MADH	MN627210	F: GCTGGTCTCATCTATTCTTTCC	98.61	0.999
MADH	MN627210	R: CGTCCAACTTCTTCCTTGAG	98.61	0.999
TUBa	MN627211	F: GGCTTGTGTCTCAGGTTATT	94.90	0.999
TUBa	MN627211	R: GTGGATATGGGACCAAGTTAG	94.90	0.999
TUBb	MN627212	F: ATATGAGGATGAGGAGGAAGG	98.35	0.999
TUBb	MN627212	R: CCCATAATCAGCCACTGTAAA	98.35	0.999
UBQ	MN627213	F: GTGGATGTTGATGGATGAAAC	98.68	0.999
UBQ	MN627213	R: GTACTTACAGAGCGTCCTTAC	98.68	0.999
UBC	MN627214	F: CCACCCAAGGTAGCATTTAG	95.95	0.982
UBC	MN627214	R: CTGGGCTCCATTGTTCTTTA	95.95	0.982

排序 Rank	Norm Finder		ΔCt
排序 Rank	基因 Gene	稳定性 Stability	基因 Gene	标准偏差 STDEV
1	GAPDH	0.121	18S rRNA	0.85
2	CYP	0.161	GADPH	0.87
3	EF1a	0.219	MDH	0.92
4	18S rRNA	0.297	EF1b	0.94
5	MDH	0.358	CYP	0.96
6	EF1b	0.468	EF1a	1.03
7	TUBb	0.657	UBC	1.16
8	Actin	0.677	Actin	1.16
9	UBC	0.737	TUBb	1.19
10	UBQ	0.742	UBQ	1.33
11	TUBa	1.799	TUBa	2.44

排序 Rank	Norm Finder		ΔCt
排序 Rank	基因 Gene	稳定性 Stability	基因 Gene	标准偏差 STDEV
1	GAPDH	0.121	18S rRNA	0.85
2	CYP	0.161	GADPH	0.87
3	EF1a	0.219	MDH	0.92
4	18S rRNA	0.297	EF1b	0.94
5	MDH	0.358	CYP	0.96
6	EF1b	0.468	EF1a	1.03
7	TUBb	0.657	UBC	1.16
8	Actin	0.677	Actin	1.16
9	UBC	0.737	TUBb	1.19
10	UBQ	0.742	UBQ	1.33
11	TUBa	1.799	TUBa	2.44

排序 Rank	Best Keeper				Ref Finder
排序 Rank	基因 Gene	相关系数 Correlation coefficient	标准差 SD	变异系数 CV	基因 Gene	几何平均数排序 Geomean of ranking values
1	MDH	0.946	0.361	1.538	MDH	1.97
2	EF1b	0.809	0.411	1.635	18S rRNA	2.06
3	GAPDH	0.946	0.531	1.844	GADPH	2.38
4	18S rRNA	0.929	0.619	3.215	EF1b	2.63
5	CYP	0.872	0.754	3.268	CYP	5.01
6	UBC	0.560	0.770	3.116	UBC	6.30
7	Actin	0.659	0.917	3.811	EF1a	6.45
8	EF1a	0.925	0.966	3.933	Actin	7.20
9	TUBb	0.843	0.994	3.708	TUBb	8.21
10	UBQ	0.731	1.108	4.594	UBQ	10.00
11	TUBa	0.825	2.380	8.200	TUBa	11.00