Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials and Stress Treatments
2.2. Total RNA Isolation and cDNA Synthesis
2.3. Selection of Candidate Reference Genes and Primer Design
2.4. Real-Time Quantitative Polymerase Chain Reaction
2.5. Analysis of Real-Time PCR Data
2.6. Evaluating Reference Genes Expression
3. Results
3.1. Verification of Primer Specificity and PCR Efficiency
3.2. Expression Profile of the Candidate Reference Genes
3.3. Expression Stability Analysis of Candidate Reference Genes
3.3.1. ΔCt Algorithm Analysis
3.3.2. geNorm Analysis
3.3.3. NormFinder Analysis
3.3.4. BestKeeper Analysis
3.3.5. Comprehensive Ranking
3.4. Validation of the Reference Genes with the SAD Gene
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
SAD | Δ9-Stearoyl-ACP desaturases |
18S | 18S ribosome RNA |
ACT | Actin |
CYP | Cyclophilin |
EF1a | Elongation factor 1-α |
EF1b | Elongation factor 1-β |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
MDH | Malate dehydrogenease |
UBC | Ubiquitin-conjugating enzyme |
TUBa | Tubulin-α |
TUBb | Tubulin-β |
UBQ | Ubiquitin-A |
FATA | fatty acid thioesterase |
FAD | fatty acid desaturation |
MeJA | jasmonate |
ABA | abscisic acid |
GA | gibberrellins |
ETH | ethyrel |
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Gene Name | Genes Symbol | Gene ID | Primers Sequences (5′-3′) | Primer Efficiency | R2 |
---|---|---|---|---|---|
18S ribosome RNA | 18S | MN650750 | F: CTGAGAAACGGCTACCACATC | 102.51 | 0.963 |
R: CGAAGAGCCCGGTATTGTTATT | |||||
ACT-β | ACT | MN627205 | F: GAGGAGAGGATCTGTCGTAAA | 91.96 | 0.998 |
R: GATAACAAGGAGAGGCCAAAG | |||||
Cyclophilin | CYP | MN627206 | F: AACAAGTTCGCCGATGAG | 95.05 | 0.999 |
R: GTCTTCGCAGTGCAAATAAAG | |||||
Elongation factor 1-α | EF1a | MN627207 | F: CCCACTTCAGGGTGTTTAC | 97.99 | 0.994 |
R: CGAAGGTGACAACCATACC | |||||
Elongation factor 1-β | EF1b | MN627208 | F: GGCTGCTAAAGCATCTACAA | 94.41 | 0.998 |
R: CGAACAGCTTCCTCTAGTTTC | |||||
Glyceraldehyde 3-phosphate dehydrogenase | GADPH | MN627209 | F: GTTGGTGACTGTAGGTCAAG | 93.73 | 0.999 |
R: AGGTCCAACACTCGGTTA | |||||
Malate dehydrogenease | MADH | MN627210 | F: GCTGGTCTCATCTATTCTTTCC | 98.61 | 0.999 |
R: CGTCCAACTTCTTCCTTGAG | |||||
Tubulin-α | TUBa | MN627211 | F: GGCTTGTGTCTCAGGTTATT | 94.9 | 0.999 |
R: GTGGATATGGGACCAAGTTAG | |||||
Tubulin-β | TUBb | MN627212 | F: ATATGAGGATGAGGAGGAAGG | 98.35 | 0.999 |
R: CCCATAATCAGCCACTGTAAA | |||||
Ubiquitin | UBQ | MN627213 | F: GTGGATGTTGATGGATGAAAC | 98.68 | 0.999 |
R: GTACTTACAGAGCGTCCTTAC | |||||
Ubiquitin-conjugating enzyme | UBC | MN627214 | F: CCACCCAAGGTAGCATTTAG | 95.95 | 0.982 |
R: CTGGGCTCCATTGTTCTTTA |
Method/Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
---|---|---|---|---|---|---|---|---|---|---|---|
(A) Cold stress | |||||||||||
Delta CT | ACT | GAPDH | CYP | EF1b | MDH | TUBb | UBC | EF1a | 18S | UBQ | TUBa |
BestKeeper | TUBb | UBC | GAPDH | EF1b | ACT | MDH | TUBa | CYP | EF1a | UBQ | 18S |
NormFinder | ACT | GAPDH | CYP | EF1b | TUBb | MDH | UBC | EF1a | 18S | TUBa | UBQ |
geNorm | ACT/EF1b | GAPDH | CYP | MDH | TUBb | UBC | 18S | EF1a | UBQ | TUBa | |
Recommended Comprehensive Ranking | ACT | EF1b | GAPDH | CYP | UBC | TUBb | MDH | EF1a | 18S | TUBa | UBQ |
(B) Heat stress | |||||||||||
Delta CT | EF1b | ACT | 18S | MDH | EF1a | CYP | TUBb | GAPDH | UBQ | TUBa | UBC |
BestKeeper | TUBb | TUBa | GAPDH | EF1b | MDH | ACT | EF1a | CYP | 18S | UBC | UBQ |
NormFinder | EF1b | ACT | EF1a | 18S | MDH | TUBb | GAPDH | CYP | UBQ | TUBa | UBC |
geNorm | ACT/EF1b | MDH | 18S | TUBb | CYP | EF1a | GAPDH | UBQ | TUBa | UBC | |
Recommended Comprehensive Ranking | EF1b | ACT | MDH | TUBb | 18S | EF1a | TUBa | CYP | GAPDH | UBQ | UBC |
(C) NaCl stress | |||||||||||
Delta CT | ACT | CYP | EF1b | GAPDH | UBC | MDH | EF1a | UBQ | 18S | TUBb | TUBa |
BestKeeper | MDH | 18S | GAPDH | EF1b | CYP | UBC | ACT | EF1a | UBQ | TUBb | TUBa |
NormFinder | CYP | ACT | EF1b | GAPDH | EF1a | UBC | UBQ | MDH | 18S | TUBb | TUBa |
geNorm | ACT/EF1b | UBC | MDH | CYP | GAPDH | UBQ | EF1a | 18S | TUBb | TUBa | |
Recommended Comprehensive Ranking | ACT | CYP | EF1b | MDH | UBC | GAPDH | 18S | EF1a | UBQ | TUBb | TUBa |
(D) PEG stress | |||||||||||
Delta CT | ACT | UBC | EF1b | MDH | CYP | EF1a | UBQ | 18S | GAPDH | TUBa | TUBb |
BestKeeper | ACT | UBC | TUBa | EF1b | TUBb | CYP | MDH | GAPDH | EF1a | UBQ | 18S |
NormFinder | UBC | ACT | EF1b | CYP | MDH | EF1a | UBQ | 18S | GAPDH | TUBa | TUBb |
geNorm | ACT/EF1b | UBC | CYP | MDH | EF1a | UBQ | GAPDH | 18S | TUBa | TUBb | |
Recommended Comprehensive Ranking | ACT | UBC | EF1b | CYP | MDH | EF1a | UBQ | 18S | TUBa | GAPDH | TUBb |
(E) ABA treatment | |||||||||||
Delta CT | ACT | CYP | TUBb | EF1b | UBC | 18S | MDH | EF1a | UBQ | GAPDH | TUBa |
BestKeeper | UBC | MDH | TUBb | EF1b | ACT | CYP | GAPDH | EF1a | 18S | UBQ | TUBa |
NormFinder | ACT | CYP | EF1b | TUBb | 18S | UBC | EF1a | UBQ | MDH | GAPDH | TUBa |
geNorm | MDH/UBC | ACT | TUBb | CYP | EF1b | 18S | EF1a | UBQ | GAPDH | TUBa | |
Recommended Comprehensive Ranking | ACT | UBC | MDH | CYP | TUBb | EF1b | 18S | EF1a | UBQ | GAPDH | TUBa |
(F) GA treatment | |||||||||||
Delta CT | UBQ | EF1b | MDH | EF1a | UBC | ACT | GAPDH | TUBb | CYP | TUBa | 18S |
BestKeeper | EF1b | UBC | MDH | ACT | UBQ | GAPDH | EF1a | TUBb | CYP | TUBa | 18S |
NormFinder | UBQ | EF1a | EF1b | GAPDH | MDH | ACT | UBC | TUBb | CYP | TUBa | 18S |
geNorm | MDH/UBC | ACT | EF1b | UBQ | EF1a | GAPDH | TUBb | CYP | TUBa | 18S | |
Recommended Comprehensive Ranking | EF1b | UBQ | MDH | UBC | EF1a | ACT | GAPDH | TUBb | CYP | TUBa | 18S |
(G) MeJA treatment | |||||||||||
Delta CT | ACT | CYP | TUBb | UBQ | MDH | GAPDH | 18S | UBC | EF1b | EF1a | TUBa |
BestKeeper | TUBb | UBC | ACT | MDH | EF1b | GAPDH | CYP | TUBa | UBQ | EF1a | 18S |
NormFinder | ACT | CYP | TUBb | UBQ | GAPDH | MDH | 18S | EF1b | UBC | EF1a | TUBa |
geNorm | ACT/MDH | UBC | TUBb | CYP | EF1b | UBQ | 18S | GAPDH | EF1a | TUBa | |
Recommended Comprehensive Ranking | ACT | MDH | CYP | TUBb | UBC | UBQ | GAPDH | EF1b | 18S | EF1a | TUBa |
(H) ETH treatment | |||||||||||
Delta CT | UBC | CYP | ACT | UBQ | MDH | TUBb | 18S | EF1b | GAPDH | TUBa | EF1a |
BestKeeper | MDH | UBC | TUBb | EF1b | ACT | GAPDH | CYP | TUBa | UBQ | EF1a | 18S |
NormFinder | CYP | UBC | UBQ | ACT | MDH | TUBb | 18S | GAPDH | EF1b | TUBa | EF1a |
geNorm | ACT/UBC | MDH | CYP | TUBb | EF1b | UBQ | GAPDH | 18S | TUBa | EF1a | |
Recommended Comprehensive Ranking | UBC | ACT | CYP | MDH | UBQ | TUBb | EF1b | 18S | GAPDH | TUBa | EF1a |
(I) Root | |||||||||||
Delta CT | CYP | EF1a | GAPDH | TUBa | EF1b | ACT | UBC | UBQ | MDH | TUBb | 18S |
BestKeeper | UBQ | CYP | TUBa | EF1a | GAPDH | ACT | EF1b | TUBb | UBC | MDH | 18S |
NormFinder | EF1a | CYP | GAPDH | TUBa | EF1b | ACT | UBC | UBQ | MDH | TUBb | 18S |
Genorm | CYP/TUBa | EF1a | GAPDH | ACT | EF1b | UBC | UBQ | MDH | TUBb | 18S | |
Recommended Comprehensive ranking | CYP | EF1a | TUBa | GAPDH | ACT | EF1b | 18S | UBQ | UBC | MDH | TUBb |
(J) Stem | |||||||||||
Delta CT | ACT | MDH | UBC | TUBb | EF1b | 18S | GAPDH | CYP | UBQ | EF1a | TUBa |
BestKeeper | UBC | TUBb | MDH | ACT | EF1b | GAPDH | CYP | TUBa | EF1a | UBQ | 18S |
NormFinder | ACT | TUBb | 18S | MDH | EF1b | UBC | GAPDH | UBQ | CYP | EF1a | TUBa |
Genorm | ACT/UBC | MDH | TUBb | EF1b | CYP | 18S | GAPDH | UBQ | EF1a | TUBa | |
Recommended Comprehensive ranking | ACT | UBC | MDH | TUBb | EF1b | 18S | CYP | GAPDH | UBQ | EF1a | TUBa |
(K) Leaf | |||||||||||
Delta CT | CYP | EF1b | ACT | UBQ | EF1a | MDH | GAPDH | TUBb | 18S | TUBa | UBC |
BestKeeper | ACT | MDH | TUBb | EF1b | CYP | UBC | GAPDH | EF1a | UBQ | TUBa | 18S |
NormFinder | CYP | EF1b | ACT | EF1a | UBQ | GAPDH | MDH | TUBb | 18S | TUBa | UBC |
Genorm | ACT/MDH | EF1b | CYP | TUBb | UBQ | EF1a | GAPDH | 18S | TUBa | UBC | |
Recommended Comprehensive ranking | ACT | CYP | EF1b | MDH | UBQ | EF1a | TUBb | GAPDH | 18S | UBC | TUBa |
(L) All sample | |||||||||||
Delta CT | ACT | EF1b | CYP | MDH | EF1a | GAPDH | UBQ | TUBb | 18S | UBC | TUBa |
BestKeeper | ACT | UBC | MDH | TUBb | EF1b | CYP | GAPDH | TUBa | EF1a | UBQ | 18S |
NormFinder | ACT | CYP | EF1b | MDH | EF1a | GAPDH | UBQ | TUBb | 18S | UBC | TUBa |
Genorm | ACT/MDH | EF1b | CYP | EF1a | UBQ | GAPDH | 18S | TUBb | UBC | TUBa | |
Recommended Comprehensive ranking | ACT | MDH | EF1b | CYP | EF1a | UBQ | GAPDH | UBC | TUBb | 18S | TUBa |
Paired Variation (Vn/Vn+1) | |||||||||
---|---|---|---|---|---|---|---|---|---|
V2/3 | V3/4 | V4/5 | V5/6 | V6/7 | V7/8 | V8/9 | V9/10 | V10/11 | |
ABA | 0.159 | 0.103 | 0.092 | 0.079 | 0.115 | 0.128 | 0.125 | 0.119 | 0.179 |
GA | 0.103 | 0.100 | 0.105 | 0.100 | 0.078 | 0.069 | 0.068 | 0.079 | 0.112 |
ET | 0.139 | 0.147 | 0.118 | 0.116 | 0.130 | 0.112 | 0.099 | 0.106 | 0.109 |
MJ | 0.141 | 0.131 | 0.120 | 0.123 | 0.127 | 0.120 | 0.105 | 0.108 | 0.133 |
NaCl | 0.125 | 0.104 | 0.094 | 0.079 | 0.097 | 0.093 | 0.099 | 0.112 | 0.178 |
PEG | 0.070 | 0.057 | 0.079 | 0.069 | 0.057 | 0.067 | 0.067 | 0.088 | 0.076 |
Heat | 0.144 | 0.143 | 0.161 | 0.144 | 0.133 | 0.208 | 0.233 | 0.207 | 0.237 |
Cold | 0.121 | 0.082 | 0.096 | 0.106 | 0.097 | 0.176 | 0.196 | 0.173 | 0.162 |
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© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
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Yang, Q.; Yang, Z.; Zeng, H.; Zou, M.; Song, X.; Wan, J.; Wang, Z.; Chen, J.; Luo, L. Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia. Forests 2024, 15, 1966. https://doi.org/10.3390/f15111966
Yang Q, Yang Z, Zeng H, Zou M, Song X, Wan J, Wang Z, Chen J, Luo L. Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia. Forests. 2024; 15(11):1966. https://doi.org/10.3390/f15111966
Chicago/Turabian StyleYang, Qian, Ziping Yang, Hui Zeng, Minghong Zou, Ximei Song, Jifeng Wan, Zhao Wang, Jing Chen, and Lianfang Luo. 2024. "Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia" Forests 15, no. 11: 1966. https://doi.org/10.3390/f15111966
APA StyleYang, Q., Yang, Z., Zeng, H., Zou, M., Song, X., Wan, J., Wang, Z., Chen, J., & Luo, L. (2024). Evaluation and Validation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Macadamia integrifolia. Forests, 15(11), 1966. https://doi.org/10.3390/f15111966