Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum
Abstract
:1. Introduction
2. Results
2.1. Selection of Candidate Reference Genes Based on Transcriptome Data
2.2. Primer Specificity and Amplification Efficiency Test of Candidate Reference Genes
2.3. Expression Profiles of Candidate Reference Genes
2.4. Expression Stability of Candidate Reference Genes
2.4.1. geNorm Analysis
2.4.2. NormFinder Analysis
2.5. The Expression Profiles of CHS and BCH Validated by the Most Stable/unstable Reference Genes
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. RNA Preparation, Reverse Transcription and qRT-PCR
4.3. Screening of Candidate Reference Genes
4.4. Primer Design and qRT-PCR Analysis of Candidate Reference Genes
4.5. Selection of Reference Genes with Stable Expression
4.6. Validation of the Candidate Reference Genes
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
qRT-PCR | Quantitative real-time polymerase chain reaction |
Cq | Cycle threshold |
ACT7 | Actin-7-like |
ACTF11 | Actin-depolymerizing factor 11-like |
ATP | ATP synthase subunit O |
EF1 δ | Elongation factor 1-delta |
MADH | Malate dehydrogenase |
g-TUB | gamma-tubulin complex component 2 |
UBC | Ubiquitin-conjugating enzyme |
26S | 26S proteasome non-ATPase regulatory subunit 2 |
40S | 40S ribosomal protein |
60S | 60S ribosomal protein |
ABP | Anthocyanins biosynthetic pathways |
CBP | Carotenoids biosynthetic pathways |
CHS | Chalcone synthase |
BCH | β-carotene hydroxylase |
FPKM | Fragments per Kilobase of transcript per millions base pairs sequenced |
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Gene Name | Description | Primer Sequence (5′-3′) | Amplicon Length (bp) | RT-qPCR Efficiency (%) | R2 |
---|---|---|---|---|---|
ACT7 | actin-7-like | F:AACTGGTATTGTGCTGGATTC R:TCATCAGTGAATCTGTAAGGTC | 128 | 88.58 | 0.9970 |
ACTF11 | actin-depolymerizing factor 11-like | F:ATGTTCATCAACAGTTGCAG R:GGCAGTGATCATCAACTC | 130 | 158.30 | 0.8844 |
ATP | ATP synthase subunit O | F:TTCACTGATCAATTACGGC R:GATGCGTAGTTTCCAGTAC | 141 | 102.44 | 0.9496 |
EF1 δ | elongation factor 1-delta | F:CTACCAAGCTTCAAAGGATG R:CTCAGATACAGTAGTAGACC | 143 | 98.32 | 0.9984 |
MADH | malate dehydrogenase | F:CTACGATATCGCTGGTACTC R:ACGAGTTCTGATCCCTCC | 123 | 105.62 | 0.9948 |
g-TUB | gamma-tubulin complex component 2 | F:ATCCATTGTGATTGAGAAGGC R:ACTGTAGTATCACCTGCCATG | 102 | 93.05 | 0.9996 |
UBC | Ubiquitin-conjugating enzyme | F:ATCTCTCAGGCAAGCATTAC R:GTAGAGGTATGGCACTAATC | 125 | 130.87 | 0.9856 |
26S | 26S proteasome non-ATPase regulatory subunit 2 | F:CAGAAGCTCGCACTAGAG R:TATGGGCAGATCATCATACTG | 162 | 102.60 | 0.9953 |
40S | 40S ribosomal protein | F:GAAGATGGTATTCCTGCAG R:TAGCCTTGGCTGCTTCATG | 139 | 93.18 | 0.9999 |
60S | 60S ribosomal protein | F:GTCCAAGTCGAATCAGTATG R:ATAGTGCGTGCCATTCTTC | 139 | 96.68 | 0.9960 |
Sample | Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
---|---|---|---|---|---|---|---|---|---|---|---|
Total | Gene | EF1δ | 60S | 40S | ACT7 | ATP | g-TUB | ACTF11 | UBC | MADH | 26S |
stability | 0.068 | 0.074 | 0.124 | 0.188 | 0.255 | 0.332 | 0.427 | 0.494 | 0.645 | 2.383 | |
Bud | Gene | 60S | EF1δ | ATP | 40S | g-TUB | ACT7 | ACTF11 | UBC | 26S | MADH |
stability | 0.143 | 0.166 | 0.193 | 0.227 | 0.287 | 0.333 | 0.360 | 0.368 | 0.385 | 0.388 | |
Flower | Gene | ATP | 60S | EF1δ | ACTF11 | ACT7 | g-TUB | 40S | MADH | UBC | 26S |
stability | 0.020 | 0.020 | 0.052 | 0.084 | 0.104 | 0.156 | 0.279 | 0.341 | 0.437 | 2.615 | |
Bud and Flower | Gene | ACT7 | 60S | EF1δ | 40S | ATP | g-TUB | UBC | ACTF11 | MADH | 26S |
stability | 0.004 | 0.004 | 0.034 | 0.081 | 0.280 | 0.303 | 0.364 | 0.465 | 0.623 | 1.519 | |
Abaxial lip | Gene | ATP | 40S | 60S | EF1δ | UBC | ACT7 | g-TUB | MADH | ACTF11 | 26S |
stability | 0.004 | 0.004 | 0.011 | 0.026 | 0.026 | 0.043 | 0.356 | 0.658 | 0.962 | 4.283 | |
Adaxial lip | Gene | ACTF11 | g-TUB | 60S | EF1δ | 40S | ACT7 | ATP | MADH | UBC | 26S |
stability | 0.012 | 0.012 | 0.124 | 0.165 | 0.192 | 0.289 | 0.558 | 0.766 | 0.822 | 2.476 | |
Petal | Gene | ACT7 | 60S | EF1δ | 40S | ATP | ACTF11 | g-TUB | UBC | MADH | 26S |
stability | 0.049 | 0.049 | 0.062 | 0.245 | 0.383 | 0.393 | 0.454 | 0.645 | 0.654 | 1.053 | |
Sepal | Gene | ACT7 | 60S | EF1δ | ATP | 40S | g-TUB | ACTF11 | UBC | MADH | 26S |
stability | 0.041 | 0.087 | 0.163 | 0.191 | 0.343 | 0.389 | 0.561 | 0.676 | 0.756 | 0.844 |
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Dong, X.-M.; Zhang, W.; Zhang, S.-B. Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum. Int. J. Mol. Sci. 2022, 23, 738. https://doi.org/10.3390/ijms23020738
Dong X-M, Zhang W, Zhang S-B. Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum. International Journal of Molecular Sciences. 2022; 23(2):738. https://doi.org/10.3390/ijms23020738
Chicago/Turabian StyleDong, Xiu-Mei, Wei Zhang, and Shi-Bao Zhang. 2022. "Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum" International Journal of Molecular Sciences 23, no. 2: 738. https://doi.org/10.3390/ijms23020738