Reliable RT-qPCR Normalization in Polypogon fugax: Reference Gene Selection for Multi-Stress Conditions and ACCase Expression Analysis in Herbicide Resistance
Abstract
1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Sample Collection Under Diverse Stresses and Growth Stages
2.2.1. Cold and Heat Stress Applications
2.2.2. Salt and Drought Stress Induction
2.2.3. Cd and Herbicide Exposure
2.2.4. Developmental Stage Sampling
2.3. Reference Gene Selection
2.4. RNA Isolation and RT-qPCR Profiling
2.5. Reference Gene Stability Assessment
2.6. ACCase Expression Profiling Across the Different Populations
3. Results
3.1. Verification of Primer Specificity and Effectiveness
3.2. Expression Stability Analysis of Candidate Reference Genes
3.2.1. Delta Ct Method Analysis
3.2.2. NormFinder Analysis
3.2.3. BestKeeper Analysis
3.2.4. geNorm Analysis
3.2.5. Comprehensive Ranking of RGs
3.3. Validation of the Recommended RGs
3.4. Expression Level of the ACCase Gene in P. fugax Under Quizalofop-p-Ethyl Exposure
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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ID | Gene Symbel | Gene Description | Primer Sequences (5′→3′) | Amplicon Length (bp) | Amplification Efficiency (%) | R2 |
---|---|---|---|---|---|---|
TRINITY_DN5506_c0_g1 | EF-1 | Elongation factor 1-alpha | GATGAAAATGCCTCCAGAACG AGCCCCACAACGAATACATC | 128 | 105.07 | 0.994 |
TRINITY_DN35447_c0_g1 | 28S | 28S ribosomal RNA | CCATCCATCTCGCAAGAAAT CGCAATATCTTCACCCGTTT | 221 | 91.88 | 0.989 |
TRINITY_DN11218_c0_g1 | ACT | Actin | GCACAATGTTGCCATACAGG AAGAACAGCTCCTCCGTTGA | 205 | 99.56 | 0.999 |
TRINITY_DN6226_c0_g1 | TUB | β-tubulin | GACTCCTTCAACTCAGCCAG CCCTACGATCTCAAGCAACAG | 125 | 103.55 | 0.986 |
TRINITY_DN51183_c0_g1 | GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | TGCCCTGTGATTTTCCGATAG AAGCCTAATTCGAGAGTTCAGAC | 158 | 95.57 | 0.993 |
TRINITY_DN15195_c1_g2 | EIF4A | Eukaryotic initiation factor 4A | TGCTTGGTTTCTGACTTCTGG TTGTGGAGTTGTCGCTAGTG | 176 | 90.52 | 0.947 |
TRINITY_DN10120_c0_g1 | UBQ | ubiquitin | AGCGGTGTCAAAGGTGTC TGGCTGAGTGGAAAGATCAAG | 180 | 97.24 | 0.993 |
TRINITY_DN3966_c0_g1 | RUB | ribulose-1,5-bisphosphate carboxylase | AACGGTGGAAGGATCAGATG GAAGATGAACCAAACCTTGCTG | 174 | 93.78 | 0.997 |
Rank | Heat Stress Subset | Drought Stress Subset | Salt Stress Subset | Cd Stress Subset | Cold Stress Subset | Quizalofop-p-ethyl Stress Subset | Growth Stage Subset | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SD | Gene | SD | Gene | SD | Gene | SD | Gene | SD | Gene | SD | Gene | SD | |
1 | EIF4A | 0.59 | EIF4A | 0.65 | EIF4A | 0.68 | EIF4A | 0.6 | EIF4A | 0.65 | EIF4A | 0.68 | EIF4A | 0.79 |
2 | TUB | 0.63 | ACT | 0.67 | EF-1 | 0.77 | ACT | 0.63 | TUB | 0.69 | TUB | 0.69 | TUB | 0.84 |
3 | EF-1 | 0.65 | TUB | 0.68 | 28S | 0.78 | EF-1 | 0.67 | ACT | 0.73 | UBQ | 0.75 | ACT | 0.97 |
4 | RUB | 0.66 | GAPDH | 0.68 | UBQ | 0.78 | GAPDH | 0.67 | EF-1 | 0.81 | GAPDH | 0.75 | 28S | 0.99 |
5 | ACT | 0.68 | EF-1 | 0.92 | TUB | 0.83 | 28S | 0.79 | RUB | 0.91 | ACT | 0.79 | EF-1 | 1.00 |
6 | 28S | 0.78 | UBQ | 0.93 | ACT | 0.89 | TUB | 0.80 | GAPDH | 1.00 | EF-1 | 0.84 | RUB | 1.16 |
7 | UBQ | 0.90 | RUB | 0.94 | GAPDH | 0.94 | UBQ | 0.85 | 28S | 1.03 | RUB | 0.90 | UBQ | 1.16 |
8 | GAPDH | 1.57 | 28S | 1.06 | RUB | 1.1 | RUB | 1.03 | UBQ | 1.09 | 28S | 1.04 | GAPDH | 1.34 |
Rank | Heat Stress Subset | Drought Stress Subset | Salt Stress Subset | Cd Stress Subset | Cold Stress Subset | Quizalofop-p-ethyl Stress Subset | Growth Stage Subset | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SV | Gene | SV | Gene | SV | Gene | SV | Gene | SV | Gene | SV | Gene | SV | |
1 | TUB | 0.047 | EIF4A | 0.165 | EIF4A | 0.269 | EIF4A | 0.243 | EIF4A | 0.181 | EIF4A | 0.344 | EIF4A | 0.254 |
2 | EIF4A | 0.226 | ACT | 0.273 | UBQ | 0.455 | ACT | 0.277 | TUB | 0.296 | TUB | 0.346 | TUB | 0.427 |
3 | EF-1 | 0.239 | TUB | 0.320 | 28S | 0.466 | EF-1 | 0.423 | ACT | 0.391 | GAPDH | 0.483 | ACT | 0.656 |
4 | RUB | 0.330 | GAPDH | 0.335 | EF-1 | 0.498 | GAPDH | 0.434 | EF-1 | 0.545 | UBQ | 0.495 | 28S | 0.684 |
5 | 28S | 0.408 | EF-1 | 0.745 | TUB | 0.627 | TUB | 0.604 | RUB | 0.650 | ACT | 0.546 | EF-1 | 0.753 |
6 | ACT | 0.410 | UBQ | 0.784 | ACT | 0.712 | 28S | 0.646 | GAPDH | 0.824 | EF-1 | 0.628 | RUB | 0.914 |
7 | UBQ | 0.803 | RUB | 0.794 | GAPDH | 0.799 | UBQ | 0.680 | 28S | 0.915 | RUB | 0.725 | UBQ | 0.939 |
8 | GAPDH | 1.538 | 28S | 0.954 | RUB | 0.942 | RUB | 0.907 | UBQ | 0.953 | 28S | 0.903 | GAPDH | 1.181 |
Rank | Heat Stress Subset | Drought Stress Subset | Salt Stress Subset | Cd Stress Subset | Cold Stress Subset | Quizalofop-p-ethyl Stress Subset | Growth Stage Subset | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SD | Gene | SD | Gene | SD | Gene | SD | Gene | SD | Gene | SD | Gene | SD | |
1 | EIF4A | 0.30 | 28S | 0.55 | EF-1 | 0.55 | EIF4A | 0.55 | 28S | 0.49 | RUB | 0.60 | RUB | 0.16 |
2 | EF-1 | 0.30 | EF-1 | 0.56 | 28S | 0.36 | ACT | 0.56 | TUB | 0.52 | ACT | 0.85 | EIF4A | 0.74 |
3 | RUB | 0.35 | ACT | 0.79 | ACT | 0.95 | EF-1 | 0.59 | ACT | 0.56 | UBQ | 0.89 | GAPDH | 0.82 |
4 | ACT | 0.39 | EIF4A | 0.91 | TUB | 0.93 | GAPDH | 0.65 | EF-1 | 0.57 | TUB | 0.93 | 28S | 0.85 |
5 | TUB | 0.40 | TUB | 1.07 | GAPDH | 0.61 | 28S | 0.73 | EIF4A | 0.70 | EIF4A | 1.06 | TUB | 0.86 |
6 | 28S | 0.41 | GAPDH | 1.07 | EIF4A | 0.53 | TUB | 0.82 | RUB | 0.74 | GAPDH | 1.10 | EF-1 | 0.96 |
7 | UBQ | 0.62 | RUB | 1.27 | UBQ | 0.94 | UBQ | 0.94 | GAPDH | 1.07 | 28S | 1.14 | ACT | 0.96 |
8 | GAPDH | 1.31 | UBQ | 1.45 | RUB | 1.05 | RUB | 1.06 | UBQ | 1.30 | EF-1 | 1.28 | UBQ | 1.29 |
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Zhao, Y.; Yang, X.; Hu, Q.; Zhang, J.; Wan, S.; Chen, W. Reliable RT-qPCR Normalization in Polypogon fugax: Reference Gene Selection for Multi-Stress Conditions and ACCase Expression Analysis in Herbicide Resistance. Agronomy 2025, 15, 1813. https://doi.org/10.3390/agronomy15081813
Zhao Y, Yang X, Hu Q, Zhang J, Wan S, Chen W. Reliable RT-qPCR Normalization in Polypogon fugax: Reference Gene Selection for Multi-Stress Conditions and ACCase Expression Analysis in Herbicide Resistance. Agronomy. 2025; 15(8):1813. https://doi.org/10.3390/agronomy15081813
Chicago/Turabian StyleZhao, Yufei, Xu Yang, Qiang Hu, Jie Zhang, Sumei Wan, and Wen Chen. 2025. "Reliable RT-qPCR Normalization in Polypogon fugax: Reference Gene Selection for Multi-Stress Conditions and ACCase Expression Analysis in Herbicide Resistance" Agronomy 15, no. 8: 1813. https://doi.org/10.3390/agronomy15081813
APA StyleZhao, Y., Yang, X., Hu, Q., Zhang, J., Wan, S., & Chen, W. (2025). Reliable RT-qPCR Normalization in Polypogon fugax: Reference Gene Selection for Multi-Stress Conditions and ACCase Expression Analysis in Herbicide Resistance. Agronomy, 15(8), 1813. https://doi.org/10.3390/agronomy15081813