Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives
Abstract
:1. Introduction
2. Results
2.1. Selection of Candidate reference genes (RGs) Based on RNA-seq Data
2.2. Expression Profile of Candidate RGs
2.3. Analysis of Gene Expression Stability in Accessions of Lactuca: Different Leaf Colour, Tissues, and Drought Stress Conditions
3. Discussion
4. Materials and Methods
4.1. Plant Material and Experimental Designs
4.2. RNA Extraction and RNA-Seq Analysis
4.3. Selection of Candidate RGs
4.4. mRNA Isolation, cDNA Synthesis and Real-Time qPCR
4.5. Stability Analysis of RGs
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Description | Primer Sequence (5′–3′) | Amplicon Length (bp) | Annealing Temperature (°C) |
---|---|---|---|---|
ADF2 | Actin-depolymerizing factor 2 | F-TTGGAGAACCAGCAGAAAC | 199 | 62 |
R-CCATCAAGCTCTCTCTTGAAC | ||||
CYB5 | Cytochrome B5 | F-GCACGCTACGAAAGAGG | 80 | 59 |
R-CAGGATGATCATCTAGAAAAGG | ||||
iPGAM | Probable 2,3-bisphosphoglycerate-independent phosphoglycerate mutase | F-GGGAGATGTTTCAATTCCAAG | 162 | 62 |
R-CCCATTAGAGAAAGATGAGCAG | ||||
SCL13 | Scarecrow-like protein 13 | F-AGTCGGTTAGCACGGTTA | 100 | 56 |
R-TTCGTGTTCGATTCTTGTT | ||||
TRXL3-3 | Thioredoxin-like protein 3-3 | F-TGGTGTCGTGTTTGTGCAGAG | 112 | 62 |
R-GTTGGGTTGTTTCTGGGCATT | ||||
VHA-H | V-type proton ATPase subunit H | F-TGCAAGTGATGATGTTTTGA | 152 | 59 |
R-TGCTTGAACAAATGAAGACC |
Gene a | Green vs. Red | Leaf vs. Stem | Drought Stress | ||
---|---|---|---|---|---|
‘Romired’ | ‘Morada de Belchite’ | L. homblei | |||
ACT | 0.564 ns | 0.938 ns | 0.421 ns | 0.018 * | 0.036 * |
α-TUB | 0.635 ns | 0.001 ** | 0.435 ns | 0.637 ns | 0.643 ns |
EEF1-α | 0.019 * | 0.066 ns | 0.976 ns | 0.089 ns | 0.696 ns |
GAPDH-2C | 0.028 * | 0.027 * | 0.407 ns | 0.302 ns | 0.593 ns |
UBC32 | 0.159 ns | 0.159 ns | 0.398 ns | 0.276 ns | 0.010 * |
UPL6 | 0.471 ns | 0.013 * | 0.003 ** | 0.013 * | 0.168 ns |
ADF2 | 0.569 ns | 0.884 ns | 0.400 ns | 0.660 ns | 0.547 ns |
CYB5 | 0.449 ns | 0.270 ns | 0.723 ns | 0.371 ns | 0.673 ns |
iPGAM | 0.833 ns | 0.773 ns | 0.128 ns | 0.418 ns | 0.210 ns |
SCL13 | 0.408 ns | 0.902 ns | 0.883 ns | 0.427 ns | 0.239 ns |
TRXL3-3 | 0.487 ns | 0.406 ns | 0.843 ns | 0.161 ns | 0.470 ns |
VHA-H | 0.198 ns | 0.576 ns | 0.116 ns | 0.195 ns | 0.741 ns |
geNorm | NormFinder | BestKeeper | Delta Ct | ||||||
---|---|---|---|---|---|---|---|---|---|
Experiment | Ranking | Gene | M | Gene | SV | Gene | SV | Gene | SV |
Green vs. red | 1 | CYB5 | 0.33 | TRXL3-3 | 0.71 | TRXL3-3 | 0.40 | CYB5 | 0.83 |
2 | ADF2 | 0.33 | VHA-H | 0.77 | CYB5 | 0.53 | ADF2 | 0.83 | |
3 | TRXL3-3 | 0.54 | CYB5 | 0.81 | SCL13 | 0.53 | SCL13 | 0.92 | |
4 | VHA-H | 1.11 | ADF2 | 1.09 | ADF2 | 0.57 | TRXL3-3 | 1.10 | |
5 | iPGAM | 1.33 | iPGAM | 1.49 | iPGAM | 0.72 | iPGAM | 1.28 | |
6 | SCL13 | 1.90 | SCL13 | 2.88 | VHA-H | 1.12 | VHA-H | 1.41 | |
Leaf vs. stem | 1 | CYB5 | 0.77 | TRXL3-3 | 0.30 | TRXL3-3 | 0.44 | TRXL3-3 | 0.93 |
2 | ADF2 | 0.77 | VHA-H | 0.30 | iPGAM | 0.47 | CYB5 | 1.03 | |
3 | TRXL3-3 | 1.01 | ADF2 | 1.18 | CYB5 | 0.77 | iPGAM | 1.08 | |
4 | VHA-H | 1.07 | CYB5 | 1.34 | ADF2 | 0.89 | ADF2 | 1.10 | |
5 | iPGAM | 1.22 | iPGAM | 1.55 | SCL13 | 0.92 | VHA-H | 1.38 | |
6 | SCL13 | 2.41 | SCL13 | 4.73 | VHA-H | 1.23 | SCL13 | 1.54 | |
Drought stress | 1 | TRXL3-3 | 1.54 | TRXL3-3 | 0.77 | TRXL3-3 | 0.85 | SCL13 | 3.89 |
2 | ADF2 | 1.54 | ADF2 | 0.77 | SCL13 | 0.92 | TRXL3-3 | 3.90 | |
3 | CYB5 | 1.98 | iPGAM | 2.81 | iPGAM | 1.11 | iPGAM | 4.01 | |
4 | iPGAM | 3.40 | CYB5 | 2.91 | ADF2 | 1.23 | ADF2 | 4.04 | |
5 | SCL13 | 4.41 | SCL13 | 6.62 | CYB5 | 1.50 | CYB5 | 4.07 | |
6 | VHA-H | 6.82 | VHA-H | 11.27 | VHA-H | 10.35 | VHA-H | 14.08 |
Ranking | Green vs. Red | Leaf vs. Stem | Drought Stress |
---|---|---|---|
1 | CYB5 | TRXL3-3 | TRXL3-3 |
2 | TRXL3-3 | CYB5 | ADF2 |
3 | ADF2 | ADF2 | SCL13 |
4 | VHA-H | VHA-H | iPGAM |
5 | SCL13 | iPGAM | CYB5 |
6 | iPGAM | SCL13 | VHA-H |
Experiment | Accession Name | Species | Group | Leaf Colour | Year | Source a | Accession Number |
---|---|---|---|---|---|---|---|
Leaf colour (green vs. red) | ‘Begoña’ | Lactuca sativa L. | Commercial variety | Green | 2018/2019 | Ramiro Arnedo Semillas S.A. | - |
‘Romired’ | Lactuca sativa L. | Commercial variety | Red | CGN | CGN24713 | ||
Tissue (leaf vs. stem) | Lactuca squarrosa | Lactuca squarrosa (Thunb.) Miq. | Wild crop relative | Semi-red (red stems) | 2020/2021 | BGHZ | BGHZ5124 |
Drought stress (C vs. DI-1 vs. DI-2) b | ‘Romired’ | Lactuca sativa L. | Commercial variety | Red | 2020/2021 | CGN | CGN24713 |
‘Morada de Belchite’ | Lactuca sativa L. | Traditional variety | Semi-red | BGHZ | BGHZ0527 | ||
Lactuca homblei | Lactuca homblei De Wild | Wild crop relative | Semi-red | BGHZ | BGHZ5322 |
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Medina-Lozano, I.; Arnedo, M.S.; Grimplet, J.; Díaz, A. Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives. Int. J. Mol. Sci. 2023, 24, 3052. https://doi.org/10.3390/ijms24033052
Medina-Lozano I, Arnedo MS, Grimplet J, Díaz A. Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives. International Journal of Molecular Sciences. 2023; 24(3):3052. https://doi.org/10.3390/ijms24033052
Chicago/Turabian StyleMedina-Lozano, Inés, María Soledad Arnedo, Jérôme Grimplet, and Aurora Díaz. 2023. "Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives" International Journal of Molecular Sciences 24, no. 3: 3052. https://doi.org/10.3390/ijms24033052