Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress
Abstract
:1. Introduction
2. Results and Discussion
2.1. Seed Germination of Chenopodium quinoa UDEC2 and UDEC4 Accessions in Response to Salt Stress
2.1.1. Anatomical and Histological Structure of Quinoa Imbibed Seeds
2.1.2. Germination Sensu Stricto in Chenopodium quinoa Seeds under Salt Stress
2.2. Selection of Candidate Reference Genes
2.3. Stability of Candidate Reference Genes during Seed Germination under 250 mM NaCl in UDEC2 and UDEC4 Accessions
2.4. Validation of Selected Reference Genes during Seed Germination by Evaluation of CqMAN7 and CqABI5 Expression
3. Materials and Methods
3.1. Plant Materials and Growth Conditions
3.2. Light Microscopy: Fixation, Embedding, Sectioning, and Polysaccharide and Protein Histological Staining
3.3. Germination Assays
3.4. Total RNA Isolation from Chenopodium quinoa Seeds and cDNA Synthesis
3.5. Selection of Candidate Reference and Target Genes
3.6. Primer Design
3.7. Quantitative PCR (qPCR)
3.8. Gene Stability Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene ID | Gene Description | Gene Abbreviation | Primer Sequence | Tm (°C) of PCR Product | Amplification Product Size (bp) | Amplification Efficiency (%) | Regression Coefficient (r2) |
---|---|---|---|---|---|---|---|
AUR62019117 | Actin-7 | CqACT7 | TGAACAGGAATCAGAGACAGCC | 83.3 | 148 | 95.1 | 0.99 |
CAGAAGACTCCATACCGACTAG | |||||||
AUR62005566 | Glyceraldehyde-3-phosphate dehydrogenase | CqGAPDH | CGGCTTCCTTCAACATCATTCCTAGC | 83.3 | 144 | 89.7 | 0.99 |
GCCTGACAGTGAGATCAACAACCG | |||||||
AUR62020295 | Monosensin activity 1 | CqMON1 | GGCGATGAACATAAGCTTGC | 79.8 | 100 | 91.5 | 0.99 |
TTCCTGCCCGAACTAACTTG | |||||||
AUR62034430 | Polypyrimidine-tract binding protein | CqPTB | CGGAGCATGTGAGTTCATGT | 80.6 | 154 | 92.7 | 0.91 |
CCAACAACAGGCTGAACAAG | |||||||
AUR62036615 | Ubiquitin-conjugating enzyme | CqUBC | TTGATCAAGGGCCCATCAGAAA | 82.3 | 103 | 90.3 | 0.99 |
AAAATCGCACTTGAGGAGGTTG | |||||||
Scaffold_4105:20989..22796 | 18S ribosomal RNA | Cq18S | GATGTTACTTTTAGGACGCCGC | 82.32 | 108 | 87 | 0.99 |
TGCCCTTCCGTCAATTCCTTTA | |||||||
AUR62007598 | Mannan endo-1,4-beta-mannosidase 7 | CqMAN7 | GGAATAGAGTCATATGGAGATGGT | 81.2 | 165 | 85.5 | 0.99 |
TCCATTCCTTATCTCCCTTGCC | |||||||
AUR62028537 | Abscisic acid-insensitive 5 | CqABI5 | TTCACCAGCAATGACAGACCAT | 82.5 | 108 | 106.5 | 0.99 |
TAGATTGAAGACTGGCGCCCC |
UDEC2 | Gene | RefFinder | geNorm | NormFinder | BestKeeper | ΔCt | |||||
Rank | GM | Rank | MV | Rank | SV | Rank | SD | Rank | SD | ||
CqACT7 | 1 | 1.19 | 1 | 0.50 | 1 | 0.50 | 2 | 0.65 | 1 | 1.13 | |
CqUBC | 2 | 1.87 | 1 | 0.50 | 2 | 0.54 | 3 | 0.70 | 2 | 1.13 | |
Cq18S | 3 | 2.28 | 2 | 0.75 | 3 | 0.68 | 1 | 0.53 | 3 | 1.26 | |
CqGAPDH | 4 | 4 | 3 | 0.89 | 4 | 0.96 | 4 | 0.80 | 4 | 1.38 | |
CqPTB | 5 | 5 | 4 | 1.07 | 5 | 1.08 | 5 | 1.17 | 5 | 1.48 | |
CqMON1 | 6 | 6 | 5 | 1.41 | 6 | 1.95 | 6 | 1.78 | 6 | 2.10 | |
UDEC4 | Gene | RefFinder | geNorm | NormFinder | BestKeeper | ΔCt | |||||
Rank | GM | Rank | MV | Rank | SV | Rank | SD | Rank | SD | ||
CqACT7 | 1 | 1.32 | 1 | 0.68 | 1 | 0.18 | 3 | 1.08 | 1 | 1.32 | |
CqUBC | 2 | 1.68 | 1 | 0.68 | 2 | 0.51 | 2 | 0.76 | 2 | 1.35 | |
Cq18S | 3 | 2.63 | 2 | 1.07 | 4 | 1.36 | 1 | 0.51 | 4 | 1.78 | |
CqPTB | 4 | 3.66 | 4 | 1.45 | 3 | 1.13 | 4 | 1.40 | 3 | 1.38 | |
CqGAPDH | 5 | 4.73 | 3 | 1.29 | 5 | 1.51 | 5 | 1.43 | 5 | 1.86 | |
CqMON1 | 6 | 6 | 5 | 1.70 | 6 | 1.96 | 6 | 2.05 | 6 | 2.20 |
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Contreras, E.; Martín-Fernández, L.; Manaa, A.; Vicente-Carbajosa, J.; Iglesias-Fernández, R. Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress. Int. J. Mol. Sci. 2023, 24, 15878. https://doi.org/10.3390/ijms242115878
Contreras E, Martín-Fernández L, Manaa A, Vicente-Carbajosa J, Iglesias-Fernández R. Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress. International Journal of Molecular Sciences. 2023; 24(21):15878. https://doi.org/10.3390/ijms242115878
Chicago/Turabian StyleContreras, Estefanía, Lucía Martín-Fernández, Arafet Manaa, Jesús Vicente-Carbajosa, and Raquel Iglesias-Fernández. 2023. "Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress" International Journal of Molecular Sciences 24, no. 21: 15878. https://doi.org/10.3390/ijms242115878