Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA Treatments
Halophyte plants, with genes responsive to abiotic stress, are promising candidates to enhance crop stress tolerance, but reliable RT-qPCR analysis requires the precise selection of candidate reference genes (CRGs) due to their inconsistent expression across tissues and stress conditions. In this st...
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2025-06-01
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| author | Seyyed Hamidreza Hashemipetroudi Ali Rezaei Markus Kuhlmann |
| author_facet | Seyyed Hamidreza Hashemipetroudi Ali Rezaei Markus Kuhlmann |
| author_sort | Seyyed Hamidreza Hashemipetroudi |
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| description | Halophyte plants, with genes responsive to abiotic stress, are promising candidates to enhance crop stress tolerance, but reliable RT-qPCR analysis requires the precise selection of candidate reference genes (CRGs) due to their inconsistent expression across tissues and stress conditions. In this study eight CRGs of <i>A. littoralis</i>, <i>AlEF1A</i>, <i>AlRPS3</i>, <i>AlGTFC</i>, <i>AlRPS12</i>, <i>AlUBQ2</i>, <i>AlTUB6</i>, <i>AlACT7</i>, and <i>AlGAPDH1</i>, were analyzed to assess their stability for the normalization of RT-qPCR data under polyethylene glycol (PEG, 20% <i>w</i>/<i>v</i> for drought simulation), abscisic acid (ABA, 100 μM), and cold stress (4 °C) treatments. The result of the algorithms suggested different CRGs for different treatments or tissue types. However the comprehensive analysis indicates that <i>AlEF1A</i> is the most stable CRG for PEG-treated leaf tissue, but <i>AlTUB6</i> is preferable for PEG-treated root tissue, while for PEG-treated leaf and root tissues, <i>AlEF1A</i> can be suggested. For cold-stressed leaf and/or root samples, <i>AlRPS3</i> was the most stable. For ABA-treated leaf and root tissues, <i>AlGTFC</i> and <i>AlEF1A</i> were the most stable CRGs, respectively, whereas <i>AlTUB6</i> was suggested for ABA-treated leaf and root tissues. Collectively, for all stresses combined (PEG, ABA, and cold), <i>AlGTFC</i> was the most stable CRG in leaf samples, while <i>AlRPS3</i> was the most stable in root samples and combined leaf and root samples. The validation analysis indicates a statistically significant difference (<i>p</i> value < 0.05) between normalization with the most and least stable CRGs. This research suggests reliable tissue-specific RGs for <i>A. littoralis</i> under abiotic stresses that can enhances the accuracy of gene expression quantification. |
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| spelling | doaj-art-038d4bc3e6a34acd9bf4fe2b4ae8281f2025-07-25T13:09:53ZengMDPI AGAgronomy2073-43952025-06-01157159610.3390/agronomy15071596Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA TreatmentsSeyyed Hamidreza Hashemipetroudi0Ali Rezaei1Markus Kuhlmann2Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University, P.O. Box 578, Sari 4818166996, IranSchool of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, CanadaRG Heterosis, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Gatersleben, GermanyHalophyte plants, with genes responsive to abiotic stress, are promising candidates to enhance crop stress tolerance, but reliable RT-qPCR analysis requires the precise selection of candidate reference genes (CRGs) due to their inconsistent expression across tissues and stress conditions. In this study eight CRGs of <i>A. littoralis</i>, <i>AlEF1A</i>, <i>AlRPS3</i>, <i>AlGTFC</i>, <i>AlRPS12</i>, <i>AlUBQ2</i>, <i>AlTUB6</i>, <i>AlACT7</i>, and <i>AlGAPDH1</i>, were analyzed to assess their stability for the normalization of RT-qPCR data under polyethylene glycol (PEG, 20% <i>w</i>/<i>v</i> for drought simulation), abscisic acid (ABA, 100 μM), and cold stress (4 °C) treatments. The result of the algorithms suggested different CRGs for different treatments or tissue types. However the comprehensive analysis indicates that <i>AlEF1A</i> is the most stable CRG for PEG-treated leaf tissue, but <i>AlTUB6</i> is preferable for PEG-treated root tissue, while for PEG-treated leaf and root tissues, <i>AlEF1A</i> can be suggested. For cold-stressed leaf and/or root samples, <i>AlRPS3</i> was the most stable. For ABA-treated leaf and root tissues, <i>AlGTFC</i> and <i>AlEF1A</i> were the most stable CRGs, respectively, whereas <i>AlTUB6</i> was suggested for ABA-treated leaf and root tissues. Collectively, for all stresses combined (PEG, ABA, and cold), <i>AlGTFC</i> was the most stable CRG in leaf samples, while <i>AlRPS3</i> was the most stable in root samples and combined leaf and root samples. The validation analysis indicates a statistically significant difference (<i>p</i> value < 0.05) between normalization with the most and least stable CRGs. This research suggests reliable tissue-specific RGs for <i>A. littoralis</i> under abiotic stresses that can enhances the accuracy of gene expression quantification.https://www.mdpi.com/2073-4395/15/7/1596abiotic stressabscisic acidpolyethylene glycolstable reference gene |
| spellingShingle | Seyyed Hamidreza Hashemipetroudi Ali Rezaei Markus Kuhlmann Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA Treatments Agronomy abiotic stress abscisic acid polyethylene glycol stable reference gene |
| title | Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA Treatments |
| title_full | Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA Treatments |
| title_fullStr | Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA Treatments |
| title_full_unstemmed | Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA Treatments |
| title_short | Validation of Reference Genes for Accurate RT-qPCR Normalization in <i>Aeluropus littoralis</i> Under Drought, Cold, and ABA Treatments |
| title_sort | validation of reference genes for accurate rt qpcr normalization in i aeluropus littoralis i under drought cold and aba treatments |
| topic | abiotic stress abscisic acid polyethylene glycol stable reference gene |
| url | https://www.mdpi.com/2073-4395/15/7/1596 |
| work_keys_str_mv | AT seyyedhamidrezahashemipetroudi validationofreferencegenesforaccuratertqpcrnormalizationiniaeluropuslittoralisiunderdroughtcoldandabatreatments AT alirezaei validationofreferencegenesforaccuratertqpcrnormalizationiniaeluropuslittoralisiunderdroughtcoldandabatreatments AT markuskuhlmann validationofreferencegenesforaccuratertqpcrnormalizationiniaeluropuslittoralisiunderdroughtcoldandabatreatments |