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 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.