Evaluation and identification of reference genes for qRT-PCR analysis in bermudagrass roots under alkaline salt stress

Quantitative real-time PCR (qRT-PCR) is a potent technique for gene expression analysis, but its accuracy relies heavily on the selection of stable reference genes. In bermudagrass (Cynodon dactylon) roots under alkaline salt stress, we sought to identify suitable reference genes. Seven candidates-EF1 alpha, PP2A, TIP41, GAPDH, Actin, beta-tubulin, and CACS-were assessed for specificity, amplification efficiency, and expression stability using geNorm, NormFinder, BestKeeper, and RefFinder. All primers exhibited high specificity and efficiency, as evidenced by single, strong bands on agarose gels and single melting peaks in qRT-PCR. Initial Delta Ct value analysis identified EF1 alpha, TIP41, and GAPDH as the most stable genes, with further analysis consistently ranking EF1 alpha as the top reference gene across all software. Validation through transcriptome data and qRT-PCR of selected core genes confirmed EF1 alpha's stability and suitability for gene expression studies in bermudagrass roots under stress. This study offers a thorough evaluation of reference genes for qRT-PCR in bermudagrass and enhances our comprehension of gene expression quantification in this species under alkaline salt stress.