Selection and Validation of Housekeeping Genes for RT-qPCR Normalization Studies Involving Potato Plants Infected with PVY

Accurate data normalization is essential for quantitative real-time polymerase chain reaction (RT-qPCR) studies. However, studies on suitable reference genes for gene expression analysis remain scarce. With RT-qPCR offering increased sensitivity, reproducibility, and a wide dynamic range, the necessity for a robust internal control gene for normalization has become more stringent. To aid in quantifying gene expressions in potato plants infected with potato virus Y (PVY), we investigated the expression profiles of five candidate housekeeping genes in potato plants, ACT (β-actin), TUB (β-tubulin), EF1α (Elongation factor 1-alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and RPL19 (ribosomal protein L19), across different strains of PVY (PVYO, PVYN, PVYNTN, and PVYN-Wi). Ct values from technical replicates were averaged using four different algorithms – geNorm, NormFinder, Comparative ∆Ct, and BestKeeper – and a comprehensive analysis using an online software, RefFinder. Our findings identified TUB and EF1α as the most stable reference genes for PVYO, TUB and GAPDH for PVYN, ACT and RPL19 for PVYNTN, and EF1α and TUB for PVYN-Wi. This research sheds light on the understanding of reference gene stability in potato plants infected with various PVY strains and provides foundational support for standardized RT-qPCR analyses on plants infected with this virus.