Screening Reference Genes for Wine Grapes for Cultivation Under Low-Temperature Stress

The harsh, cold, and dry winters in northern China necessitate burying wine grapevines (Vitis vinifera) for winter protection. In this study, we screened for stably expressed reference genes in wine grapes (V. vinifera) under low-temperature stress at 4 °C (chilling) and −15 °C (freezing). A cold-resistant line “Hanniang 1301” and its cold-sensitive parent ‘Cabernet Sauvignon’ were treated at 4 °C and −15 °C for varying durations. Nineteen candidate reference genes were selected for qPCR analysis. Gene stability under chilling and freezing stress was evaluated using the following five algorithms: Delta CT (ΔCt), geNorm, NormFinder, BestKeeper, and RefFinder. The optimal reference genes under chilling (4 °C) and freezing (−15 °C) conditions were pairs with dual-reference combinations. However, the genes selected differed between chilling and sub-freezing temperatures. For chilling stress (4 °C), EF1α-1 and EF1α-2 were the most stable. Meanwhile, for freezing stress (−15 °C), GAPDH and Actin were optimal. We identified suitable reference genes for gene expression studies in wine grapes under low-temperature stress; this establishes a theoretical foundation for optimizing reference gene selection in plants under other abiotic stresses.