Selection and Evaluation of Reference Genes for Quantitative Real-Time Reverse-Transcriptase PCR under Cold Stress in Eggplant (Solanum melongena L.) during Fruit Development

Abstract Background Eggplant (Solanum melongena L.) is a thermophilic vegetable, and its yield and quality are often affected by cold stress. Therefore, identifying the key genes and mechanisms of cold tolerance has become a significant topic in eggplant. qRT-PCR has been widely used to analyse gene expression patterns, and reliable reference genes are necessary for this technique. Methods To select and evaluate suitable reference genes for qRT-PCR, 18 candidate genes selected from transcriptome sequence data were subjected to analysis of their expression stability under natural cold and normal temperature conditions. Four commonly used programs (geNorm, NormFinder, BestKeeper and RefFinder) were used to determine the stabilities of these genes. Results The results showed that D5, D4 and D1 were the three most stable reference genes among the 18 genes. Then, D5, D4 and D1 were compared with commonly used reference genes. The results showed that D5 was still the most stable gene, followed by APRT, D4, and Actin was the least stable gene. Conclusion D5, APRT and D4 were recommended as a reference gene combination for gene expression normalization under cold stress and at normal temperature during fruit development. Our results provide a molecular foundation for further research on the cold tolerance mechanism of eggplant.