Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek-Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.