Selection of reliable reference genes for gene expression studies in Botrytis cinerea

- This study first evaluates reference genes systematically in Botrytis cinerea.
- UBQ gene is identified as the most reliable reference gene in B. cinerea.
- The expressions of OA related genes don't change much during confrontation test.

Botrytis cinerea is an important plant pathogen causing grey mold disease in a wide range of plant species. The aim of this study was to identify reliable reference genes that can be used for the analysis of relative gene expression in B. cinerea with quantitative real-time reverse transcription PCR (qRT-PCR). Six commonly used housekeeping genes including actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UCE), α-tubulin (α-TUB) and β-tubulin (β-TUB) were selected to test their expression stabilities in B. cinerea treated with different concentration of oxalic acid (1, 5 and 10 mM) and confronted with antagonistic Trichoderma afroharzianum. Four in silico algorithms (geNorm, BestKeeper, NormFinder and Comparative ΔCt) were applied to evaluate the expression stabilities of these genes, and the UBQ gene was identified as the most stably expressed. It was used to normalize the expression levels of three genes related to oxalic acid production (NADPH, VEL1 and OAH) when B. cinerea was challenged by T. afroharzianum. The results of this study are useful for gene expression analysis in B. cinerea.