Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

Clonostachys rosea is a potential biocontrol fungus that can produce highly resistant chlamydospores under specific conditions. To investigate the genes related to chlamydospore formation, we identified reliable reference genes for quantification of gene expression in C. rosea 67-1 during sporulation. In this study, nine reference genes, actin (ACT), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone (HIS), RNA polymerase II CTD phosphatase Fcp1 (RPP), succinate-semialdehyde dehydrogenase (SSD), TATA-binding protein (TBP), ubiquitin (UBQ), and ubiquitin-conjugating enzyme (UCE), were selected and cloned from 67-1, and their expression stability during chlamydospore formation was determined using reverse transcription quantitative PCR and assessed using the software geNorm, NormFinder and BestKeeper. The Ct values of the candidates ranged from 19.9 to 29.7, among which HIS, ACT and SSD exhibited high expression levels. The statistical analysis showed that ACT and SSD were most stably expressed, while UBQ and GAPDH showed relatively large variations under different culture conditions. Calculation of pairwise variation value indicated that two reference genes were required for precise quantification. Finally, ACT and SSD were selected to normalize gene expression during chlamydospore production in C. rosea 67-1. To the best of our knowledge, this is the first report of SSD as a reference gene. This study will facilitate the accurate quantification of differentially expressed genes during the generation of chlamydospores and contribute to the investigation of the molecular mechanism underlying chlamydospore formation in C. rosea.