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The development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris. Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris. Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long periods, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable RGs for cold treatments in short and long periods, respectively. Moreover, target genes, two-component-system histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as RGs leads to biased results. Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research., Competing Interests: The authors have declared that no competing interests exist.

Cordyceps militaris is a famous traditional edible and medicinal fungus in Asia, and its fruiting body has rich medicinal value. The molecular mechanism of fruiting body development is still not well understood in C. militaris. In this study, phylogenetically analysis and protein domains prediction of the 14 putative chitinases were performed. The transcription level and enzyme activity of chitinase were significant increased during fruiting body development of C. militaris. Then, two chitinase genes (Chi1 and Chi4) were selected to construct gene silencing strain by RNA interference. When Chi1 and Chi4 genes were knockdown, the differentiation of the primordium was blocked, and the number of fruiting body was significantly decreased approximately by 50% compared to wild-type (WT) strain. The length of the single mature fruiting body was shortened by 27% and 38% in Chi1- and Chi4-silenced strains, respectively. In addition, the chitin content and cell wall thickness were significantly increased in Chi1- and Chi4-silenced strains. These results provide new insights into the biological functions of chitinase in fruiting body development of C. militaris. [ABSTRACT FROM AUTHOR]