Selection and validation of internal control genes for quantitative real-time RT–_qPCR normalization ofPhlebopus portentosusgene expression under different conditions

AbstractPhlebopus portentosus(Berk. and Broome) Boedijin is an attractive edible mushroom and considered as the unique bolete to have achieved artificial cultivationin vitro. Gene expression analysis has become widely used in researches of edible fungi and is important to uncover the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable results of gene expression analysis by RT–qPCR. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on the expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated usinggeNorm, NormFinderandRefFinderunder different conditions and developmental stages. The results revealed that MSF1-domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA binding protein 1 (TBP1) and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1),Actinand ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The geneSYBwas selected based on the transcriptome results and was identified as a novel reference gene inP. portentosus. This is the first detailed study for identification reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.