Selection and validation of internal control genes for quantitative real-time RT-qPCR normalization of Phlebopus portentosus gene expression under different conditions

Abstract

Phlebopus portentosus (Berk. and Broome) Boedijin is an attractive edible mushroom and considered as the unique bolete to have achieved artificial cultivation in 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 using geNorm, NormFinder and RefFinder under 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), Actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. 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.