Validation of reference genes for quantitative gene expression analysis in Auricularia cornea

Auricularia cornea Ehrenb., previously named A. polytricha (Mont.) Sacc, has become one of the most widely cultivated mushrooms in China. Considerable research has been conducted on its cultivation, pathogen identification, proteomics, and more. However, to the best of our knowledge, no studies have been performed on reference-gene validation in this species. Formerly, reference genes were selected for their expression levels only relied upon from others species, owing to the fact that the gene stability in this species is unknown. In this study, nine candidate genes, including tubulin alpha-1A chain (TUBA1A), β-tubulin (Btu), phosphoglucomutase (Pgm), actin 1 (Act1), protein phosphatase 2A regulatory subunit (PP2A), polyubiquitin (UBQ), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), 18S ribosomal protein (18S) and 28S ribosomal protein (28S), were evaluated among different strains and developmental stages. Four algorithms (i.e., geNorm, NormFinder, BestKeeper and RefFinder) were used to analyze candidate genes. The results revealed that UBQ was the most stable reference gene, while 18S was the least stable. Despite these results, the candidate genes were largely inadequate and only two were considered suitable. Based on candidate gene stability, PP2A and UBQ were identified as a set of usable interior control genes for future analyses in this species. This is the first systematic study conducted for selecting reference genes in A. cornea, and lays the foundation for identifying genes and quantifying gene expression in this species.