Screening and Identification of Reference Genes Under Different Conditions and Growth Stages of Lyophyllum decastes

Internal reference genes are a prerequisite for ensuring the accuracy of gene verification experiments, but few relevant studies on <i>Lyophyllum decastes</i> have investigated the growth cycle and different environmental conditions. In this study, the qPCR results of 22 house-keeping genes were analyzed using <i>GeNorm</i>, <i>BestKeeper</i>, <i>NormFinder</i> and <i>RefFinder</i>. The results revealed that the most stable gene differed under different conditions. Across all developmental stages and under hot, cold, acidic, alkaline, and salt conditions, <i>UBCE</i> gene displays the greatest expression stability. However, <i>EF1b</i>, <i>β-ACT</i>, <i>HSD17B3</i>, and <i>Cyb</i> presented the greatest stability under cold, heat, and acidic conditions, and heavy metal exposure, respectively. To screen for genes suitable for all conditions, <i>RefFinder</i>'s ranking results revealed that <i>UBCE</i> and <i>EF1b</i> ranked in the top 2, demonstrating the highest gene expression stability. In contrast, <i>Cyb</i> was positioned at the bottom of the comprehensive ranking table. This study not only revealed potential factors affecting the suitability of reference genes but also identified optimal reference genes from a set of candidate genes across diverse conditions.