Selection and Validation of Stable Reference Genes for RT-qPCR in Scotogramma trifolii (Lepidoptera: Noctuidae)

The clover cutworm, <i>Scotogramma trifolii</i> Rottemberg (Lepidoptera: Noctuidae), is a globally distributed polyphagous pest causing significant economic losses to agricultural crops. RT-qPCR is a gold-standard technique for gene expression analysis, yet its accuracy depends critically on stable reference genes for data normalization. To address the lack of validated reference genes in <i>S. trifolii</i>, we evaluated six candidate genes (<i>β-actin</i>, <i>RPL9</i>, <i>GAPDH</i>, <i>RPL10</i>, <i>EF1-α</i>, and <i>TUB</i>) across four developmental stages (egg, larva, pupa, and adult) and six adult tissues (head, thorax, abdomen, wings, legs, and antennae) using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Stability analysis identified <i>β-actin</i>, <i>RPL9</i>, and <i>GAPDH</i> as the most reliable reference genes for developmental stage normalization, while <i>RPL10</i>, <i>GAPDH</i>, and <i>TUB</i> were validated for adult tissues. Functional validation using the odorant receptor gene <i>StriOR20</i> revealed significant discrepancies in relative expression levels when normalized with unstable reference genes (<i>TUB</i> and <i>RPL9</i>), emphasizing the necessity of rigorous reference gene selection. This study establishes the first comprehensive reference gene panel for <i>S. trifolii</i>, providing a robust foundation for gene expression studies in this agriculturally important pest.