Reference genes selection for qRT-PCR analysis in Dendroctonus rufipennis

Dendroctonus rufipennis (Coleoptera: Curculionidae) is a major forest pest in North America, yet molecular studies on this species remain limited due to the lack of validated reference genes for quantitative gene expression analysis. This study systematically evaluated eight candidate reference genes (AK, EF1A, RPL32, RPS18, SDHA, TUBA, TUBB, and UBIQ) across diverse experimental conditions and developmental stages of the insect. The expression stability of these genes was assessed using four widely accepted algorithms: geNorm, NormFinder, BestKeeper, and ΔCt method. Our findings demonstrate that RPL32, RPS18, and SDHA exhibit the highest stability across developmental stages; TUBB and RPS18 are optimal for sex-specific and larval facultative diapause studies; RPL32 and RPS18 perform best for adult obligatory diapause termination and short-term cold exposure experiments. The relative expression levels of target gene kr-h1 significantly varied according to normalization with recommended combinations and the least suited reference genes. These findings provide a robust set of reference genes for normalizing gene expression in D. rufipennis, offering a valuable foundation for future molecular research on this ecologically significant pest.