Selection of stable reference genes for accurate reverse-transcription quantitative PCR in cotton-herbivore studies...

Virus-induced gene silencing (VIGS) has been a crucial tool for elucidating gene function in Upland cotton (Gossypium hirsutum) due to its complex allotetraploid genome. Reverse-transcription quantitative PCR is routine for measuring gene expression in VIGS studies, yet reference gene stability has not been adequately evaluated when using VIGS especially under biotic stress in cotton. Here, we employed several statistical methods (∆Ct, geNorm, BestKeeper, NormFinder, and weighted rank aggregation) to evaluate the stability of six candidate reference genes (GhACT7, GhPP2A1, GhUBQ7, GhUBQ14, GhTMN5, and GhTBL6) in wild-type and VIGS-infiltrated plants under cotton aphid herbivory stress over time in a fully factorial experiment. Ranked stability analyses overwhelmingly indicated that the frequently used reference genes GhUBQ7 and GhUBQ14 were the least stable whereas GhACT7 and GhPP2A1 were the most stable under VIGS and cotton aphid herbivory stress. Results were validated by comparing normalization methods of the phytosterol biosynthesis gene GhHYDRA1 in response to aphid herbivory. Normalization using GhACT7/GhPP2A1 revealed significant upregulation of GhHYDRA1 in aphid-infested plants. Conversely, normalization using GhUBQ7 reduced sensitivity to detect expression changes, highlighting the importance of stable reference gene selection for accurate expression normalization and interpretation. This study will facilitate future investigations of the genetics underpinning cotton-herbivore interactions necessary for novel pest management biotechnology discovery.