Selection and validation of reference genes for quantifying gene expression in the Gill's mealybug.

The Gill's mealybug, Ferrisia gilli Gullan, (Hemiptera: Pseudococcidae) has emerged as a major pest of pistachio in California. Because F. gilli is only relatively recently described, there are no validated reference genes to normalize the expression data from real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in this species. We selected and validated 8 commonly used reference genes (RPS8, TBP, UBQE2, RPL7, RPL5, RPL40, RPLP1, and HEL) for expression stability in F. gilli. These genes were evaluated in 5 different geographical populations of F. gilli collected from organic and conventionally grown pistachio orchards. Candidate reference genes were also evaluated in F. gilli fed with 4 plant hosts: pistachio, almond, grapes, and lima beans. The stability of candidate genes was analyzed using 4 software algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Three genes RPS8, RPL40, and RPL7 encoding for ribosomal proteins were identified as the most stable across the treatments and thus were recommended for normalizing the qRT-PCR data. These findings will support resistance monitoring, molecular toxicology, and functional genomics research in F. gilli.