Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED

The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading to most parts of the world owing to its strong ecological adaptability, which is considered as a model insect for stress tolerance studies under rapidly changing environments. Selection of a suitable reference gene for quantitative stress-responsive gene expression analysis based on qRT-PCR is critical for elaborating the molecular mechanisms of thermotolerance. To obtain accurate and reliable normalization data in MED, eight candidate reference genes (beta-act, GAPDH, beta-tub, EF1-alpha, GST, 18S, RPL13A and alpha-tub) were examined under various thermal stresses for varied time periods by using geNorm, NormFinder and BestKeeper algorithms, respectively. Our results revealed that beta-tub and EF1-alpha were the best reference genes across all sample sets. On the other hand, 18S and GADPH showed the least stability for all the samples studied. beta-act was proved to be highly stable only in case of short-term thermal stresses. To our knowledge this was the first comprehensive report on validation of reference genes under varying temperature stresses in MED. The study could expedite particular discovery of thermotolerance genes in MED. Further, the present results can form the basis of further research on suitable reference genes in this invasive insect and will facilitate transcript profiling in other invasive insects.