Reference gene validation in Eotetranychus sexmaculatus (Acari: Tetranychidae) feeding on mite-susceptible and mite-resistant rubber tree germplasms.

Reliable reference genes are quite important in calculating gene transcript levels by using real-time quantitative reverse transcription-PCR (RT-qPCR). Eotetranychus sexmaculatus is known as a dangerous mite causing significant yield reduction of rubber tree latex; however, selection of appropriate reference genes for validation of target gene expression in E. sexmaculatus has not been conducted yet. In the present study, nine candidate reference genes were analyzed for their expression stability in different life stages of E. sexmaculatus by using common algorithms including comparative ΔCq method, geNorm, NormFinder and BestKeeper. In addition, a comprehensive analysis software (RefFinder) was used to assign an overall final rank for each candidate gene. The results showed that ß-actin and ß-TUB were the best two reference genes and were subjected to evaluate expression of two protective enzyme genes (EsCu/ZnSOD and EsCAT1) in E. sexmaculatus. We found that the expression of EsCu/ZnSOD and EsCAT1 in E. sexmaculatus feeding on mite-resistant rubber tree germplasm was significantly lower compared with those feeding on mite-susceptible germplasm. These results will facilitate research in revealing molecular mechanisms underlying rubber tree resistance to the spider mite.