Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but for normalization of data requires the use of stable reference genes. However, suitable reference genes are still not known in the case of Phenacoccus solenopsis under variable experimental treatments. The present study focused on the identification of stable housekeeping genes as a reference for analysis under different abiotic and biotic factors in P. solenopsis. We analyzed the relative expression of six commonly used candidate reference genes in different developmental stages, host-feeding assay, temperature treatments and field distribution conditions. Expression stabilities were analyzed by geNorm, NormFinder, and RefFinder. Under developmental and field distribution conditions, β-Tubulin was found to be most stable reference genes followed by rpl32 and α-Tubulin. In the case host feeding treatment conditions, β-Tubulin and α-tubulin identified to be the most stable reference genes, while in temperature stress, a combination of α-Tubulin and rpl32 found to be suitable for normalizing the RT-qPCR data. Further, the above-identified genes were validated using RT-qPCR based gene expression analysis of four objective genes namely, Myoinhibitory peptides (MIPs), Zinc_metalloprotease (Zn_Mp), fatty acid synthase (fas) and alpha-glucosidase. Identified reference genes will facilitate gene expression studies in future under different stress treatments in P. solenopsis.