Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

The brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae) can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens, including two different treatments (resistant or susceptible rice) and three virulent N. lugens populations. Three software programs (BestKeeper, Normfinder and geNorm) were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, β-ACT, β-TUB and α-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and α-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and α-TUB were the most suitable reference genes in N. lugens. These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.