Screening of reference genes in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae) under heavy metal, pesticide, and plant virus stress

The study aims to identify stable reference genes for Sogatella furcifera under the stress of heavy metals, pesticides, and the Southern rice black-streaked dwarf virus (SRBSDV) that it transmits, thereby standardizing gene expression analysis under these adverse conditions.

Using qRT-PCR technology, we evaluated the expression stability of eight candidate reference genes (EF1-α, RPS18, ACT, TUB, GAPDH, RPL9, RPL10, 18S) under the stress of heavy metal cadmium (Cd), the pesticides Flupyrimin and Dinotefuran, and SRBSDV through GeNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder software.

The results demonstrated that all primers for the eight candidate reference genes exhibited high amplification specificity across different stress conditions. Under Cd and Flupyrimin stress, RPL9 and RPL10 were the most stable reference genes; Dinotefuran stress, ACT and RPL9 showed the highest stability; and SRBSDV stress, TUB and RPL9 were the most stable. The stability of the selected reference genes was further validated using gstd1 and Vg1 as target genes.

The results indicated that the combined use of multiple reference genes resulted in a lower standard error compared to single reference genes, providing the optimal normalization standard. This study provides a set of stable reference genes for qRT-PCR-based gene expression analysis of S. furcifera under the stress of Cd, the pesticides Flupyrimin and Dinotefuran, and SRBSDV, laying a foundation for subsequent research on gene function.