In order to precisely assess gene expression level, a suitable internal reference gene must be chosen to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For greenbug, Schizaphis graminum, a suitable reference gene for assessing the level of transcriptional expression of target genes has yet to be explored. In our study, eight reference genes, elongation fator 1 beta (Ef1β), TATA box binding protein (TBP), alpha-tubulin (α-TUB), 18S ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), actin (ACT), and ribosomal protein L18 (RPL18) were evaluated in S. graminum at different developmental stages, tissues, and insecticide treatments. To further explore whether these genes are suitable to serve as internal control, three software-based approaches (geNorm, BestKeeper, and NormFinder), ΔCt method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized gene expression data of three target genes, heat shock protein gene (HSP70), cytocrome P450 gene (SgraCYP18A1), and glutathione S-transferase (GST). We found that the most suitable reference genes varied considerably under different experimental conditions. For developmental stages, α-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACT were suitable reference genes; for insecticide treatments, 28S and α-TUB were suitable for normalizations of expression data. In addition, 28S and α-TUB were the suitable reference gene as they had the most stable expression among different developmental stages, tissues and insecticide treatments. This should be useful for the selection of the suitable reference genes to obtain reliable RT-qPCR data in the gene expression of S. graminum.

To explore resistant mechanism of wild oat to fenoxaprop-p-ethyl, the susceptibility of Acetyl-CoA Carboxylase (ACCase) from 24 wild oat populations to fenoxaprop-p-ethyl, the level of gene expression, and mutation site of ACCase were conducted. In vitro ACCase activities were solated and measured by enzyme-linked immunosorbent assay kit (ELISA) assays, the results indicated that the IC ₅₀ value of the ACCase of the most unsusceptible to fenoxaprop-p-ethyl in the wild oat population from Yexian2017 (W24) was 7206.557-fold compared to that of the ACCase of most susceptible to fenoxaprop-p-ethyl in the wild oat population from Queshan (W11). The differential expression of genes in wild oat treated by the IC ₅₀ fenoxaprop-p-ethyl concentration (6.9 mg/L) for 24 hours using RNA-seq, digital gene expression (DGE) profling was conducted. We found that 8 unigenes annotated as ACCase genes, 0 up-regulaed expression and 3 down-regulated expression were observed. The down-regulaed expressed ACCase was selected for qPCR in the relative susceptible population were significantly more suppressed than the three relative resistant populations. The mutations point of ACCase, Ile-1781-Leu, Trp-1999-Cys, Trp-2027-Cys, Ile-2041-Asn, Asp-2078-Gly, Cys-2088-Arg published were not found in the populations tested by multiple sequence alignment with a model complete ACCase sequence of Alopecurus myosuroides . These findings suggest that ACCase plays a critical role in the development of wild oat resistance to fenoxaprop-p-ethyl.