Abstract
Hawthorn spider mite, Amphitetranychus viennensis Zacher, is an economically important arthropod pest for fruit trees and woody ornamental plants. Extensive and repetitive use of synthetic acaricides has led to the development of resistance in A. viennensis. To understand the molecular basis of pesticide resistance, and to develop genetic-based control alternatives (e.g., RNAi-based biopesticides), a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) is needed. In the proceeding phase of this research, we screened for the internal references for RT-qPCR analysis from a pool of A. viennensis housekeeping genes under the intrinsic conditions, including developmental stage, sex, and diapause. Here, we continued our efforts to search for the reference genes under an array of extrinsic conditions, including temperature, humidity, photoperiod, host plant, and dietary RNAi. The stability of these candidate reference genes was investigated using geNorm, NormFinder, BestKeeper, and ΔCt method, respectively. Finally, RefFinder, a statistical platform integrating all four algorisms, provided a comprehensive list of genes for each extrinsic condition: (1) EF1A, α-tubulin and Actin3 were the best candidates for temperature, (2) GAPDH, 18S, and Actin3 were the most stable genes for humidity, (3) V-ATPase B, Actin3, and 18S were the top reference genes for photoperiod, (4) GAPDH, V-ATPase B, and α-tubulin were recommended for host plants, and (5) GAPDH, V-ATPase B, and RPS9 were the top choices for dietary RNAi. Overall, V-ATPase B, GAPDH, and Actin3 were the most commonly selected reference genes in A. viennensis regardless of the experimental conditions, including both intrinsic and extrinsic. Information present here lays the foundation for the genomic and functional genomic research in A. viennensis.
Highlights
A better understanding of gene expression profiles can provide molecular insight into complex mechanisms underlying the effects of stressors on organisms (Nolan et al, 2006)
The R2 ranged between 0.98 and 1.00, and efficiency of real-time quantitative reverse transcription PCR (RT-qPCR) ranged between 90 and 110% (Supplementary Table S1). These results suggest that all primer sets met the standard requirement for RT-qPCR analyses (Taylor et al, 2010)
The reliability and accuracy of RT-qPCR analysis are often affected by the quality of RNA and the efficiency of reverse transcription
Summary
A better understanding of gene expression profiles can provide molecular insight into complex mechanisms underlying the effects of stressors on organisms (Nolan et al, 2006). Realtime quantitative reverse transcription PCR (RT-qPCR) is one of the most effective methods for quantifying gene expression levels because of its specificity, reproducibility, and sensitivity (Ginzinger, 2002; Derveaux et al, 2010). The accuracy of this technique, can be affected by PCR amplification, primer performance and sample quantity (Bustin, 2002; Bustin et al, 2005; Strube et al, 2008). Reliable endogenous reference genes should be used to eliminate bias and reduce errors (Vandesompele et al, 2002). The stability of reference genes should be independent of experimental conditions. Housekeeping genes (HKGs), which are involved in basic metabolism and cellular maintenance, have been used extensively as reference genes for RT-qPCR analysis (Lü et al, 2018)
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