Abstract
Hawthorn spider mite, Amphitetranychus viennensis Zacher, is one of the most devastating pests of deciduous fruit trees. The overall goal of this research is to develop a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) analysis in A. viennensis following the MIQE (minimum information for publication of Quantitative real time PCR experiments) guidelines. Based on the previous knowledge, we hypothesized that internal references for RT-qPCR analysis reside in housekeeping genes (HKGs). To test this hypothesis, we examined the stability of nine HKGs from A. viennensis, including 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), Elongation factor 1-alpha (EF1A), Actin3, V-ATP vacuolar-type H+-ATPase (V-ATPase), α-tubulin (α-tubulin), Ribosomal protein L13 (RPL13), 40S ribosomal protein S9 (RPS9), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The expression profile of these candidates under intrinsic conditions was evaluated by a panel of computational programs, including geNorm, Normfinder, BestKeeper, and ΔCt method. Based on RefFinder, a comprehensive software integrating all four above-mentioned algorithms, V-ATPase, Actin3, and GAPDH are the top three reference genes, which are stably expressed across all the intrinsic conditions, including developmental stage, sex, and diapause. In addition, we compared reference genes recommended for different developmental stages among the nine cell-content feeding arthropods, including four spider mites, A. viennensis, Tetranychus urticae, Tetranychus cinnabarinus, and Panonychus citri, and five hemipterans, Myzus persicae, Aphis gossypii, Toxoptera citricida, Lipaphis erysimi, and Sogatella furcifera. Not surprisingly, rRNAs and ribosomal proteins, the most abundant RNA species, is the top choice, and follows by EF1A, Actin, GAPDH, and tubulin. Information present here lays the foundation for the genomic and functional genomic research in cell-content feeding arthropods in general and A. viennensis in particular.
Highlights
Analyzing differential gene expression in various biosynthesis pathways is an integral part of the genomics research (Ginzinger, 2002; Van Guilder et al, 2008)
We examined the stability of nine Housekeeping genes (HKGs) identified from A. viennensis transcriptome under different experimental conditions
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% (Table 1), which are consistent with previous studies (Taylor et al, 2010)
Summary
Analyzing differential gene expression in various biosynthesis pathways is an integral part of the genomics research (Ginzinger, 2002; Van Guilder et al, 2008). Reproducibility, and sensitivity, real-time quantitative reverse transcription PCR (RT-qPCR) is considered by many as one of the best methods to quantify gene expression (Valasek and Repa, 2005; Park et al, 2008). To ensure reliability and accuracy, one critical step in RT-qPCR analysis is to use reference genes as internal controls from the same samples (Vandesompele et al, 2002). Housekeeping genes (HKGs), which are constitutively expressed and maintain the basic cellular functions in the cell, have been used extensively as internal references for RT-qPCR analysis (Yang et al, 2015b, 2018). Reference genes should be stably expressed across different biotic and abiotic conditions. We have not yet identify a reference gene can be stably expressed across any given experimental conditions. A systematic and customized study of reference genes for each tested species is critically important (Gutierrez et al, 2008; Hruz et al, 2011; Kozera and Rapacz, 2013)
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