AUTHOR=Yang Jing , Gao Yue , Liu Zhongfang , Lu Junjiao , Zhang Yuying , Zhang Pengjiu , Fan Jianbin , Zhou Xuguo , Fan Renjun 

TITLE=Selection of Reference Genes for RT-qPCR Analysis Under Intrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis (Acarina: Tetranychidae)

JOURNAL=Frontiers in Physiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2019.01427

DOI=10.3389/fphys.2019.01427

ISSN=1664-042X

ABSTRACT=<p>Hawthorn spider mite, <italic>Amphitetranychus viennensis</italic> 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 <italic>A. viennensis</italic> 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 <italic>A. viennensis</italic>, including <italic>18S ribosomal RNA</italic> (<italic>18S</italic>), <italic>28S ribosomal RNA</italic> (<italic>28S</italic>), <italic>Elongation factor 1-alpha</italic> (<italic>EF1A</italic>), <italic>Actin3</italic>, <italic>V-ATP vacuolar-type H<sup>+</sup>-ATPase</italic> (<italic>V-ATPase</italic>), α<italic>-tubulin</italic> (α<italic>-tubulin</italic>), <italic>Ribosomal protein L13</italic> (<italic>RPL13</italic>), <italic>40S ribosomal protein S9</italic> (<italic>RPS9</italic>), and <italic>Glyceraldehyde 3-phosphate dehydrogenase</italic> (<italic>GAPDH</italic>). 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, <italic>V-ATPase</italic>, <italic>Actin3</italic>, and <italic>GAPDH</italic> 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, <italic>A. viennensis, Tetranychus urticae, Tetranychus cinnabarinus</italic>, and <italic>Panonychus citri</italic>, and five hemipterans, <italic>Myzus persicae, Aphis gossypii, Toxoptera citricida, Lipaphis erysimi</italic>, and <italic>Sogatella furcifera</italic>. Not surprisingly, rRNAs and ribosomal proteins, the most abundant RNA species, is the top choice, and follows by <italic>EF1A, Actin, GAPDH</italic>, and <italic>tubulin.</italic> Information present here lays the foundation for the genomic and functional genomic research in cell-content feeding arthropods in general and <italic>A. viennensis</italic> in particular.</p>