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Front. Physiol. | doi: 10.3389/fphys.2019.01427

Selection of reference genes for RT-qPCR analysis under intrinsic conditions in the hawthorn spider mite, Amphitetranychus viennensis (Acarina: Tetranychidae)

 Xuguo Zhou1*,  Jing Yang2, Yue Gao2, Zhongfang Liu2, Junjiao Lu2,  Yuying Zhang2, Pengjiu Zhang2, Jianbin Fan2 and Renjun Fan2*
  • 1University of Kentucky, United States
  • 2Institute of Plant Protection, Shanxi Academy of Agricultural Science, China

Hawthorn spider mite, Amphitetranychus viennensis Zacher, is one of the most devastating sap-sucking pest of deciduous fruit trees. Most recently, we sequenced the genome of A. viennensis. To take advantage of the newly established genomic resources, however, we need to develop a standardized protocol for Real-Time Quantitative Reverse Transcription PCR (RT-qPCR), one of the most effective and broadly adopted methods to quantify gene expression. Following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines, internal references are required to counter the innate technical errors and biases in RT-qPCR analysis. Based on previous knowledge, we hypothesized that internal references for RT-qPCR analysis reside in housekeeping genes. To examine this hypothesis, we assessed the stability of nine housekeeping genes from A. viennensis transcriptome. These candidates, 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), have been used extensively as the reference genes for RT-qPCR analysis. The expression of these candidates under intrinsic conditions, across developmental stage, sex, and diapause, was evaluated by a panel of computational programs, including geNorm, Normfinder, BestKeeper, and ΔCt method. Finally, a specific set of reference genes is recommended for each intrinsic conditions, respectively, based on RefFinder, a comprehensive analytical platform integrating all four above-mentioned algorisms.
Overall, V-ATPase, Actin3, and GAPDH are consistently stably expressed across all the intrinsic conditions in A. viennensis. In addition, we compared reference genes recommended for different developmental stages among the nine sap-sucking 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 for the reference gene(s), and follows by EF1A, Actin, GAPDH, and tubulin, which all exceed 10% chance of been selected. Information present here lays the foundation for the genomic and functional genomic research in sap-sucking arthropods in general and A. viennensis in particular.

Keywords: RT-qPCR, Reference genes, housekeeping genes, hawthorn spider mites, Amphitetranychus viennensis, cell-content feeding

Received: 06 Aug 2019; Accepted: 04 Nov 2019.

Copyright: © 2019 Zhou, Yang, Gao, Liu, Lu, Zhang, Zhang, Fan and Fan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Xuguo Zhou, University of Kentucky, Lexington, United States, xuguozhou@uky.edu
Prof. Renjun Fan, Institute of Plant Protection, Shanxi Academy of Agricultural Science, Taiyuan, 030031, Shanxi Province, China, rjfan@163.com