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

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

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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.

Original languageEnglish
Article number1427
JournalFrontiers in Physiology
Volume10
DOIs
StatePublished - Nov 19 2019

Bibliographical note

Funding Information:
This project was supported by the National Key R&D Program of China (No. 2016YFD0201134), the Special Fund for Outstanding Research Group in Shanxi Academy of Agricultural Sciences (No. YCX2018D2YS08), the Science and Technology Development Program of Shanxi Province (Agricultural Sector) (No. 2015-TN-03-09), the Science and Technology Leading Project for Agricultural Industry Development in Shanxi Academy of Agricultural Sciences (No. 2019CYYL-07), and an Enrichment Grant, KTRDC, College of Agriculture, Food, and Environment, University of Kentucky.

Publisher Copyright:
© Copyright © 2019 Yang, Gao, Liu, Lu, Zhang, Zhang, Fan, Zhou and Fan.

Keywords

  • Amphitetranychus viennensis
  • RT-qPCR
  • cell-content feeding
  • hawthorn spider mites
  • housekeeping genes
  • reference genes

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

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