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Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis
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 pest...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187807/ https://www.ncbi.nlm.nih.gov/pubmed/32372977 http://dx.doi.org/10.3389/fphys.2020.00378 |
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author | Yang, Jing Zhang, Yuying Zhao, Jin Gao, Yue Liu, Zhongfang Zhang, Pengjiu Fan, Jianbin Zhou, Xuguo Fan, Renjun |
author_facet | Yang, Jing Zhang, Yuying Zhao, Jin Gao, Yue Liu, Zhongfang Zhang, Pengjiu Fan, Jianbin Zhou, Xuguo Fan, Renjun |
author_sort | Yang, Jing |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7187807 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-71878072020-05-05 Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis Yang, Jing Zhang, Yuying Zhao, Jin Gao, Yue Liu, Zhongfang Zhang, Pengjiu Fan, Jianbin Zhou, Xuguo Fan, Renjun Front Physiol Physiology 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. Frontiers Media S.A. 2020-04-21 /pmc/articles/PMC7187807/ /pubmed/32372977 http://dx.doi.org/10.3389/fphys.2020.00378 Text en Copyright © 2020 Yang, Zhang, Zhao, Gao, Liu, Zhang, Fan, Zhou and Fan. http://creativecommons.org/licenses/by/4.0/ 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. |
spellingShingle | Physiology Yang, Jing Zhang, Yuying Zhao, Jin Gao, Yue Liu, Zhongfang Zhang, Pengjiu Fan, Jianbin Zhou, Xuguo Fan, Renjun Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis |
title | Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis |
title_full | Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis |
title_fullStr | Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis |
title_full_unstemmed | Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis |
title_short | Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis |
title_sort | selection of reference genes for rt-qpcr analysis under extrinsic conditions in the hawthorn spider mite, amphitetranychus viennensis |
topic | Physiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187807/ https://www.ncbi.nlm.nih.gov/pubmed/32372977 http://dx.doi.org/10.3389/fphys.2020.00378 |
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