Hostname: page-component-77f85d65b8-9nbrm Total loading time: 0 Render date: 2026-04-12T22:15:04.300Z Has data issue: false hasContentIssue false
  • English
  • Français

Screening of reference genes using real-time quantitative PCR for gene expression studies in Neoseiulus barkeri Hughes (Acari: Phytoseiidae)

Published online by Cambridge University Press:  29 October 2018

C. Wang ,
J. Yang ,
Q. Pan ,
S. Yu ,
R. Luo ,
H. Liu ,
H. Li ,
L. Cong  and
C. Ran
C. Wang
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
J. Yang
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
Q. Pan
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
S. Yu
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
R. Luo
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
H. Liu
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
H. Li
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
L. Cong
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
C. Ran*
Affiliation:
Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712, China
*
*Author for correspondence Phone: +86-23-6834-9798 Fax: 023-68349005 E-mail: ranchun@cric.cn
Get access Rights & Permissions [Opens in a new window]

Abstract

A stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms. Neoseiulus barkeri Hughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACT and Hsp40 were the top two stable reference genes in developmental stages. The Hsp60 and Hsp90 were the most stable reference genes in various acaricides stress. For alterations in temperature, Hsp40 and α-TUB were the most suitable reference genes. About UV stress, EF1α and α-TUB were the best choice, and for the different prey stress, β-ACT and α-TUB were best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression in N. barkeri.

Keywords

RT-qPCRNeoseiulus barkerireference genesgene expression

Information

Type
Research Papers
Information
Bulletin of Entomological Research , Volume 109 , Issue 4 , August 2019 , pp. 443 - 452
Check for updates
Copyright
Copyright © Cambridge University Press 2018 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

Footnotes

†

These authors contributed equally to this study.

References

Andersen, C.L., Jensen, J.L. & Ørntoft, T.F. (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64, 5245–5250.Google Scholar
Bagnall, N.H. & Kotze, A.C. (2010) Evaluation of reference genes for real-time PCR quantification of gene expression in the Australian sheep blowfly, Lucilia cuprina. Medical and Veterinary Entomology 24, 176–181.Google Scholar
Bansal, R., Mamidala, P., Mian, M.A.R., Mittapalli, O. & Michel, A.P. (2012) Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae). Journal of Economic Entomology 105, 1432–1438.Google Scholar
Bas, A., Forsberg, G., Hammarström, S. & Hammarström, M.L. (2004) Utility of the housekeeping genes 18S rRNA, β-actin and glyceraldehyde-3-phosphate- dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scandinavian Journal of Immunology 59, 566–573.Google Scholar
Bonde, J. (1989) Biological studies including population growth parameters of the predatory mite Amblyseius barkeri [Acarina.: Phytoseiidae] at 25°C in the laboratory. Biocontrol 34, 275–287.Google Scholar
Bustin, S.A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25, 169–193.Google Scholar
Bustin, S.A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology 29, 4021–4022.Google Scholar
Bustin, S.A., Benes, V., Nolan, T. & Pfaffl, M.W. (2005) Quantitative real-time RT-PCR – a perspective. Journal of Molecular Endocrinology 34, 597–601.Google Scholar
Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. & Wittwer, C.T. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55, 611–622.Google Scholar
Chapuis, M.P., Tohidi-Esfahani, D., Dodgson, T., Blondin, L., Ponton, F., Cullen, D., Simpson, S.J. & Sword, G.A. (2011) Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyzes of density-dependent behavioural plasticity in the Australian plague locust. BMC Molecular Biology 12, 7.Google Scholar
Chervoneva, I., Li, Y.Y., Schulz, S., Croker, S., Wilson, C., Waldman, S.A. & Hyslop, T. (2010) Selection of optimal reference genes for normalization in quantitative RT-PCR. BMC Bioinformatics 11, 253.Google Scholar
De Boer, M.E., De Boer, T.E., Mariën, J., Timmermans, M.J.T.N., Nota, B., Van Straalen, N.M., Ellers, J. & Roelofs, D. (2009) Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola). BMC Molecular Biology 10, 54.Google Scholar
De Boer, M.E., Berg, S., Timmermans, M.J., Den Dunnen, J.T., Van Straalen, N.M., Ellers, J. & Roelofs, D. (2011) High throughput nano-liter RT-qPCR to classify soil contamination using a soil arthropod. BMC Molecular Biology 12, 11.Google Scholar
Derveaux, S., Vandesompele, J. & Hellemans, J. (2010) How to do successful gene expression analysis using real-time PCR. Methods 50, 227–230.Google Scholar
Feng, L.Y., Yu, Q., Li, X., Ning, X.H., Wang, J., Zou, J.J., Zhang, L.L., Wang, S., Hu, J.J., Hu, X. L. & Bao, Z.M. (2013) Identification of reference genes for qRT-PCR analysis in Yesso Scallop Patinopecten yessoensis. PLoS ONE 8, e75609.Google Scholar
Ginzinger, D.G. (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Experimental Hematology 30, 503–512.Google Scholar
Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. (1996) Real time quantitative PCR. Genome Research 6, 986–994.Google Scholar
Hessein, N.A. & Parrella, M.P. (1990) Predatory mites help control thrips on floriculture crops. California Agriculture 44, 19–21.Google Scholar
Horňáková, D., Matoušková, P., Kindl, J., Valterová, I. & Pichová, I. (2010) Selection of reference genes for real-time polymerase chain reaction analysis in tissues from Bombus terrestris and Bombus lucorum of different ages. Analytical Biochemistry 397, 118–120.Google Scholar
Huggett, J., Dheda, K., Bustin, S. & Zumla, A. (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity 6, 279–284.Google Scholar
Jiang, H.B., Liu, Y.H., Tang, P. A., Zhou, A.W. & Wang, J.J. (2010) Validation of endogenous reference genes for insecticide-induced and developmental expression profiling of Liposcelis bostsrychophila (Psocoptera: Liposcelididae). Molecular Biology Reports 37, 1019–1029.Google Scholar
Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, M., Jona´k, J., Lind, K., Sindelka, R., Sjöback, R., Sjögreen, R., Strömbom, L., Stählberg, A. & Zoric, N. (2006) The real-time polymerase chain reaction. Molecular Aspects of Medicine 27, 95–125.Google Scholar
Li, R.M., Xie, W., Wang, S.L., Wu, Q.J., Yang, N., Yang, X., Pan, H.P., Zhou, X.M., Bai, L.Y., Xu, B.Y., Zhou, X.G. & Zhang, Y.J. (2013) Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS ONE 8, e53006.Google Scholar
Lopez-Pardo, R., de Galarreta, J.I.R. & Ritter, E. (2013) Selection of housekeeping genes for qRT-PCR analysis in potato tubers under cold stress. Molecular Breeding 31, 39–45.Google Scholar
Lord, J.C., Hartzer, K., Toutges, M. & Oppert, B. (2010) Evaluation of quantitative PCR reference genes for gene expression studies in Tribolium castaneum after fungal challenge. Journal of Microbiological Methods 80, 219–221.Google Scholar
Lourenco, A.P., Mackert, A., Cristino, A.D. & Simoes, Z.L.P. (2008) Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie 39, 372–U333.Google Scholar
Lu, Y., Yuan, M., Gao, X., Kang, T., Zhan, S., Hu, W. & Li, G.H. (2013). Identification and validation of reference genes for the normalization of gene expression data in qRT-PCR analysis in Aphis gossypii (Hemiptera: Aphididae). PLoS ONE 8, e68059.Google Scholar
Ma, K.S., Li, F., Liang, P.Z., Chen, X.W., Liu, Y. & Gao, X.W. (2016) Identification and validation of reference genes for the normalization of gene expression data in qRT-PCR analysis in Aphis gossypii (Hemiptera: Aphididae). Journal of Insect Science 16, 1–9.Google Scholar
Mamidala, P., Rajarapu, S.P., Jones, S.C. & Mittapalli, O. (2011) Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius. Journal of Economic Entomology 48, 947–951.Google Scholar
Mariany, A.M., Bianca, M.M., Laura, C.L., Mark, D.L., Douglas, B.W. & Zhu, F. (2016) Selection of reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae. International Journal of Biological Sciences 12, 1129–1139.Google Scholar
Mehta, R., Birerdinc, A., Hossain, N., Afendy, A., Chandhoke, V., Younossi, Z. & Baranova, A. (2010) Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples. BMC Molecular Biology 11, 39.Google Scholar
Moraes, G.J.D., McMurtry, J.A., Denmark, H.A. & Campos, C.B. (2004) A revised catalog of the mite family Phytoseiidae. Zootaxa 434, 1–494.Google Scholar
Niu, J.Z., Dou, W., Ding, T.B., Yang, L.H., Shen, G.M. & Wang, J.J. (2012) Evaluation of suitable reference genes for quantitative RT-PCR during development and abiotic stress in Panonychus citri (McGregor) (Acari: Tetranychidae). Molecular Biology Reports 39, 5841–5849.Google Scholar
Peng, R., Zhai, Y.F., Ding, H., Di, T.Y., Zhang, T., Li, B., Shen, W.D. & Wei, Z.G. (2012) Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm Bombyx mori. Acta Biochimica Et Biophysica Sinica 44, 614–622.Google Scholar
Petrushov, A.Z. (1992) Pyrethroid resistance in the predacious mite Amblyseius barkeri. Bulletin OEPP/EPPO Bulletin 22, 471–473.Google Scholar
Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29, e45.Google Scholar
Pfaffl, M.W., Tichopad, A., Prgomet, C. & Neuvians, T.P. (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – excel-based tool using pair-wise correlations. Biotechnology Letters 26, 509–515.Google Scholar
Ponton, F., Chapuis, M.P., Pernice, M., Sword, G.A. & Simpson, S.J. (2011) Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. Journal of Insect Physiology 57, 840–850.Google Scholar
Radonić, A., Thulke, S., Mackay, I.M., Landt, O., Siegert, W. & Nitsche, A. (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications 313, 856–862.Google Scholar
Scharlaken, B., De Graaf, D.C., Goossens, K., Brunain, M., Peelman, L.J. & Jacobs, F.J. (2008) Reference gene selection for insect expression studies using quantitative real-time PCR: the head of the honeybee, Apis mellifera, after a bacterial challenge. Journal of Insect Science 8, 1–10.Google Scholar
Schmittgen, T.D. & Livak, K.J. (2008) Analyzing real-time PCR data by the comparative CT method. Nature Protocols 3, 1101–1108.Google Scholar
Shen, G.M., Jiang, H.B., Wang, X.N. & Wang, J.J (2010) Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). BMC Molecular Biology 11, 76.Google Scholar
Shen, G.M., Huang, Y., Jiang, X.Z., Dou, W. & Wang, J.J. (2013) Effect of β-cypermethrin exposure on the stability of nine housekeeping genes in Bactrocera dorsalis (Diptera: Tephritidae). Florida Entomologist 96, 442–450.Google Scholar
Snell, T.W., Brogdon, S.E. & Morgan, M.B. (2003) Gene expression profiling in ecotoxicology. Ecotoxicology 12, 475–483.Google Scholar
Steinau, M., Rajeevan, M.S. & Unger, E.R. (2006) DNA and RNA references for qRT-PCR assays in exfoliated cervical cells. Journal of Molecular Diagnostics 8, 113–118.Google Scholar
Sun, W., Jin, Y., He, L., Lu, W. & Li, M.L. (2010) Suitable reference gene selection for different strains and developmental stages of the carmine spider mite, Tetranychus cinnabarinus, using quantitative real-time PCR. Journal of Insect Science 10, 208.Google Scholar
Tabunoki, H., Ode, H., Banno, Y., Katsuma, S., Shimada, T., Mita, K., Yamamoto, K., Sato, R., Ishii-Nozawa, R. & Satoh, J. (2011) BmDJ-1 is a key regulator of oxidative modification in the development of the silkworm, Bombyx mori. PLoS ONE 6, e17683.Google Scholar
Thellin, O., Zorzi, W., Lakaye, B., Hennen, G., Coumans, B., Hennen, G., Grisar, T., Lgout, A. & Heinen, E. (1999) Housekeeping genes as internal standards: use and limits. Journal of Biotechnology 75, 291–295.Google Scholar
Tong, Z.G., Gao, Z.H., Wang, F., Zhou, J. & Zhang, Z. (2009) Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Molecular Biology 10, 71.Google Scholar
Toutges, M.J., Hartzer, K., Lord, J. & Oppert, B. (2010) Evaluation of reference genes for quantitative polymerase chain reaction across life cycle stages and tissue types of Tribolium castaneum. Journal of Agricultural and Food Chemistry 58, 8948–8951.Google Scholar
Tunbridge, E. M., Eastwood, S.L. & Harrison, P.J. (2011) Changed relative to what? Housekeeping genes and normalization strategies in human brain gene expression studies. Biological Psychiatry 69, 173–179.Google Scholar
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Roy, N.V., De Paepe, A. & Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, 0034.1–0034.11.Google Scholar
VanGuilder, H.D., Vrana, K.E. & Freeman, W.M. (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44, 619–626.Google Scholar
Veazey, K.J. & Golding, M.C. (2011) Selection of stable reference genes for quantitative RT-PCR comparisons of mouse embryonic and extra-embryonic stem cells. PLoS ONE 6, e27592.Google Scholar
Veerman, A. (1992) Diapause in phytoseiid mites: a review. Experimental and Applied Acarology 14, 1–60.Google Scholar
Wan, H.J., Zhao, Z.G., Qian, C.T., Sui, Y.H., Malik, A.B. & Chen, J.F. (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Analytical Biochemistry 399, 257–261.Google Scholar
Xia, B., Zou, Z.W., Li, P.X. & Lin, P. (2012) Effect of temperature on development and reproduction of Neoseiulus barkeri (Acari: Phytoseiidae) fed on Aleuroglyphus ovatus. Experimental and Applied Acarology 56, 33–41.Google Scholar
Xie, F., Sun, G., Stiller, J.W. & Zhang, B. (2011) Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database. PLoS ONE 6, e26980.Google Scholar
Xu, J., Xu, Z.C., Zhu, Y.J., Luo, H.M., Qian, J., Ji, A., Hu, Y., Sun, W., Wang, B., Song, J., Sun, C. & Chen, S. (2014) Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum. Current Microbiology 68, 120–126.Google Scholar
Yang, C.G., Wang, X.L., Tian, J., Liu, W., Wu, F., Jiang, M & Wen, H. (2013) Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus). Gene 527, 183–192.Google Scholar
Zhu, F., Xu, J., Palli, R., Ferguson, J. & Palli, S.R. (2011) Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata. Pest Management Science 67, 175–182.Google Scholar
Supplementary material: File

Wang et al. supplementary material

Table S1

Download Wang et al. supplementary material(File)
File 48.1 KB