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Journal of Integrative Agriculture  2019, Vol. 18 Issue (4): 854-864    DOI: 10.1016/S2095-3119(18)61973-2
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Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development
ZHENG Lu-ying1, 2*, ZHANG Zhi-jun2*, ZHANG Jin-ming2, LI Xiao-wei2, HUANG Jun2, LIN Wen-cai2, LI Wei-di2, LI Chuan-ren1, LU Yao-bin2 
1 College of Agriculture, Yangtze University, Jingzhou 434025, P.R.China
2 Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R.China
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Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic.  Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species.  A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR.  However, the accuracy and consistency of this technique depend on the reference gene(s) selected.  In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder.  The results showed that EF1-β, RP-L32 and RP-18S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm.  RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis.  Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase (JHAMT) gene.  Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages.  The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.
Keywords:  Phenacoccus solenopsis        RT-qPCR        sexual dimorphic development        reference gene        gene stability  
Received: 16 October 2017   Accepted:
Fund: This work was supported by the National Natural Science Foundation of China (31270580), the Key Research and Development Program of Zhejiang Province, China (2018C02036), the Key Technologies R&D Program of China during the 12th Five-Year Plan period (2012BAD19B06) and the Special Fund for Agro-scienti?c Research in the Public Interest of China (201103026).
Corresponding Authors:  Correspondence ZHANG Zhi-jun, E-mail:; LU Yao-bin, E-mail:   
About author:  * These authors contributed equally to this study.
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ZHENG Lu-ying
ZHANG Zhi-jun
ZHANG Jin-ming
LI Xiao-wei
LIN Wen-cai
LI Wei-di
LI Chuan-ren
LU Yao-bin

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ZHENG Lu-ying, ZHANG Zhi-jun, ZHANG Jin-ming, LI Xiao-wei, HUANG Jun, LIN Wen-cai, LI Wei-di, LI Chuan-ren, LU Yao-bin. 2019. Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development. Journal of Integrative Agriculture, 18(4): 854-864.

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