Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae)

PLOS ONE, Dec 2019

Liriomyza trifolii is a highly-invasive leafmining insect that causes significant damage to vegetables and horticultural crops worldwide. Relatively few studies have quantified gene expression in L. trifolii using real-time quantitative PCR (RT-qPCR), which is a reliable and sensitive technique for measuring gene expression. RT-qPCR requires the selection of reference genes to normalize gene expression data and control for internal differences between samples. In this study, nine housekeeping genes from L. trifolii were selected for their suitability in normalizing gene expression using geNorm, Normfinder, BestKeeper, the ΔCt method and RefFinder. HSP21.7, which encodes heat shock protein 21.7, was used as a target gene to validate the expression of candidate reference genes. Results indicated that ACTIN and 18S were optimal for developmental stage and low temperature, TUB and 18S showed the most stable expression for sex, and GAPDH and ACTIN were the best reference genes for monitoring gene expression at high temperature. Selection and validation of appropriate reference genes are critical steps in normalizing gene expression levels, which improve the accuracy and quality of expression data. Results of this study provide vital information on reference genes and is valuable in developing a standardized RT-qPCR protocol for functional genomics research in L. trifolii.

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Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae)

July Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) Ya-Wen Chang 0 1 2 Jing-Yun Chen 0 1 2 Ming-Xing Lu 0 1 2 Yuan Gao 0 2 Zi-Hua Tian 0 2 Wei- Rong Gong 0 2 Wei Zhu 0 2 Yu-Zhou Du 0 1 2 0 Funding: This research was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the National Science and Technology Support Program [ 2012BAD19B06 (YZD) ( 1 School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University , Yangzhou , China , 2 Laboratory for Prevention and Control of Alien Pests, Suzhou Entry-Exit Inspection and Quarantine Bureau , Suzhou , China , 3 Joint International Research Laboratory of Agriculture and Agri- Product Safety, Yangzhou University , Yangzhou , China , 4 Plant Protection and Quarantine Station of Jiangsu Province , Nanjing , China , 5 Agricultural Technology Extension Service Center of Guangling District , Yangzhou , China 2 Editor: Yulin Gao, Chinese Academy of Agricultural Sciences Institute of Plant Protection , CHINA Liriomyza trifolii is a highly-invasive leafmining insect that causes significant damage to vegetables and horticultural crops worldwide. Relatively few studies have quantified gene expression in L. trifolii using real-time quantitative PCR (RT-qPCR), which is a reliable and sensitive technique for measuring gene expression. RT-qPCR requires the selection of reference genes to normalize gene expression data and control for internal differences between samples. In this study, nine housekeeping genes from L. trifolii were selected for their suitability in normalizing gene expression using geNorm, Normfinder, BestKeeper, the ΔCt method and RefFinder. HSP21.7, which encodes heat shock protein 21.7, was used as a target gene to validate the expression of candidate reference genes. Results indicated that ACTIN and 18S were optimal for developmental stage and low temperature, TUB and 18S showed the most stable expression for sex, and GAPDH and ACTIN were the best reference genes for monitoring gene expression at high temperature. Selection and validation of appropriate reference genes are critical steps in normalizing gene expression levels, which improve the accuracy and quality of expression data. Results of this study provide vital information on reference genes and is valuable in developing a standardized RT-qPCR - Data Availability Statement: All relevant data are within the paper. protocol for functional genomics research in L. trifolii. Introduction Liriomyza (Diptera: Agromyzidae) is a phyletic genus exhibiting replacement that continues to spread throughout the world [1±3]. L. trifolii Burgess is an invasive pest in China that has Science and Technology Program of Yangzhou [YZ2014171 (YZD)](http://kjj.yangzhou.gov.cn/), and the Basic Research Program of Agricultural application of Suzhou [SNG201602 (JYC)] (http:// szkj.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. caused great losses in agricultural and horticultural crops [2±5]. In mainland China, it was initially discovered in Guangdong Province in 2005 [6±7] and has now been reported in more than ten provinces [8±10]. Both the larval and adult stages of L. trifolii damage crop plants. The larvae feed and damage the foliage, and female adults puncture plant tissue during ovipo sition. Both activities can reduce photosynthesis, increase defoliation, and result in yield loss [11±12]. Due to the rampant use of pesticides and onset of insecticide resistance, the development of more environmentally favorable pest management strategies is imperative. For example, strategies employing RNA interference (RNAi) or gene knockout approaches have been successfully developed for many pests [13±15]. Genetic approaches show great promise in pest management but require functional studies to identify suitable target genes and expression profiles in insects [16±19]. To successfully implement genetic strategies for control of L. trifolii, the use of standardized, real-time quantitative PCR (RT-qPCR) protocols using MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) is critical [ 20 ]. RT-qPCR is widely used to analyze gene expression because of its accuracy, sensitivity, reproducibility and quantitative ability [21±22]. To accurately calculate gene expression using RT-qPCR, it is vital to use appropriate reference genes to normalize the data. The expression of ideal reference genes should be constant in different tissues and experimental conditions. However, recent research has indicated that widely-used reference genes were differentially expressed and only stable during specific conditions [ 23 ]. ACTIN was one of the most stable reference genes that has been used for different d (...truncated)


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Ya-Wen Chang, Jing-Yun Chen, Ming-Xing Lu, Yuan Gao, Zi-Hua Tian, Wei-Rong Gong, Wei Zhu, Yu-Zhou Du. Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae), PLOS ONE, 2017, Volume 12, Issue 7, DOI: 10.1371/journal.pone.0181862