Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri

Abstract

Tamarixia radiata (Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in T. radiata, the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for T. radiata has yet been reported. Here, we selected seven housekeeping genes of T. radiate and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using RefFinder software, which contains four different programs (geNorm, ΔCt, BestKeeper, and NormFinder). Pairwise variation was analyzed by geNorm software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: 18S and EF1A for the developmental stage; PRS18 and EF1A for sex, PRS18 and RPL13 for different tissues (head, thorax, abdomen); EF1A and ArgK between two populations; β-tubulin and EF1A for different temperatures (5, 15, 25, 35 °C); and ArgK and PRS18 for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of HSP70 (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from T. radiata for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP.

Selection and validation of reference genes for RT-qPCR-based analyses of Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae)

“…To date, reliable reference genes have been identified for many Hymenoptera species ( Table 3 ), including Cotesia chilonis ( Li et al, 2019 ), Apis mellifera ( Lourenço et al, 2008 ), Lysiphlebia japonica ( Gao et al, 2017a ), Aphidius gifuensis ( Gao et al, 2017b ), Tamarixia radiate ( Guo et al, 2020 ), and Solenopsis invicta ( Cheng et al, 2013 ). A. japonicus is an important natural predator of a range of agricultural pests, and further molecular analyses of this species may thus support further biocontrol efforts.…”

Silencing of V‐ATPase‐E gene causes midgut apoptosis of Diaphorina citri and affects its acquisition of Huanglongbing pathogen

“…The PCR protocol followed that outlined by Guo et al. (2020). The purified PCR product was cloned into the pClone007 Blunt vector (TSINGKE, Beijing, China) for sequencing.…”

“…The PCR reaction system comprised a 25 μL reaction mixture containing LA Taq DNA polymerase (Takara, Shiga, Japan). The PCR protocol followed that outlined by Guo et al (2020). The purified PCR product was cloned into the pClone007 Blunt vector (TSINGKE, Beijing, China) for sequencing.…”

Physiological and Molecular Response Modifications by Ultraviolet-C Radiation in Plutella xylostella and Its Compatibility with Cordyceps fumosorosea

“…RPS13 was used as the reference gene. The relative gene expression was computed using the 2-ΔΔCt method [ 84 , 85 , 86 ]. Details concerning the primers used are given in Table 1 .…”