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. 2020 Oct 10;11(10):1178.
doi: 10.3390/genes11101178.

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

Chang-Fei Guo  1   2 , Hui-Peng Pan  1   2 , Li-He Zhang  1   2 , Da Ou  1   2 , Zi-Tong Lu  1   2 , Muhammad Musa Khan  1 , Bao-Li Qiu  1   2   3
Affiliations

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

Chang-Fei Guo et al. Genes (Basel). .

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.

Keywords: RT-qPCR analysis; RefFinder; Tamarixia radiata; gene expression; reference gene.

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Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1
Agarose gel electrophoresis of these seven candidate reference genes. M, Molecular marker. Templates in the polymerase chain reactions (PCRs) were as follows: (1) 18S; (2) Actin; (3) ArgK; (4) EF1A; (5) RPL13; (6) RPS18; and (7) β-tubulin.
Figure 2
Figure 2
Melting curves of the seven candidate reference genes in Tamarixia radiata. (A) 18S, (B) Actin, (C) ArgK, (D) EF1A, (E) RPL13, (F) RPS18, and (G) β-tubulin.
Figure 3
Figure 3
Standard curves of the seven candidate reference genes. (A) 18S, (B) Actin, (C) ArgK, (D) EF1A, (E) RPL13, (F) RPS18, and (G) β-tubulin.
Figure 4
Figure 4
Expression profiles of the seven candidate reference genes in all six experiments for T. radiata. The expression levels of the reference genes are shown in terms of the Ct-value for each experimental condition. (A) Developmental stage, (B) sex, (C) tissue, (D) population, (E) temperature, and (F) diet.
Figure 5
Figure 5
Pairwise variation (V) values using geNorm based on different comparisons: developmental stage, sex, tissue, population, temperature and diet.
Figure 6
Figure 6
Stability of the seven candidate reference gene expressions in T. radiata under different treatment conditions analyzed using RefFinder. A lower Geomean value indicates a more stable expression based on RefFinder. (A) Developmental stage, (B) sex, (C) tissue, (D) population, (E) temperature, and (F) diet.
Figure 7
Figure 7
Relative gene expression of HSP70 in different temperatures. The relative abundance of HSP70 in different temperatures were normalized to the most stable (A: β-tubulin and EF1A) and least stable (B: RPL13 and ArgK) reference genes, respectively. The values are means ± SE (Standard error). The significant differences are indicated by different letters, e.g., a, b, c (p < 0.05).
Figure 8
Figure 8
Relative gene expression of HSP70 in different tissues of T. radiata. The relative abundance of HSP70 in the head, thorax and abdomen were normalized to the most stable (A: RPL13 and RPS18) and least stable (B: β-tubulin and ArgK) reference genes, respectively. The values are means + SE. Normalized to head. The significant differences are indicated by different letters, e.g., a, b, c (p < 0.05).
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References

    1. Bove J.M. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 2006;88:7–37.
    1. Wang N., Pierson E.A., Setubal J.C., Xu J., Levy J.G., Zhang Y.Z., Li J.Y., Rangel L.T., Martins J.J. The Candidatus Liberibacter-host interface: Insights into pathogenesis mechanisms and disease control. Annu. Rev. Phytopathol. 2017;55:451–482. doi: 10.1146/annurev-phyto-080516-035513. - DOI - PubMed
    1. Zhou C.Y. The status of citrus Huanglongbing in China. Trop. Plant Pathol. 2020;45:279–284. doi: 10.1007/s40858-020-00363-8. - DOI
    1. Bassanezi R.B., Lopes S.A., De Miranda M.P., Wulff N.A., Volpe H.X.L., Ayres A.J. Overview of citrus huanglongbing spread and management strategies in Brazil. Trop. Plant Pathol. 2020;45:251–264. doi: 10.1007/s40858-020-00343-y. - DOI
    1. Grafton-Cardwell E.E., Stelinski L.L., Stansly P.A. Biology and management of asian citrus psyllid, vector of the Huanglongbing pathogens. Annu. Rev. Entomol. 2013;58:413–432. doi: 10.1146/annurev-ento-120811-153542. - DOI - PubMed

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