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Suitable reference genes for RT-qPCR analysis in Dichelops melacanthus (Hemiptera: Pentatomidae)

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Abstract

The relative quantification of gene expression is mainly realized through reverse transcription-quantitative PCR (RT-qPCR). However, the accuracy of this technique is deeply influenced by the expression stability of the reference genes used for data normalization. Therefore, the selection of suitable reference genes for a given experimental condition is a prerequisite in gene expression studies. Dichelops melacanthus (Hemiptera: Pentatomidae) is an important phloem sap-sucking insect pest of soybean, wheat, and maize in Brazil. Most of the genetic and molecular biology studies require gene expression analysis. Nevertheless, there are no reports about reference genes for RT-qPCR data normalization in D. melacanthus. In this study, we evaluated the expression stability of nine candidate reference genes (nadh, sdhb, gapdh, fau, ef1a, rpl9, ube4a, gus and rps23) in different developmental stages, body parts, sex, starvation-induced stress and dsRNA exposure by RefFinder software that integrates the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt method. Our results showed that ef1a and nadh are the most stable reference genes for developmental stages, fau and rps23 for sex, ube4a and rps23 for body parts, rpl9 and fau for starvation stress, and nadh and sdhb for dsRNA exposure treatment. The reference genes selected in this work will be useful for further RT-qPCR analyses on D. melacanthus, facilitating future gene expression studies that can provide a better understanding of the developmental, physiological, and molecular processes of this important insect pest. Moreover, the knowledge gained from these studies can be helpful to design effective and sustainable pest management strategies.

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Data availability

The expression datasets generated during the current study are available from the corresponding author on reasonable request. All other data generated or analyzed during this study are included in this published article and its Supplementary Information file.

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Acknowledgements

The authors would like to acknowledge the funding agencies Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Banco Nacional do Desenvolvimento (BNDES) and Embrapa Milho e Sorgo.

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Author notes

  1. Daniele Heloísa Pinheiro

    Present address: Laboratory of Plant–Pest Molecular Interaction, Embrapa Genetic Resources and Biotechnology, Brasília, DF, 70770-917, Brazil

  2. Raquel Oliveira Moreira

    Present address: School of Agrarian and Veterinary Sciences, São Paulo State University–UNESP, Jaboticabal, SP, 14884-900, Brazil

  3. Natália Alves Leite

    Present address: Department of Crop Protection, Federal University of Rio Grande Do Sul, Porto Alegre, RS, 91540-00, Brazil

  4. André da Silva Xavier

    Present address: Department of Agronomy, Federal University of Espírito Santo, Alegre, ES, 29500-000, Brazil

Authors and Affiliations

  1. Nucleus of Applied Biology, Embrapa Maize and Sorghum, Sete Lagoas, MG, 35701-970, Brazil

    Daniele Heloísa Pinheiro, Raquel Oliveira Moreira, Natália Alves Leite, Ana Carolina Redoan, André da Silva Xavier, Beatriz de Almeida Barros & Newton Portilho Carneiro

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Contributions

DHP, ROM, BB and NPC conceived and designed the experiments. DHP, ROM, NAL and ACR performed the experiments. DHP and NAL performed the statistical analysis. DHP interpreted the data and composed the manuscript. DHP, NAL, BB, AX and NPC reviewed the manuscript. All authors approved the manuscript.

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Correspondence to Newton Portilho Carneiro.

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Pinheiro, D.H., Moreira, R.O., Leite, N.A. et al. Suitable reference genes for RT-qPCR analysis in Dichelops melacanthus (Hemiptera: Pentatomidae). Mol Biol Rep 47, 4989–5000 (2020). https://doi.org/10.1007/s11033-020-05550-z

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