Defining Suitable Reference Genes for qRT-PCR in Plagiodera versicolora (Coleoptera: Chrysomelidae) under Different Biotic or Abiotic Conditions

Abstract

Plagiodera versicolora (Coleoptera: Chrysomelidae) is one of the most destructive pests of the Salicaceae worldwide, which has established complex interactions with surrounding organisms. Uncovering the molecular mechanisms of some antagonistic interactions would facilitate the development of environmentally friendly pest insect management strategies. Suitable reference genes are essential for reliable qPCR and gene expression analysis in molecular studies; however, a comprehensive assessment of reference genes in P. versicolora is still lacking. In this study, the stability of seven housekeeping genes (including Actin, EF1A, α-tubulin, RPL13a, RPS18, RPL8 and UBC) was investigated under both biotic (developmental stages, tissues, sex and pathogen treatment) and abiotic (RNA interference treatment, temperature treatment) conditions. The geNorm, NormFinder, BestKeeper, and ΔCt programs were used to analyze gene expression data. The RefFinder synthesis analysis was applied to suggest a handful of appropriate reference genes for each experimental condition. RPS18 and EF1A were the most reliable reference genes in different development stages; RPS18 and RPL8 were most stable in female and male adults, different tissues, different temperatures, and pathogen treatment; α-tubulin and RPL13a were most stable after dietary RNAi treatment. The research provides a strong basis for future research into the molecular biology of P. versicolora.

Transcriptome analysis and identification of chemosensory genes in the larvae of Plagiodera versicolora

“…The RPS18 gene (accession number: OM885975) was used as the reference gene for normalizing the expression of various samples [ 66 ]. And RPS18 gene has been proven stable in different development stages, female and male adults, different tissues, different temperatures, and pathogen treatment [ 67 ]. The gene-specific primers were designed using Beacon Designer 8.0 (amplicon length: 100 ± 20 bp; Tm: 60 ± 2 °C; GC%: 45–55%) (PRIMER Biosoft International, CA, USA), and were listed in Supplementary Table S 2 .…”

Selection and Evaluation of Reference Genes for Quantitative Real-Time PCR in Tomato (Solanum lycopersicum L.) Inoculated with Oidium neolycopersici

“…In the molecular breeding of tomato resistance to PM, it is of great significance to study the differential expression of tomato PM-related functional genes at different growth stages or in specific environments. The expression stability of the reference genes determines the authenticity of the target gene's expression [58,59], but there is no such report on the systematic screening of reference genes in tomatoes under PM stress. Therefore, mining the most stable reference genes of tomatoes under PM stress will also be more helpful in revealing the internal law of the expression of tomato PM-related functional genes and lay the foundation for the molecular breeding of tomato resistance to PM.…”

Validation of Appropriate Reference Genes for qRT–PCR Normalization in Oat (Avena sativa L.) under UV-B and High-Light Stresses

“…In BestKeeper analysis, the stability of the expression levels of reference genes is evaluated using both the coefficient of variation (CV) and SD; the smaller the CV value is, the greater the stability. Finally, RefFinder ( , accessed on 15 March 2022) was used for the comprehensive evaluation of suitable reference genes, which is a comprehensive analysis tool for evaluating gene expression stability when the results of geNorm, NormFinder and BestKeeper were inconsistent [ 54 , 55 ].…”