Abstract

The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.

Highlights

  • The brassica leaf beetle, Phaedon brassicae Baly, is a notorious defoliator of crucifers and is widely distributed in East and South Asia with high fecundity [1, 2]

  • The results indicated that RPL19 and TATA-Box binding protein (TBP) were considered as the best reference genes across different developmental stages, RPL32 and EF-1α were recommended for tissue expression analysis, RPL32 and TBP were identified as the optimal candidates for the temperature experiment, and RPL32 and RPL19 were selected for the insecticide treatment

  • Several concerns have been raised regarding the variations in RT-qPCR method, and normalization of RT-qPCR data with reference genes is a commonly used strategy to calibrate experimental errors introduced by RT-qPCR [10]

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Summary

Introduction

The brassica leaf beetle, Phaedon brassicae Baly, is a notorious defoliator of crucifers and is widely distributed in East and South Asia with high fecundity [1, 2]. In the Yangtze River Valley, there are two distinct infestation peaks in the field: the single spring generation and the two generations in autumn, which undergo aestivating and hibernating imaginal diapause in soil, separately [1, 4, 5].

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