Selection and validation of reference genes for RT-qPCR analysis of the Longan psyllid Cornegenapsylla sinica (Hemiptera: Psyllidae)

Cornegenapsylla sinica is a devastating pest of longan that vectors the longan pathogen witches' broom virus (LgWB), leading to significant agricultural losses. Efficient control strategies targeting this pest are imperative for sustainable longan production. However, the genetic research on C. sinica is relatively limited, which may hinder the discovery of effective control strategies. Accurate gene expression analysis under various conditions using RT-qPCR is essential for advancing our understanding of this pest and for identifying potential targets for management. In this study, a comprehensive array of specific algorithms, including geNorm, Normfinder, BestKeeper, and the Delta Ct method, was applied to assess the stability of 8 candidate reference genes under 4 distinct experimental conditions: developmental stages, sex, tissue, and temperature. Through the application of RefFinder software, a ranking of expression stability among the candidate genes was established. The results indicated that RPL13 and RPL6 were the most stable reference genes under varying developmental stages and temperatures, ATPB and RPL13 were the top choices for different sexes, and RPL13 and EF1 alpha were the most stable in different tissues. Additionally, heat shock protein 70 (Hsp70) served as a reporter gene to validate the selected reference genes. This study is the first to report detailed data on comprehensive reference genes suitable for RT-qPCR in C. sinica, laying the groundwork for biological control and functional target gene research in this species, which is crucial for preventing the spread of longan witches' broom virus in longan trees.