Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses.

Simple Summary: The dark black chafer, Holotrichia parallela Motschulsky (Coleoptera: Scarabaeidae), is an important subterranean insect in China due to its wide distribution and the high degree of damage it causes. Selecting stable reference genes is crucial for accurate quantitative polymerase chain reaction (qPCR) and gene expression analysis. This study evaluated the expression stability of 11 candidate reference genes in H. parallela under various biological conditions and environmental stresses. Our findings suggest that the optimum reference genes were as follows: RPL18 and RPL13a for developmental stages and RNAi conditions, RPL13a, RPL18, and RPL32 for female and male adults, RPL13a and RPS3 for different tissues, RPL32, RPL13a, and RPS3 for varying photoperiod conditions, and Actin and RPL13a for different temperatures. These discoveries will serve as a foundation for subsequent precise qPCR and gene expression studies in H. parallela and other closely related insect species. Holotrichia parallela is among the world's most destructive pests. For accurate qPCR and gene expression studies, the selection of stable and appropriate reference genes is crucial. However, a thorough evaluation of potential reference genes for use in H. parallela research is lacking. In this study, 11 reference genes (GAPDH, RPL32, RPL7A, RPS18, RPL13a, RPL18, Actin, RPS7, RPS3, VATB,and EF1A) were evaluated under different biological conditions and environmental stresses. The stability of 11 potential reference gene transcripts was evaluated through various computational tools, including geNorm, BestKeeper, NormFinder, theΔCt method, and the RefFinder program. Under various developmental stages and RNAi conditions, RPL18 and RPL13a exhibited the greatest stability. RPL13a, RPL18, and RPL32 were the most stable genes in both male and female adults. Under differing tissue conditions, RPL13a and RPS3 stood out as the most reliable. Moreover, under varying photoperiod conditions, RPL13a, RPS3 and RPL32 were the most stable genes. Lastly, Actin and RPL13a were the most stable genes across different temperatures. These findings offer essential criteria for selecting suitable reference genes across diverse experimental settings, thereby establishing a solid basis for accurate gene expression studies in H. parallela using RT-qPCR. [ABSTRACT FROM AUTHOR]