Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions

Abstract

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

Keywords: RT-qPCR; South American tomato leafminer; biological factors; environmental factors; expression stability; reference genes.

Figure 1

Expression levels of seven candidate reference genes in T. absoluta. Line across the box shows the median value, while inside the box indicates Ct values. The top and bottom whiskers represent the 25th and 75th percentages, respectively. Developmental stages (A), adult tissues (B), insecticides (C), temperatures (D), plant VOCs (E), and total (F) based on C_t values of all samples exhibited in the five experimental conditions.

Figure 2

Stability analysis of the seven candidate reference genes expression of T. absoluta under the following experimental conditions: developmental stage (A), adult tissue (B), insecticide (C), temperature (D), and plant VOC (E), as calculated by four different software programs: geNorm (1), NormFinder (2), BestKeeper (3), and RefFinder (4).

Figure 3

Optimal number of reference genes for normalization in RT-qPCR of T. absoluta under different conditions. The pairwise variation (V_n/V_n+1, n is the number of reference genes) analyzed using the geNorm software was used to determine the optimal number of reference genes required for accurate normalization. Black dotted lines indicate the threshold for paired variation values (V_n/V_n+1 = 0.15). V_n/n+1 < 0.15 indicates that the optimal number of reference genes for normalization is n, and the V_n/n+1 > 0.15 indicates that the optimal number is n+1.