Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression in Goji Fruit Fly (Neoceratitis asiatica Becker) under Developmental Stages and Five Abiotic Stresses

Abstract

Goji fruit fly, Neoceratitis asiatica, is a major pest on the well-known medicinal plant Lycium barbarum. Dissecting molecular mechanisms of infestation and host selection of N. asiatica will contribute to the determination of best management practices for pest fly control. Gene expression normalization by Real-time quantitative PCR (qPCR) requires the selection and validation of appropriate reference genes (RGs). Hence, 15 candidate RGs were selected from transcriptome data of N. asiatica. Their expression stability was evaluated with five algorithms (∆Ct, Normfinder, GeNorm, BestKeeper, and RefFinder) for sample types differing in the developmental stage, sex, tissue type, and in response to five different abiotic stresses. Our results indicated that the RGs β-Actin + GST for sex, RPL32 + EF1α for tissue type, RPS13+ EF1α for developmental stages along with odor stimulation, color induction, and starvation-refeeding stresses, EF1α + GAPDH under insecticide stress, RPS13 + RPS18 under temperature stress, respectively, were selected as the most suitable RGs for qPCR normalization. Overall, RPS18 and EF1α were the two most stable RGs in all conditions, while RPS15 and EF1β were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target odorant-binding protein OBP56a gene in male and female antennae, different tissues, and under odor stimulation. The results of OBP56a expression were consistent with transcriptome data. Our study is the first research on the most stable RGs selection in N. asiatica, which will facilitate further studies on the mechanisms of host selection and insecticide resistance in N. asiatica.

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)

“…RefFinder is a comprehensive evaluation tool that generates stability scores by estimating the geometric mean of internal control genes to lessen the effects of a single algorithm’s limitations [ 36 ]. Many species, including Neoceratitis asiatica [ 18 ] and Anastatus japonicus [ 17 ], were studied using a similar strategy. The expression of HSP90 under different temperature treatments was evaluated to authenticate our findings.…”

“…Finally, TATA and ACTIN were recognized as excellent for evaluating distinct diapause conditions [ 17 ]. RPS18 and EF1α were the two trustiest genes in Neoceratitis asiatica , whereas RPS15 and EF1β were the most untrustworthy [ 18 ]. Instead of employing generic reference genes, choose appropriate housekeeping genes under the circumstances for specific insect species.…”

Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions

“…However, stable reference genes were a precondition for measuring the accuracy of RT-qPCR results [28]. Numerous published studies have been dedicated to the identification of reference genes in organisms [16,[29][30][31][32][33][34][35], among which some house-keeping genes were certified to be the unstable expression under various experimental conditions [16,[33][34][35]. So it was urgently necessary to recruit the optimal reference genes for RT-qPCR analysis.…”

Chromosome-level genome assembly of an important wolfberry fruit fly (Neoceratitis asiatica Becker)

“…Neoceratitis asiatica Becker is a major pest on the well-known traditional Chinese medicinal plant wolfberry ( Lycium barbarum ), which is limited distributed in Ningxia, Tibet and Xinjiang, China 8 . The outbreaks of N. asiatica affected the yield and quality of wolfberry seriously in recent years, especially in the organic wolfberry orchards that have not been exposed to any pesticides in Ningxia, resulting in fruit-damage rates of up to 80% every year 9 . Faced with the flies that feed only on wolfberries, people are inexperienced in the prevention and control of N. asiatica .…”