Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Shu, Benshui; Yu, Huilin; Dai, Jinghua; Zi-ge, Xie; Qian, Wanqiang; Lin, Jintian

doi:10.1016/s2095-3119(20)63298-1

Cited by 19 publications

(6 citation statements)

References 41 publications

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“…In our research, α-TUB showed instability under PWN treatment conditions. COX responds to a wide variety of metabolic states and is also considered a novel reference gene for different tissues in A. hygrophila and Spodoptera frugiperda ( Guo et al, 2021 ; Shu et al, 2021 ), while was inconsistent with our results. In M. saltuarius subjected to several experimental conditions (different developmental stages, adults treated with PWN, and all samples), COX7 was particularly unstable reference gene.…”

Section: Discussioncontrasting

confidence: 99%

Identification and Validation of Reference Genes for Gene Expression Analysis in Monochamus saltuarius Under Bursaphelenchus xylophilus Treatment

Ren

et al. 2022

Front. Physiol.

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A special mutual relationship exists between the pine wood nematode (PWN) Bursaphelenchus xylophilus and its vector beetles of genus Monochamus, which enables PWN to spread, at the same time provides longhorned beetles with more weak hosts. PWN are attracted to the pupal chambers and then carried inside the trachea of beetle adults, which is a necessary part to complete the B. xylophilus infection cycle. The growth and immune responses of the vector beetle will affect this carrying process, however, they were rarely studied in Monochamus saltuarius. Real-time quantitative polymerase chain reaction (RT-qPCR), one of the most common methods for quantitative gene expression analysis, was performed to explore the key genes and pathways involved in the growth, development and immune responses of M. saltuarius at different developmental stages associated with infection of PWN and PWN treatment conditions. To enhance the accuracy of RT-qPCR data, the expression of target genes needs to be normalized with reference genes, which are stably expressed under varied experimental conditions. In our study, the stability of 14 candidate reference genes in M. saltuarius samples at different developmental stages associated with infection of PWN or PWN treatment conditions was evaluated using delta Ct, geNorm, NormFinder, BestKeeper and RefFinder algorithms. Moreover, KLF gene was used to validate the stability of the selected reference genes. Under experimental conditions of this study, RPL7 and TER were suitable reference genes at different developmental stages associated with infection of PWN. RPL7 and RPS5 were considered the most stable reference genes in the pupae treated with PWN. RPS5 and SNX6 could be used as reference genes in the adults treated with PWN. RPL7, EF1-γ, and RPS5 could be used as stable reference genes in all the samples. This work is the first to evaluate reference genes in M. saltuarius, laying a foundation for further gene expression experimental procedures and understanding the phoretic relationship between M. saltuarius and B. xylophilus.

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Section: Discussioncontrasting

confidence: 99%

Identification and Validation of Reference Genes for Gene Expression Analysis in Monochamus saltuarius Under Bursaphelenchus xylophilus Treatment

Ren

et al. 2022

Front. Physiol.

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“…The total volume of the reaction mixture was 20 μL with 10 μL of 2 ×SuperMix, 1.5 μL of each primer, 1 μL of cDNA template, and 6 μL of RNase-free water. The protocol program was as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 30 s. The reference gene was the elongation factor 1 alpha (EF1α) for S. frugiperda (Shu et al, 2020), and the experiment was performed for three technical replicates with three independent biological replicates. The 2 −ΔΔCt method (Livak and Schmittgen, 2001) was used for calculating the relative expression levels of target genes.…”

Section: Qrt-pcrmentioning

confidence: 99%

Resistance monitoring and mechanism in the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) for chlorantraniliprole from Sichuan Province, China

et al. 2023

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The fall armyworm, Spodoptera frugiperda (Noctuidae: Lepidoptera), is a wide-reaching notorious insect pest of important cereal crops, which has developed resistance to multiple classes of insecticides. It invaded the Sichuan Province of China in 2019. In this study, we performed resistance monitoring of insecticides for 11 field-collected populations from Sichuan, and all the populations were susceptible to emamectin benzoate and chlorpyrifos. The variations in resistance level to indoxacarb (resistance ratio (RR), 9.23–45.53-fold), spinetoram (RR, 4.32–18.05-fold), and chlorantraniliprole (RR, 2.02–10.39-fold) were observed among these populations. To investigate the resistance mechanism of chlorantraniliprole, synergism tests were performed and showed that piperonyl butoxide had a slight synergistic effect on chlorantraniliprole for the QJ-20 population (1.43-fold) in moderate resistance (RR, 10.39-fold) compared with the treatment group without synergist. Furthermore, the expression scanning for resistance-related genes showed that five P450 genes (CYP6AE43, CYP321A8, CYP305A1, CYP49A1, and CYP306A1) and the ryanodine receptor gene (Ryr, chlorantraniliprole target) were overexpressed in the QJ-20 population. These results indicated that the fall armyworm in Sichuan has exhibited diverse susceptibilities to several classes of insecticides, and the overexpression of Ryr and several P450 genes may contribute to the development of resistance in S. frugiperda to chlorantraniliprole.

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“…Four programs (geNorm, NormFinder, BestKeeper, and RefFinder) based on different algorithms were used to select stable reference genes in the various species and under different experimental conditions [11,43,44]. Divergent results between the different programs were also observed due to the different statistical analysis algorithms underlying the software.…”

Section: Discussionmentioning

confidence: 99%

Stable Reference Gene Selection for qRT-PCR Normalization in Strawberry (Fragaria × ananassa) Leaves under Different Stress and Light-Quality Conditions

Lü

Wang

et al. 2021

Horticulturae

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Selecting an appropriate reference gene is of crucial importance for improving the accuracy of qRT-PCR analyses. In this study, strawberry (Fragaria ananassa) seedlings were subjected to different environmental conditions including heat, cold, drought, salt, white-light, blue-light, and red-light treatments. The expression levels of seven candidate reference genes, including Fa18S, FaGAPDH, FaPIRUV, FaDBP, FaHISTH4, FaACTIN1, and FaACTIN2, in the strawberry leaves were measured by qRT-PCR. Then, four programs (geNorm, NormFinder, BestKeeper, and RefFinder) were employed as tools to evaluate the expression stability of the candidate reference genes. The results showed that the expression stability of the reference genes varied under different conditions. For the cold stress and white-light treatments, FaACTIN2 was evaluated to be the most stable reference gene. FaGAPDH should be used as the reference gene under salt-stress condition and red-light treatment. For the data normalization under drought-stress treatment, FaDBP is the recommended reference gene with the highest expression stability. FaHISTH4 was observed to be the best reference gene for data normalization under heat stress and blue-light treatment. This work provides information on selecting reference genes for accurate gene expression analyses of target genes in strawberry leaves under various abiotic stress and light-quality conditions.

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Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Cited by 19 publications

References 41 publications

Identification and Validation of Reference Genes for Gene Expression Analysis in Monochamus saltuarius Under Bursaphelenchus xylophilus Treatment

Identification and Validation of Reference Genes for Gene Expression Analysis in Monochamus saltuarius Under Bursaphelenchus xylophilus Treatment

Resistance monitoring and mechanism in the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) for chlorantraniliprole from Sichuan Province, China

Stable Reference Gene Selection for qRT-PCR Normalization in Strawberry (Fragaria × ananassa) Leaves under Different Stress and Light-Quality Conditions

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