Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions

Arya, Shashi; Jain, Gourav; Upadhyay, Santosh Kumar; Sarita, .; Singh, Harpal; Dixit, S. K.; Verma, Praveen Chandra

doi:10.1038/s41598-017-13925-9

Cited by 35 publications

(39 citation statements)

References 40 publications

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“…In this study, host plants caused the most variations for the Ct values in A. viennensis. Similar studies in other arthropods, however, have not found such dramatically variations in Ct values (the cotton mealybug, P. solenopsis, Arya et al, 2017; and the green peach aphid, M. persicae, Kang et al, 2017). Our results suggest that host plants can cause significant changes on arthropods at the molecular level.…”

Section: Discussionsupporting

confidence: 86%

“…Consequently, the selection of appropriate reference genes is critical in offsetting variations and biases (Lü et al, 2018). Recently, there has been a dramatic increase in studies of reference gene selection in the cell-content feeding arthropods, including Hemiptera ( Supplementary Table S3; Li et al, 2013;Yang et al, 2014Yang et al, , 2015bYuan et al, 2014;Shang et al, 2015;Koramutla et al, 2016;Ma et al, 2016;An et al, 2016;Yu et al, 2016;Arya et al, 2017;Kang et al, 2017), and three spider mites species (Sun et al, 2010;Niu et al, 2012;Yue et al, 2013;Yang et al, 2015a). Most of these studies in Tetranychidae species have focused on the expression patterns of reference genes under the impact of insecticide and developmental stage.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, selection of reference genes has been conducted for an array of cell-content feeding arthropods, including three spider mites species, Tetranychus urticae (Yang et al, 2015a), Tetranychus cinnabarinu (Sun et al, 2010), and Panonychus citri (Niu et al, 2012), and many Hemiptera species, Myzus persicae (Kang et al, 2017), Aphis gossypii (Ma et al, 2016), Toxoptera citricida (Shang et al, 2015), Lipaphis erysimi (Koramutla et al, 2016), Sogatella furcifera , Aphis craccivora (Yang et al, 2015b), Bemisia tabaci (Li et al, 2013), Phenacoccus solenopsis (Arya et al, 2017), Nilaparvata lugens (Yuan et al, 2014), Acyrthosiphon pisum (Yang et al, 2014), and Ericerus pela (Yu et al, 2016). In addition, we previously screened for the internal references under intrinsic conditions from a pool of A. viennensis HKGs (Yang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis

et al. 2020

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Hawthorn spider mite, Amphitetranychus viennensis Zacher, is an economically important arthropod pest for fruit trees and woody ornamental plants. Extensive and repetitive use of synthetic acaricides has led to the development of resistance in A. viennensis. To understand the molecular basis of pesticide resistance, and to develop genetic-based control alternatives (e.g., RNAi-based biopesticides), a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) is needed. In the proceeding phase of this research, we screened for the internal references for RT-qPCR analysis from a pool of A. viennensis housekeeping genes under the intrinsic conditions, including developmental stage, sex, and diapause. Here, we continued our efforts to search for the reference genes under an array of extrinsic conditions, including temperature, humidity, photoperiod, host plant, and dietary RNAi. The stability of these candidate reference genes was investigated using geNorm, NormFinder, BestKeeper, and Ct method, respectively. Finally, RefFinder, a statistical platform integrating all four algorisms, provided a comprehensive list of genes for each extrinsic condition:(1) EF1A, α-tubulin and Actin3 were the best candidates for temperature, (2) GAPDH, 18S, and Actin3 were the most stable genes for humidity, (3) V-ATPase B, Actin3, and 18S were the top reference genes for photoperiod, (4) GAPDH, V-ATPase B, and α-tubulin were recommended for host plants, and (5) GAPDH, V-ATPase B, and RPS9 were the top choices for dietary RNAi. Overall, V-ATPase B, GAPDH, and Actin3 were the most commonly selected reference genes in A. viennensis regardless of the experimental conditions, including both intrinsic and extrinsic. Information present here lays the foundation for the genomic and functional genomic research in A. viennensis.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis

et al. 2020

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“…α-TUB is the main component of microtubules that form the cytoskeleton structure, which plays an important role in several eukaryotic cellular processes, including cell mobility and division, and intracellular trafficking 29 . Our results are consistent with previous studies reporting that α-TUB was stable for developmental stages, sexes, tissues, temperature and photoperiod stresses in Empoasca onukii 8 , among various tissues from Coleomegilla maculata 30 and Mythimna separata 24 , for different temperatures in Phenacoccus solenopsis 31 as well as for developmental stages, tissues and sexes in Hermetia illucens 32 . The RPL12 gene encodes a structural protein of ribosomes and is involved in protein translation 33 .…”

Section: Experimental Conditionsupporting

confidence: 93%

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Pinheiro

Siegfried

2020

Sci Rep

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The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the main insect pest of peppers (Capsicum spp.) throughout the southern U.S. and a potential target for novel control methods that may require gene expression analyses. careful selection of adequate reference genes to normalize Rt-qpcR data is an important prerequisite for gene expression studies since the expression stability of reference genes can be affected by the experimental conditions leading to biased or erroneous results. the lack of studies on validation of reference genes for Rt-qpcR analysis in A. eugenii limits the investigation of gene expression, therefore it is needed a systematic selection of suitable reference genes for data normalization. In the present study, three programs (BestKeeper, geNorm and normfinder) were used to analyze the expression stability of candidate reference genes (β-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) in A. eugenii under different experimental conditions. our results revealed that the most stably expressed reference genes in A. eugenii varied according to the experimental condition evaluated: developmental stages (EF1-α, 18S and RPL12), sex (RPS23 and RPL12), low temperature (GAPDH and α-TUB), high temperature (α-TUB and RPS23), all temperatures (α-TUB and GAPDH), starvation (RPL12 and α-TUB), and dsRNA exposure (α-TUB and RPL12). Our study provides for the first time valuable information on appropriate reference genes that can be used in the analysis of gene expression by Rt-qpcR in biological experiments involving A. eugenii.Reverse-transcription quantitative PCR (RT-qPCR) is widely used in gene expression studies due to its simplicity, reproducibility, high sensitivity, accuracy and cost-effectiveness 1,2 . Although RT-qPCR is considered a highly accurate technique, several experimental factors can lead to results that are not reliable measurements of gene expression. These factors include purity and integrity of RNA, quantity of starting RNA and cDNA, reverse transcription and PCR efficiency, and pipetting errors 3 . Thus, in RT-qPCR analysis is necessary to use reference genes to normalize the data in order to eliminate or at least reduce the technical variation among the tested samples and precisely estimate the expression of the target genes 4 .Usually, housekeeping genes related to basic cellular functions are used as reference genes in the normalization strategy because these genes are supposed to have constitutive and stable expression under a variety of physiological conditions and experimental treatments. However, several studies have demonstrated that the expression of housekeeping genes is not always stable and can be influenced by developmental stage, tissue, sex, and biotic or abiotic stresses that the organism is subjected 5-9 . Therefore, the selection of suitable reference genes according to the specific experimental conditions is essential to ensure accurate results.The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the most economica...

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“…For example, GAPDH, β-TUB, CYP2, and 18s rRNA are often selected as reference genes. However, the expression levels of HKGs may vary between different species, and experimental conditions [21][22][23][24][25][26][27]. Thus, the selection of a stable reference gene has become increasingly important.…”

Section: Introductionmentioning

confidence: 99%

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

Zhang

et al. 2020

Genes

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Papaver somniferum L. is an important medical plant that produces analgesic drugs used for the pain caused by cancers and surgeries. Recent studies have focused on the expression genes involved in analgesic drugs biosynthesis, and the real-time quantitative polymerase chain reaction (RT-qPCR) technique is the main strategy. However, no reference genes have been reported for gene expression normalization in P. somniferum. Herein, nine reference genes (actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin 2 (CYP2), elongation factor 1-alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic (GAPC2), nuclear cap-binding protein subunit 2 (NCBP2), protein phosphatase 2A (PP2A), TIP41-like protein (TIP41), and tubulin beta chain (TUB)) of P. somniferum were selected and analyzed under five different treatments (cold, drought, salt, heavy metal, and hormone stress). Then, BestKeeper, NormFinder, geNorm, and RefFinder were employed to analyze their gene expression stability. The results reveal that NCBP2 is the most stable reference gene under various experimental conditions. The work described here is the first report regarding on reference gene selection in P. somniferum, which could be used for the accurate normalization of the gene expression involved in analgesic drug biosynthesis.

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Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions

Cited by 35 publications

References 40 publications

Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis

Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

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