Abstract
The eggplant shoot and fruit borer, Leucinodes orbonalis Guenée (Lepidoptera: Crambidae) is a monophagous and destructive insect pest of eggplant. L. orbonalis developed multiple insecticide resistance that led to frequent field control failures in many countries. The possible reversal of resistance in L. orbonalis requires an understanding of the expression dynamics of individual genes conferring resistance to insecticides through gene expression analysis. The availability of reference genes (RG) to normalize transcript data of target genes, achieved by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), is mandatory. Hence, the present study investigated the stable expression pattern of nine putative reference genes viz., actin, 28S ribosomal protein S18c, mitochondrial isoform X2 (28SM), calnexin, β-tubulin, 28S ribosomal protein S3 mitochondrial (28SR3), TATA box binding protein (TATA), elongation factor-1α (EF-1α), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and ubiquitin 60S ribosomal protein (Ubiquitin) to select the most stable genes that could be used as reference genes for target transcript gene normalization. Different algorithms such as ΔCt, geNorm, NormFinder and BestKeeper were used to assess gene expression stability. By integrating these data into online RefFinder tool, 28SR3 and GAPDH, were chosen as highly suitable RGs based on their stable expression pattern.
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Abbreviations
- 28SM :
-
28S ribosomal protein S18c, mitochondrial isoform X2
- 28SR3 :
-
28S ribosomal protein S3 mitochondrial
- TATA :
-
TATA box binding protein
- EF-1α :
-
Elongation factor-1α
- GAPDH :
-
Glyceraldehyde-3-phosphate-dehydrogenase
- Ubiquitin :
-
Ubiquitin 60S ribosomal protein
- RG:
-
Reference gene
- M value:
-
Expression stability value
- Cq:
-
Qquantification cycle
- RT-qPCR:
-
Quantitative reverse-transcription polymerase chain reaction
- GST:
-
Glutathione S-transferase
- NTC:
-
No template control
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Acknowledgements
The authors profusely thank Indian Council of Agricultural Research for generous funding under CRP Genomics project. The first author acknowledges the award of DST-Inspire fellowship (2016/IF160900). The facilities extended by Director, ICAR-NBAIR, and Director, ICAR-IIHR, Bengaluru are gratefully acknowledged.
Funding
The present research was supported by Indian Council of Agricultural Research- CRP Genomics project and Department of Science & Technology, Ministry of Science & Technology, Government of India, DST/INSPIRE Fellowship/2016/IF160900.
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Kariyanna B. Conducted experiment, Analysis, Manuscript writing, Material and methods, Hypothesis. Prabhuraj A. Constructing experiment, Analysis, Material methods, corrections. Asokan, R. Analysis and Technical suggestion, Manuscript Correction, Resource supply. Prasad Babu Materials and Methods, Analysis, Reviews and literatures. Jalali S. K. Resources supply, technical guiding and suggestions, primary preview of manuscript. Venkatesan T. Resources supply, technical guiding and suggestions, Reviews and literatures. Gandhi Gracy R. Analysis, technical guiding and suggestions, Reviews and literatures. Mohan M. Designing experiment, Resources supply, Manuscript corrections, analysis, Material and methods, technical guiding and suggestions.
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Kariyanna, B., Prabhuraj, A., Asokan, R. et al. Identification of suitable reference genes for normalization of RT-qPCR data in eggplant fruit and shoot borer (Leucinodes orbonalis Guenée). Biologia 75, 289–297 (2020). https://doi.org/10.2478/s11756-019-00346-4
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DOI: https://doi.org/10.2478/s11756-019-00346-4