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Validation of Reference Genes for Gene Expression Studies in Aphis glycines (Hemiptera: Aphididae)

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Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs. We evaluated seven potential HKGs (SDFS, succinate dehydrogenase flavoprotein subunit; EF1a, elongation factor-1α; HEL, helicase; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; RPS9, ribosomal protein S9; TBP, TATA-box binding protein; and UBQ, ubiquitin-conjugating protein) to determine the most efficient HKGs that have stable expression among tissues, developmental stages, and aphids fed on susceptible and host plant‐resistant soybean. HKG stability was determined using GeNorm and NormFinder. Results from three different experimental conditions revealed high stability of TBP compared with the other HKGs profiled across the samples assayed. RPS9 showed stable expression among aphids on susceptible and resistant plants, whereas EF1a showed stable expression in tissues and developmental stages. Therefore, we recommend the TBP as a suitable HKG for efficient normalization among treatments, tissues, and developmental stages of A. glycines. In addition, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate and reliable normalization of qRT-PCR data in A. glycines.

Keywords: Aphis glycines; housekeeping gene; normalization; quantitative real-time polymerase chain reaction

Document Type: Research Article

DOI: http://dx.doi.org/10.1603/EC12095

Publication date: August 1, 2012

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  • Journal of Economic Entomology is published bimonthly in February, April, June, August, October, and December. The journal publishes articles on the economic significance of insects and is divided into the following sections: apiculture & social insects; arthropods in relation to plant disease; forum; insecticide resistance and resistance management; ecotoxicology; biological and microbial control; ecology and behavior; sampling and biostatistics; household and structural insects; medical entomology; molecular entomology; veterinary entomology; forest entomology; horticultural entomology; field and forage crops, and small grains; stored-product; commodity treatment and quarantine entomology; and plant resistance. In addition to research papers, Journal of Economic Entomology publishes Letters to the Editor, interpretive articles in a Forum section, Short Communications, Rapid Communications, and Book Reviews.
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