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Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea

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Abstract

Quantitative real-time RT-PCR (qPCR) has been widely used to investigate gene expression during seed germination, a process involving seed transition from dry/physiologically inactive to hydrated/active state. This transition may result in altered expression of many housekeeping genes (HKGs), conventionally used as internal controls, thereby posing a challenge about selection of HKGs in such scenarios. The objectives of this study included identifying valid reference genes for seed priming and germination studies, both of which involve the transition of seed hydration status, and assessing whether or not findings derived from the “seed model” used in this study would also be applicable to other plant species. Eight commonly used HKGs were evaluated in maize seeds during hydropriming and germination. Using Bestkeeper, geNorm, and NormFinder, we provided a rank of stability for these HKGs. Actdf, UBQ, βtub, 18S, Act, and GAPDH were adjudged as valid internal controls by geNorm and NormFinder. Under the second objective, we conducted a case study with spinach seeds collected during osmopriming and germination. Our results indicate that the conclusions derived from maize were applicable to spinach as well, in that 18S exhibited greater expression stability than GAPDH in osmoprimed and germinated seeds; this held true even under stress conditions. While both of these genes were rejected by BestKeeper, we found that 18S exhibited stable expression when “dry” and “hydrated” seeds were analyzed as separate data sets. Although this approach precludes the comparison between “hydrated” and “dry” seeds, it still provides effective comparison among samples of same hydration status.

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Abbreviations

HKG:

Housekeeping genes

18S:

18S rRNA

αtub:

alpha-Tubulin

βtub:

belta 2-Tubulin

Actdf:

Actin depolymerizing factor

Act:

Actin

GAPDH:

Glyceraldehydes-3-phosphate dehydrogenase

Glub:

Globulin

UBQ:

Ubiquitine

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Acknowledgments

This paper of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project no. 3601 was supported by Hatch Act and State of Iowa funds. We thank Dr. Manjit Misra (Director, Seed Science Center), Plant Science Institute, and the Department of Horticulture at Iowa State University for their financial assistance to support this work.

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Correspondence to Rajeev Arora.

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Chen, K., Fessehaie, A. & Arora, R. Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea . Plant Mol Biol Rep 30, 478–487 (2012). https://doi.org/10.1007/s11105-011-0354-x

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