Evaluation of duplicated reference genes for quantitative real-time PCR analysis in genome unknown hexaploid oat ( Avena
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(2020) 16:138 Yang et al. Plant Methods https://doi.org/10.1186/s13007-020-00679-1
Open Access
RESEARCH
Evaluation of duplicated reference genes for quantitative real‑time PCR analysis in genome unknown hexaploid oat (Avena sativa L.) Zheng Yang, Kai Wang, Usman Aziz, Cuizhu Zhao* and Meng Zhang*
Abstract Background: Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. However, no suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented for oat yet. Single-copy gene is often selected as RG, which is challengeable or impactable in unexplored polyploids. Results: In this study, eleven candidate RGs, including four duplicated genes, were selected from oat transcriptome. The stability and the optimal combination of these candidate RGs were assessed in 18 oat samples by using four statistical algorithms including the ΔCt method, geNorm, NormFinder and BestKeeper. The most stable RGs for “all samples”, “shoots and roots of seedlings”, “developing seeds” and “developing endosperms” were EIF4A (Eukaryotic initiation factor 4A-3), UBC21 (Ubiquitin-Conjugating Enzyme 21), EP (Expressed protein) and EIF4A respectively. Among these RGs, UBC21 was a four-copy duplicated gene. The reliability was validated by the expression patterns of four various genes normalized to the most and the least stable RGs in different sample sets. Conclusions: Results provide a proof of concept that the duplicated RG is feasible for qPCR in polyploids. To our knowledge, this study is the first systematic research on the optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat. Keywords: Oat, Reference gene, qPCR, Duplicated gene, Polyploid, Gene expression, Endosperm Background Oat (Avena sativa L.) is an allohexaploid (2n = 6 × = 42) cereal crop with estimated 13 Gb genome [1]. With an upswing in food and industrial utilization, oats are now cultivated worldwide and form an important dietary staple on a global scale [2, 3]. As a wholegrain product, rolled oats are a rich source of minerals, starch and lipids, and they are a predominant supply of soluble fiber β-glucan [4, 5]. Particularly, unlike other cereals, most *Correspondence: [email protected]; [email protected] College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China
of the lipids in oat seeds are deposited in cells of oat endosperms which also accumulate starch [6, 7]. Due to the rich constituents, oats also possess different pharmacological purposes like antioxidative, immunomodulatory, antidiabetic and anti-cholesterolaemic effects [8, 9]. Additionally, oat plants are more adapted to severe weather compared to other monocot crops, and they comparatively require fewer pesticide and fertilizers than other food cereals [10, 11]. These features boost oat as an eco-friendly crop with valuable nutrition and pharmaceutical applications. Many classic breeding approaches are already underway to explore and impr
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