Abstract
BackgroundOat (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.ResultsIn 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.ConclusionsResults 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.
Highlights
Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses
Enzyme 21 (UBC21), Elongation factor 1-Alpha (EF1A), Glyceraldehyde-3-phosphate Dehydrogenase C Subunit 1 (GAPDH1), 18S Ribosomal RNA (18S) ribosomal RNA (18S), Heterogeneous nuclear ribonucleoprotein 27C (HNR), Expressed protein (EP), TBC1 domain family member 22A (TBC), Tubulin alpha-6 chain (TUA6) and Eukaryotic initiation factor 4A-3 (EIF4A), were identified as candidates for quantitative real-rime polymerase chain reaction (qPCR) primer designing and their sequences were listed in Additional file 1
In this study, eleven candidate Reference gene (RG) were screened for qPCR in 18 samples of hexaploid oat, four of which were duplicated genes
Summary
Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. No suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented for oat yet. Oat (Avena sativa L.) is an allohexaploid (2n = 6 × = 42) cereal crop with estimated 13 Gb genome [1]. Gene expression analysis is becoming increasingly important for exploring functions of candidate genes in biological research. Quantitative real-rime polymerase chain reaction (qPCR) is more commonly used for measuring mRNA levels of specific genes for its specificity, sensitivity, flexibility, scalability, and most importantly its potential for high throughput [15, 16]. The amounts of qPCR products are generally calculated by the relative quantification compared with stably expressed genes, which is the most robust and straightforward method of accurately quantifying subtle changes [17]. The validity of an RG is critical for generating reliable and accurate qPCR results [19, 20]
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