Abstract
Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.
Highlights
Sacha inchi (Plukenetia volubilis L.), a member of the Euphorbiaceae, is native to the rain forest of South America [1,2]
The melting temperatures of the PCR products all ranged between 78.74 °C for ubiquitin-conjugating enzyme (UCE) and 82.95 °C for CYC
The results indicated that the candidate reference genes evaluated in this study encompassed a wide range of cycle threshold (Ct) values, ranging from 8 to 30, with the majority ranging from 16 to 27 (Figure 2, Supplementary Table S1)
Summary
Sacha inchi (Plukenetia volubilis L.), a member of the Euphorbiaceae, is native to the rain forest of South America [1,2]. Because its seed oil is rich in polyunsaturated fatty acids (PUFAs) and lipovitamins, Sacha inchi has great potential economic value to the food and pharmaceutical industries [3,4]. Sacha inchi oil is a good feedstock for biodiesel production [5]. To promote gene function studies in Sacha inchi, transcriptomic analysis has been performed for the period of seed development, and numerous key genes involved in the regulation of seed oil biosynthesis have been identified [6]. A good characterization of expression profiles of these key genes will facilitate a better understanding of gene function in seed oil biosynthesis.
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