title: Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR.
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.
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei 230027, China. niulongjian@126.com.|Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. niulongjian@126.com.
authors.forename: Longjian
authors.initials: L
authors.lastname: Niu
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. taoyanbin@xtbg.ac.cn.
authors.forename: Yan-Bin
authors.initials: YB
authors.lastname: Tao
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. chenms@xtbg.org.cn.
authors.forename: Mao-Sheng
authors.initials: MS
authors.lastname: Chen
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. qtfu2002@163.com.
authors.forename: Qiantang
authors.initials: Q
authors.lastname: Fu
authors.affiliation: Department of Life Sciences, College of Life Science and Agriculture, Zhoukou Normal University, Zhoukou 466001, China. lcq2013@aliyun.com.
authors.forename: Chaoqiong
authors.initials: C
authors.lastname: Li
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. yuling-dong@163.com.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. wangxiulan@xtbg.ac.cn.
authors.forename: Xiulan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. hhy@xtbg.org.cn.
authors.forename: Huiying
authors.initials: H
authors.lastname: He
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei 230027, China. zfxu@xtbg.ac.cn.|Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China. zfxu@xtbg.ac.cn.
authors.forename: Zeng-Fu
authors.initials: ZF
authors.lastname: Xu
mesh_terms: D000465:Algorithms; D018556:Crops, Agricultural / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D019665:Euphorbiaceae / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development*; D035264:Flowers / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D009928:Organ Specificity; D010938:Plant Oils; D010940:Plant Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D012639:Seeds / Q000235:genetics
chemical_list: D010938:Plant Oils; D010940:Plant Proteins
keywords: Plukenetia volubilis; RT-qPCR; biofuels; flower development; reference gene; seed development