Research Article: Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

Date Published: March 31, 2017

Publisher: Public Library of Science

Author(s): Feifan Hou, Sen Li, Jinyao Wang, Xiuping Kang, Yiqun Weng, Guoming Xing, Yuan Huang.


Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the ‘Datong’ landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species.

Partial Text

The long yellow daylily (LYD hereinafter), or yellow flower vegetable (huang hua cai in Chinese), Hemerocallis citrina Baroni., is a perennial herb in the family Liliaceae, which is native to central and northern China, the Korea Peninsula, and Japan [1]. LYD is widely cultivated in this region and used as an ornamental plant, vegetable or medicinal plant because of its beautiful flower, pleasant flavor, and beneficial secondary metabolites; thus it is also considered as a functional vegetable crop [2]. The consumed part of this plant is mainly its flower buds, which are harvested before flowering. The LYD returns green in March, blooms from June to September in the summer. This crop is easily adaptable to various growth environments.

RT-qPCR is one of the most common molecular biology research tools, and its reliability and accuracy strongly depends on appropriate normalization using stably expressed reference genes [30]. Although several reference genes have been proposed for two Liliaceae species Lilium brownie [13] and Lilium davidii var. unicolor [20], their usefulness in LYD was not known. Reference genes suitable for one species are not necessarily working for others, and there are no one-fit-all reference genes with constantly stable expression across all plants species [31]. Our results indicate that the expression stability of reference genes was affected by developmental stages, tissues, landraces, and physiological status, which emphasizes the necessity of selecting suitable reference genes for RT-qPCR.




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