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ARS Home » Midwest Area » Madison, Wisconsin » Vegetable Crops Research » Research » Publications at this Location » Publication #339599
Title: Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

Author
item HOU, FEIFAN - Shanxi University
item LI, SEN - Shanxi University
item WANG, JINYAO - Shanxi University
item KANG, XIUPING - Shanxi University
item Weng, Yiqun
item XING, GUOMING - Shanxi University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/18/2017
Publication Date: 3/31/2017
Citation: Hou, F., Li, S., Wang, J., Kang, X., Weng, Y., Xing, G. 2017. Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani. PLoS One. 12(3). doi: 10.1371/journal.pone.0174933.

Interpretive Summary: 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 con'rmed the results of selected reference genes. This is the 'rst 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.

Technical Abstract: Gene expression analysis requires the use of reference genes in the target species. The long yellow daylily is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asia. However, reference genes for use in RT-qPCR in this specialty crop are not available. In the present study, six potential reference genes, ACT, AP4, TUB, UBQ, 18S and 60S rRNA were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages. 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, HcAOC3 and HcTAT which play important roles in alkaloid biosynthesis were also examined in different organs of the ‘Datong’ landrace, which further con'rmed the results of selected reference genes. This is the 'rst 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.