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

Abstract

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.

Fig 1. Commercial stage flower buds of different landraces and flower buds at different developmental stages of ‘Datong’.

A, B and C are commercial-stage flower buds of ‘Panlong’, ‘Changzuizi’ and ‘Datong’ landraces, respectively. D and E are flower buds of ‘Datong’ landrace at 0-5cm and 5-10cm stage, respectively.

Fig 2. Box-whiskers plot showing Ct variation of six candidate reference genes in the LYD samples.

(A) flower buds at different developmental stages of ‘Datong’ landrace; (B) different organs of ‘Datong’ landrace; (C) commercial flower buds of different landraces; and (D) all samples. The horizontal line within each box represents the median value. For each box, the upper and lower edges indicate the 25th and 75th percentiles, while whiskers represent the maximum and minimum values.

Fig 3. Pairwise Variation (V) analyses of six candidate reference genes in LYD samples.

Pairwise variation (V_n/V_n+1) values were analyzed using the geNorm program. (A) flower buds at different developmental stages of ‘Datong’ landrace; (B) different organs of ‘Datong’ landrace; (C) commercial flower buds of different landraces; and (D) all samples. When the pairwise variation (V_n/V_n+1) is less than 0.15, it is recommended that no additional genes are required for the normalization.

Fig 4. Relative expression level of (A) HcAOC3 and (B) HcTAT in different organs of ‘Datong’ landrace plants.

The most or the least stable reference genes were used for analysis. The error bars represent standard errors, and t-test statistics were generated from an ANOVA model characterizing among-relative expression levels in the same sample. * P < 0.05.