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

<div><p>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, <i>Hemerocallis citrina</i> 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 <i>H</i>. <i>citrina</i> are not available. In the present study, six potential reference genes, <i>actin</i> (<i>ACT</i>), <i>AP-4 complex subunit</i> (<i>AP4</i>), <i>tubulin</i> (<i>TUB</i>), <i>ubiquitin</i> (<i>UBQ</i>), <i>18S</i> and <i>60S ribosomal RNA</i>, 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 <i>60S</i>, <i>TUB</i>, and <i>AP4</i> was appropriate whereas <i>ACT</i> and <i>60S</i> was suitable for normalization of different organs. In addition, <i>AP4</i> exhibited the most stable expression in flower buds among different developmental stages. <i>UBQ</i> was less stable than the other reference genes under the experimental conditions except under different organs was <i>18S</i>. The relative expression levels of two genes, <i>primary-amine oxidase</i> (<i>HcAOC3</i>) and <i>tyrosine aminotransferase</i> (<i>HcTAT</i>) 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.</p></div