Open Access

ARTICLE

Reference Gene Selection for qRT-PCR Normalization in Iris germanica L.

by Yinjie Wang, Yongxia Zhang, Qingquan Liu, Liangqin Liu, Suzhen Huang, Haiyan Yuan^*

Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China

* Corresponding Author: Haiyan Yuan. Email:

Phyton-International Journal of Experimental Botany 2021, 90(1), 277-290. https://doi.org/10.32604/phyton.2020.011545

Received 15 May 2020; Accepted 07 July 2020; Issue published 20 November 2020

Abstract

Quantitative real-time PCR (qPCR) is an effective and widely used method to analyze expression patterns of target genes. Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression by qRT-PCR. In Iris germanica L., no studies have yet been published regarding the evaluation of potential reference genes. In this study, nine candidate reference genes were assessed at different flower developmental stages and in different tissues by four different algorithms (GeNorm, NormFinder, BestKeeper, and RefFinder). The results revealed that ACT11 (Actin 11) and EF1α (Elongation factor 1 alpha) were the most stable reference genes in different tissues, whereas TUA (Tubulin alpha) and UBC9 (Ubiquitin-protein ligase 9) were the most stable ones in different flower developmental stages. UBC9 and ACT11 were the most stable reference genes in all of the tested samples, while the SAMDC (S-Adenosylmethionine decarboxylase) showed the least stability. Finally, to validate the suitability of the selected reference genes, the relative expression level of IgTPS (beta-caryophyllene synthase) was assessed and highlighted the importance of suitable reference gene selection. This work constitutes the first systematic evaluation of potential reference genes in I. germanica and provides guidelines for future research on gene function and molecular mechanisms on I. germanica and related species.

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