Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum.

Xiu-Mei Dong,Shi-Bao Zhang,Wei Zhang

doi:10.3390/ijms23020738

Abstract

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

Highlights

Flower color is a pivotal plant phenotype for ornamentation and population diversity [1,2]
To identify reference genes that can be used to examine gene expression changes in diverse tissues, we evaluated the stability of candidate reference gene expression in several tissues at the same stage of development, i.e., in the abaxial lip, adaxial lip, petal, and sepal of the bud; and in the abaxial lip, adaxial lip, petal, and sepal of the flower
To identify reference genes that can be used to examine gene expression changes in a single tissue across developmental stages, we evaluated the stability of candidate reference gene expression in each individual tissue across developmental stages, i.e., abaxial lip of bud and flower; adaxial lip of bud and flower petal of bud and flower; sepal of bud and flower; and all tissues of bud and flower

Summary

Introduction

Flower color is a pivotal plant phenotype for ornamentation and population diversity [1,2]. Investigating the formation of flower color is key to understand the evolution of color variation in plant populations and may facilitate ornamental plant breeding [3,4]. (Orchidaceae) is an important ornamental plant that possess flowers with a deep red V-shape on the abaxial lip in flower stage [5,6]. In the bud stage, the color of sepal and petal are green (Figure 1A). Sepal and petal turned yellow green, and reddish brown on some longitudinal veins (Figure 1B). The molecular mechanisms of flower color formation in C. lowianum remains unclear. Gene expression analysis is fundamental to understanding gene function and the molecular mechanisms of developmental processes [7]

Methods

Results

Conclusion