Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

Tingting Zhou,Fangfang Fu,Fuliang Cao,Xiaoming Yang,Guibin Wang

doi:10.3390/f11111217

Abstract

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.

Highlights

Quantitative real-time polymerase chain reaction is a commonly used method to analyze the expression level of functional genes and has become the preferred method to quantify mRNA, as this method meets the requirements for quantitative analyses of data in molecular medical, biotechnological, microbiological, and diagnostic-type studies [1,2]
Primer specificity was evaluated by RT-PCR products
The quantitative real-time polymerase chain reaction (qRT-PCR) E-values of the thirteen candidate reference genes ranged from 95.76% (HAS28) to 107.28% (ADSS), and the R2 ranged from 0.9801 (RPII) to 0.999 (ACT)

Summary

Introduction

Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used method to analyze the expression level of functional genes and has become the preferred method to quantify mRNA, as this method meets the requirements for quantitative analyses of data in molecular medical, biotechnological, microbiological, and diagnostic-type studies [1,2]. The benefits of this technique compared with traditional RNA measurement methods include its good specificity, high accuracy, wide detection range, simple operation, and safety. Several methods, including the delta cycle threshold (∆Ct) method [12] and the use of software programs such as geNorm [13], BestKeeper [14], NormFinder [15], and RefFinder [16], have been commonly used to select the appropriate reference genes

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Discussion

Conclusion