Determination of reliable reference genes for gene expression studies in Chinese chive (Allium tuberosum) based on the transcriptome profiling

Jing Tong,Mingchi Liu,Beibei Han,Baoju Wang,Ning Liu,Yanhai Ji,Hao Liang,Zhanhui Wu,Manman Hu

doi:10.1038/s41598-021-95849-z

Jing Tong, Mingchi Liu + Show 7 more

Open Access

https://doi.org/10.1038/s41598-021-95849-z

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Abstract

Chinese chive (Allium tuberosum) is widely cultivated around the world for its unique flavor, nutrient, and medicinal values, yet its molecular mechanism on flavor formation and other metabolic pathways remains intangible. The elucidation of these complex processes begins with investigating the expression of the genes of interest, however the appropriate reference genes (RGs) for normalizing the gene expression are still unavailable in A. tuberosum. To fill this lacuna, transcriptome-wide screening was undertaken to identify the most stable genes according to the analysis of their FPKM values. The expression stability of the RGs was further evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The comprehensive analysis showed that GLY1 and SKP1, instead of two traditionally used RGs (eIF1α and ACT2), were the most stable genes across diverse A. tuberosum tissues, indicating the necessity to carefully validate the stability of RGs prior to their use for normalizations. As indicated by geNorm, the normalizations with at least two RGs could give more accurate results. qRT-PCR experiments were conducted with randomly selected genes, demonstrating that normalization with a combination of GLY1 and SKP1 resulted in reliable normalization results. Our finding represents the first attempt toward establishing a standardized qRT-PCR analysis in this economically important vegetable.

Highlights

Chinese chive (Allium tuberosum Rottler ex Spr), a perennial herb plant native to North China, has become more and more popular in the food inventory owing to its garlicky flavor, abundant nutrient, and great medicinal properties[1,2,3]
Two commonly used traditional reference genes (RGs), eIF1α, and ACT2 in Chinese chive were included in the comprehensive analysis by algorithms such as geNorm, NormFinder, and BestKeeper that assess the stability of genes based on the variance of quantification cycle values in each tissue samples
Commonly used as RGs, are expressed at relatively high levels, the first step was attempted to identify the genes with high expressions in all A. tuberosum tissues

Summary

Introduction

Chinese chive (Allium tuberosum Rottler ex Spr), a perennial herb plant native to North China, has become more and more popular in the food inventory owing to its garlicky flavor, abundant nutrient, and great medicinal properties[1,2,3]. The ideal RGs, in the relative quantifications, should be expressed constantly and stably in all examined samples regardless of experimental conditions, such as different developmental stages, biological processes, treatments, and even different organs or tissues[19,20]. It is a huge challenge, in reality, to identify such an RG that meets the criterion. We propose GLY1 and SKP1 are the preferred internal control genes for normalizing gene expression across diverse tissues of Chinese chive. This study is the first analysis of the validation of RGs for the accurate determination of transcriptional patterns in Chinese chive and will facilitate expression studies on the genes associated development in this Allium crop

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