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. 2023 Feb 8;12(4):763.
doi: 10.3390/plants12040763.

Screening and Verification of Reference Genes for Analysis of Gene Expression in Garlic (Allium sativum L.) under Cold and Drought Stress

Qizhang Wang  1   2 , Chunqian Guo  1 , Shipeng Yang  1 , Qiwen Zhong  1 , Jie Tian  1
Affiliations

Screening and Verification of Reference Genes for Analysis of Gene Expression in Garlic (Allium sativum L.) under Cold and Drought Stress

Qizhang Wang et al. Plants (Basel). .

Abstract

The principal objective of this study was to screen and verify reference genes appropriate for gene expression evaluation during plant growth and development under distinct growth conditions. Nine candidate reference genes were screened based on garlic transcriptome sequence data. RT-qPCR was used to detect the expression levels of the aforementioned reference genes in specific tissues under drought and cold stress. Then, geNorm, NormFinder, BestKeeper, and ReFinder were used to consider the consistency of the expression levels of candidate reference genes. Finally, the stress-responsive gene expression of ascorbate peroxidase (APX) was quantitatively evaluated to confirm the chosen reference genes. Our results indicated that there were variations in the abundance and stability of nine reference gene transcripts underneath cold and drought stress, among which ACT and UBC-E2 had the highest transcript abundance, and 18S rRNA and HIS3 had the lowest transcript abundance. UBC and UBC-E2 were the most stably expressed genes throughout all samples; UBC and UBC-E2 were the most stably expressed genes during cold stress, and ACT and UBC were the most stably expressed genes under drought stress. The most stably expressed genes in roots, pseudostems, leaves, and cloves were EF1, ACT, HIS3, UBC, and UBC-E2, respectively, while GAPDH was the most unstable gene during drought and cold stress conditions and in exclusive tissues. Taking the steady reference genes UBC-E2, UBC, and ACT as references during drought and cold stress, the reliability of the expression levels was further demonstrated by detecting the expression of AsAPX. Our work thereby offers a theoretical reference for the evaluation of gene expression in garlic in various tissues and under stress conditions.

Keywords: RT-qPCR; garlic; gene expression; normalization; reference gene.

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Conflict of interest statement

All authors have read and approved this version of the article. No conflicts of interest exist in the submission of this manuscript.

Figures

Figure 1
Figure 1
Garlic RNA samples (A) and amplification products of candidate reference genes (B) detected by agarose electrophoresis.
Figure 2
Figure 2
RT−qPCR melting curves of the nine candidate reference genes in garlic.
Figure 3
Figure 3
Distribution of Ct values of the nine candidate reference genes in garlic among all the tested samples. The boxes indicate the 25th and 75th percentiles. The line across the box and the inner square in each box indicate the median and mean Ct values, respectively.
Figure 4
Figure 4
Mean expression stability values (M) of candidate reference genes. Note: The most stable genes are listed on the right and the most unstable genes are listed on the left.
Figure 5
Figure 5
Determination of optimal parameters for candidate reference genes.
Figure 6
Figure 6
AsAPX expression in different parts of garlic under drought(A) and cold(B)stress.
See this image and copyright information in PMC

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References

    1. Ried K., Frank O., Stocks N. Aged garlic extract reduces blood pressure in hypertensives: A dose–response trial. Eur. J. Clin. Nutr. 2013;67:64–70. doi: 10.1038/ejcn.2012.178. - DOI - PMC - PubMed
    1. Zhu J.-K. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 2002;53:247–273. doi: 10.1146/annurev.arplant.53.091401.143329. - DOI - PMC - PubMed
    1. Farahani H.A., Valadabadi S.A., Daneshian J., Shiranirad A.H., Khalvati M.A. Medicinal and aromatic plants farm-ing under drought conditions. J. Hortic. For. 2009;1:086–092.
    1. Ji L., Zhou P., Zhu Y., Liu F., Li R., Qiu Y. Proteomic Analysis of Rice Seedlings Under Cold Stress. Protein J. 2017;36:299–307. doi: 10.1007/s10930-017-9721-2. - DOI - PubMed
    1. Feng D.-R., Liu B., Li W.-Y., He Y.-M., Qi K.-B., Wang H.-B., Wang J.-F. Over-expression of a cold-induced plasma membrane protein gene (MpRCI) from plantain enhances low temperature-resistance in transgenic tobacco. Environ. Exp. Bot. 2009;65:395–402. doi: 10.1016/j.envexpbot.2008.12.009. - DOI