Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

Guanglong Wang; Chang Tian; Yunpeng Wang; Faxiang Wan; Laibao Hu; Aisheng Xiong; Jie Tian

doi:10.7717/peerj.7319

Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

PeerJ. 2019 Jul 16:7:e7319. doi: 10.7717/peerj.7319. eCollection 2019.

Authors

Guanglong Wang^#¹, Chang Tian^#², Yunpeng Wang¹, Faxiang Wan¹, Laibao Hu¹, Aisheng Xiong³, Jie Tian⁴

Affiliations

¹ School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, Jiangsu, China.
² Key Laboratory of Landscape Agriculture, Ministry of Agriculture, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, China.
³ State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, China.
⁴ Academy of Agriculture and Forestry Sciences of Qinghai University (Qinghai Academy of Agriculture and Forestry Sciences), Qinghai Key Laboratory of Vegetable Genetics and Physiology, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Qinghai, China.

^# Contributed equally.

Abstract

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

Keywords: Garlic; Gene expression; Reference genes; Salt stress; qRT-PCR.

Grants and funding

This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20170460), Scientific Research Foundation for Doctor from Huaiyin Institute of Technology (No. Z301B16531), the Fundamental Research Project of Qinghai Science & Technology Department (No. 2017-ZJ-925Q), the Project of Qinghai Science & Technology Department (No. 2016-ZJ-Y01), and the Open Project of State Key Laboratory of Plateau Ecology and Agriculture of Qinghai University (No. 2016-ZZ-06). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.