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Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses

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Abstract

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This study identified stable reference genes for normalization of gene expression data in qRT-PCR analysis of leaf and root tissues in creeping bentgrass under four abiotic stresses.

Abstract

Examination of gene expression using quantitative real-time PCR (qRT-PCR) in plant responses to abiotic stresses can provide valuable information for stress-tolerance improvement. Selecting stable reference genes for qRT-PCR analysis is critically important. The objective of this study was to determine the stability of expression for eight candidate reference genes (ACT, EF1a, TUB, UPL7, GAPDH, PP2A, PEPKR1, and CACS) in two tissues (roots and leaves) of a perennial grass species under four abiotic stresses (salt, drought, cold, and heat) using four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). The results showed that (1) the combinations of CACS and UPL7 or PP2A and ACT were stably expressed in salt-treated roots or leaves; (2) the combinations of GAPDH and CACS or PP2A and PEPKR1 were stable in roots and leaves under drought stress; (3) CACS and PP2A exhibited stable expression in cold-treated roots and the combination of EF1a and UPL7 was also stable in cold-treated leaves; and (4) CACS and PP2A were the two most stable reference genes in heat-stressed roots and UPL7 combined with GAPDH and PP2A was stably expressed in heat-stressed leaves. The qRT-PCR analysis of a target gene, AsSAP expression patterns in response to salinity and drought stress, confirmed the reliability of those selected and stable reference genes. Identification of stable reference genes in creeping bentgrass will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mechanisms conferring stress tolerance in this species.

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Acknowledgments

This work was supported by the China Postdoctoral Science Foundation (2014M551612) and Jiangsu Postdoctoral Science Foundation (1302018B).

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    Authors and Affiliations

    1. College of Agro-grassland Science, Nanjing Agricultural University, Nanjing, 210095, China

      Yu Chen, Baoyun Hu, Jun Liu, Zhimin Yang & Zhihua Li

    2. College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China

      Zhiqun Tan

    3. Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ, 08901, USA

      Bingru Huang

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    1. Yu Chen
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    Correspondence to Zhimin Yang or Bingru Huang.

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    Communicated by Z.-Y. Wang.

    Y. Chen and B. Hu contributed equally to the article.

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    299_2015_1830_MOESM1_ESM.tif

    Fig S1 Primer specificity and amplicon size. Agarose gel (1.8 %) electrophoresis indicates amplification of a single PCR product of the expected size for 9 genes (Number 1-9: ACT, EF1α, TUB, PP2A, UPL7, GAPDH, PEPKR1, CACS, and AsSAP). Melting curves of 9 genes show single peaks. M represents 100 bp DNA marker. (TIFF 3658 kb)

    299_2015_1830_MOESM2_ESM.tif

    Fig S2 Pairwise variation (V) of the candidate reference genes calculated by GeNorm. Vn/Vn+1 values were used for decision of the optimal number of reference genes. (TIFF 268 kb)

    Table S1 Amplification Efficiency of qRT-PCR of eight reference genes and a target gene. (DOC 36 kb)

    Table S2 Stability analysis of reference genes assayed by NormFinder software. (DOCX 19 kb)

    Table S3 Stability analysis of reference genes assayed by BestKeeper software. (DOCX 19 kb)

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    Chen, Y., Hu, B., Tan, Z. et al. Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses. Plant Cell Rep 34, 1825–1834 (2015). https://doi.org/10.1007/s00299-015-1830-9

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    • DOI: https://doi.org/10.1007/s00299-015-1830-9

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