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Volume 14
Issue 19
10.3390/plants14193101
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Article

EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions

by
Lei Huang
1,
Yuyi Zhang
2,
Fei Gao
1,
Yu Fu
1,
Jing Sun
1,
Jie Zhou
3,
Jun Tao
1,
Xudong He
3,* and
Nan Guo
1,*
1
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
2
Jiangsu Key Laboratory for Conservation and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Memorial Sun Yat-Sen), Nanjing 210014, China
3
National Willow Engineering Technology Research Center, Jiangsu Academy of Forestry, Nanjing 211153, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(19), 3101; https://doi.org/10.3390/plants14193101
Submission received: 26 August 2025 / Revised: 30 September 2025 / Accepted: 7 October 2025 / Published: 8 October 2025
(This article belongs to the Section Plant Molecular Biology)
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Abstract

Salix suchowensis is an ideal model organism for investigating nitrogen (N) transport mechanisms due to its low N-input requirements. Accurate quantification of gene expression is essential for elucidating these processes, with quantitative real-time PCR (RT-qPCR) being the preferred method. However, the identification of stable reference genes for normalization in Salix suchowensis under varying N conditions remains unresolved. In this study, thirteen commonly employed candidate reference genes were evaluated across root, stem, and leaf tissues, under four N treatments (NH4NO3, NH4+, NO3−, and N deficiency). Five genes (UBQ1, UBQ3, 18S, H2A2, and H2B2) were excluded due to poor amplification efficiency or irregular melting curves. The remaining eight genes were further assessed for expression stability using the geNorm, NormFinder, and BestKeeper algorithms. Integrated ranking via RefFinder identified EF1α, EFβ, and αTUB as the most stable reference genes. GeNorm analysis suggested that two reference genes were sufficient for reliable normalization. Validation using the N-responsive gene SsAMT1 and SsNRT2 confirmed the stability of EF1α, EFβ, and αTUB as suitable reference genes. Based on comprehensive stability assessments and experimental validation, we recommended EF1α + αTUB as optimal reference gene pairs for RT-qPCR normalization under varying N conditions. Furthermore, the consistent expression of EF1α and αTUB across nine willow genotypes highlighted their broader applicability within Salix species. This study provides valuable methodological guidance for advancing molecular research on N transport in woody perennial plants.
Keywords: Salix suchowensis; nitrogen; reference gene; RT-qPCR Salix suchowensis; nitrogen; reference gene; RT-qPCR

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MDPI and ACS Style

Huang, L.; Zhang, Y.; Gao, F.; Fu, Y.; Sun, J.; Zhou, J.; Tao, J.; He, X.; Guo, N. EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions. Plants 2025, 14, 3101. https://doi.org/10.3390/plants14193101

AMA Style

Huang L, Zhang Y, Gao F, Fu Y, Sun J, Zhou J, Tao J, He X, Guo N. EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions. Plants. 2025; 14(19):3101. https://doi.org/10.3390/plants14193101

Chicago/Turabian Style

Huang, Lei, Yuyi Zhang, Fei Gao, Yu Fu, Jing Sun, Jie Zhou, Jun Tao, Xudong He, and Nan Guo. 2025. "EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions" Plants 14, no. 19: 3101. https://doi.org/10.3390/plants14193101

APA Style

Huang, L., Zhang, Y., Gao, F., Fu, Y., Sun, J., Zhou, J., Tao, J., He, X., & Guo, N. (2025). EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions. Plants, 14(19), 3101. https://doi.org/10.3390/plants14193101

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