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Background <p>Banana (<i>Musa</i> spp.) production in the Philippines is hampered by drought, underscoring the need for drought-tolerant cultivars. RT-qPCR (quantitative real-time polymerase chain reaction) is a powerful tool for analyzing gene expression but requires stable internal control genes for accurate normalization. However, there is limited data regarding reference gene stability in local banana cultivars under drought stress. Identifying a reliable internal control gene is essential for accurate differential gene expression analysis, which can support breeding efforts for drought-tolerant cultivars. This study aims to select the most stably expressed internal control gene among five candidates (<i>25S</i>, <i>ACT1</i>, <i>EF1</i>-<i>α</i>, <i>L2</i>, and <i>RPS2</i>) in drought-stressed ‘Lakatan’ (AAA) and ‘Saba’ (ABB/BBB) cultivars.</p> Methods and results <p>Under controlled greenhouse conditions, biological triplicates per cultivar were subjected to well-watered and drought-imposed treatments. After drought imposition, total RNA was extracted from the leaf tissues, followed by complementary DNA (cDNA) synthesis. Primer efficiency (<i>E</i>) per candidate gene was evaluated for optimization. Quantification cycle (C<sub>q</sub>) data was determined by RT-qPCR in technical triplicates using a universal SYBR<sup>®</sup> Green supermix, then subjected to statistical analyses using boxplot and RefFinder for gene stability evaluation. Candidate genes yielded <i>E</i> values of 99.3–101.8% and C<sub>q</sub> values of 15.6–32.0, adhering to MIQE (Minimum Information for Publication of qPCR Experiments) guidelines. Boxplot analysis and comprehensive gene stability values from RefFinder (integrating geNorm, NormFinder, BestKeeper, and △C<sub>q</sub> algorithms) identified <i>L2</i> as the most stable internal control gene followed by <i>RPS2</i>, <i>ACT1</i>, <i>EF1</i>-<i>α</i>, and <i>25</i><i>S</i> with resulting geometric mean rankings of 1.41, 1.57, 2.28, 4, and 5, respectively.</p> Conclusions <p>This study identifies <i>L2</i>, encoding the ribosomal protein L2, as the most stable internal control gene in drought-stressed bananas, a crucial prerequisite for differential gene expression analysis in drought-related studies. These findings allow for more accurate measurement of gene expression, thereby reinforcing the role of potential drought response genes that can be used for breeding climate-smart banana cultivars through marker-assisted selection and new breeding techniques.</p>

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Selection of stable internal control genes for quantitative real-time PCR (RT-qPCR) in banana (Musa spp.) under short-term drought stress

  • 期刊论文

  • 发表日期：

    2025年5月31日

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  • Mary Krizelle Veronica A. Paragas,
  • Elaine P. Laurio,
  • Jen Daine L. Nocum,
  • Marc Lawrence E. Francisco,
  • Darlon V. Lantican,
  • Jose Arnel O. Reyes,
  • Anna Mariel U. Toledo,
  • Anand Noel C. Manohar

摘要

Background

Banana (Musa spp.) production in the Philippines is hampered by drought, underscoring the need for drought-tolerant cultivars. RT-qPCR (quantitative real-time polymerase chain reaction) is a powerful tool for analyzing gene expression but requires stable internal control genes for accurate normalization. However, there is limited data regarding reference gene stability in local banana cultivars under drought stress. Identifying a reliable internal control gene is essential for accurate differential gene expression analysis, which can support breeding efforts for drought-tolerant cultivars. This study aims to select the most stably expressed internal control gene among five candidates (25S, ACT1, EF1-α, L2, and RPS2) in drought-stressed ‘Lakatan’ (AAA) and ‘Saba’ (ABB/BBB) cultivars.

Methods and results

Under controlled greenhouse conditions, biological triplicates per cultivar were subjected to well-watered and drought-imposed treatments. After drought imposition, total RNA was extracted from the leaf tissues, followed by complementary DNA (cDNA) synthesis. Primer efficiency (E) per candidate gene was evaluated for optimization. Quantification cycle (Cq) data was determined by RT-qPCR in technical triplicates using a universal SYBR® Green supermix, then subjected to statistical analyses using boxplot and RefFinder for gene stability evaluation. Candidate genes yielded E values of 99.3–101.8% and Cq values of 15.6–32.0, adhering to MIQE (Minimum Information for Publication of qPCR Experiments) guidelines. Boxplot analysis and comprehensive gene stability values from RefFinder (integrating geNorm, NormFinder, BestKeeper, and △Cq algorithms) identified L2 as the most stable internal control gene followed by RPS2, ACT1, EF1-α, and 25S with resulting geometric mean rankings of 1.41, 1.57, 2.28, 4, and 5, respectively.

Conclusions

This study identifies L2, encoding the ribosomal protein L2, as the most stable internal control gene in drought-stressed bananas, a crucial prerequisite for differential gene expression analysis in drought-related studies. These findings allow for more accurate measurement of gene expression, thereby reinforcing the role of potential drought response genes that can be used for breeding climate-smart banana cultivars through marker-assisted selection and new breeding techniques.