Paragas Mary Krizelle Veronica A, Laurio Elaine P, Nocum Jen Daine L, Francisco Marc Lawrence E, Lantican Darlon V, Reyes Jose Arnel O, Toledo Anna Mariel U, Manohar Anand Noel C
Institute of Plant Breeding, College of Agriculture and Food Science, University of the Philippines Los Baños, 4031, College, Laguna, Philippines.
Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños, 4031, College, Laguna, Philippines.
Mol Biol Rep. 2025 May 31;52(1):526. doi: 10.1007/s11033-025-10617-w.
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.
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 (C) 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 C 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 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.
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.
菲律宾的香蕉（Musa spp.）生产受到干旱的阻碍，这凸显了对耐旱品种的需求。逆转录定量聚合酶链反应（RT-qPCR）是分析基因表达的有力工具，但需要稳定的内参基因进行准确的标准化。然而，关于干旱胁迫下当地香蕉品种中参考基因稳定性的数据有限。确定一个可靠的内参基因对于准确的差异基因表达分析至关重要，这可以支持耐旱品种的育种工作。本研究旨在从五个候选基因（25S、ACT1、EF1-α、L2和RPS2）中筛选出在干旱胁迫下的‘拉卡坦’（AAA）和‘萨巴’（ABB/BBB）品种中表达最稳定的内参基因。
在可控的温室条件下，每个品种设置三个生物学重复，分别进行充分浇水和干旱胁迫处理。干旱胁迫处理后，从叶片组织中提取总RNA，然后进行互补DNA（cDNA）合成。评估每个候选基因的引物效率（E）以进行优化。使用通用的SYBR Green混合试剂通过RT-qPCR对技术重复样本测定定量循环（C）数据，然后使用箱线图和RefFinder进行统计分析以评估基因稳定性。候选基因的E值在99.3%至101.8%之间，C值在15.6至32.0之间，符合MIQE（qPCR实验发表的最低信息）指南。箱线图分析和RefFinder（整合geNorm、NormFinder、BestKeeper和△C算法）得出的综合基因稳定性值确定L2是最稳定的内参基因，其次是RPS2、ACT1、EF1-α和25S，其几何平均排名分别为1.41、1.57、2.28、4和5。
本研究确定编码核糖体蛋白L2的L2是干旱胁迫香蕉中最稳定的内参基因，这是干旱相关研究中差异基因表达分析的关键前提条件。这些发现有助于更准确地测量基因表达，从而加强潜在干旱响应基因的作用，这些基因可用于通过标记辅助选择和新的育种技术培育适应气候变化的香蕉品种。
