Selection of Suitable Reference Genes in <i>Pinus massoniana</i> Lamb. Under Different Abiotic Stresses for qPCR Normalization

• Main Authors: Peihuang Zhu, Yinyan Ma, Lingzhi Zhu, Yu Chen, Rong Li, Kongshu Ji
• Format: Article
• Language: English
• Published: MDPI AG 2019-07-01
• Series: Forests
• Subjects: <i>Pinus massoniana</i>; qPCR; reference gene; abiotic stresses
• Online Access: https://www.mdpi.com/1999-4907/10/8/632

The normalization of data by choosing suitable reference genes is fundamental for obtaining accurate and reliable results in quantitative real-time polymerase chain reaction (qPCR) analyses. In this study, the expression stability of 12 candidate reference genes of <i>Pinus massoniana</i> under different abiotic stresses was evaluated using four statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The results indicate that the following genes could be used as reference genes under different treatments: Actin 2 (<i>ACT2</i>) and F-box family gene (<i>F-box</i>) for salinity treatment, cyclophilin (<i>CYP</i>) and alpha-tubulin (<i>TUA</i>) for ABA treatment, actin 7 (<i>ACT7</i>) and <i>CYP</i> for drought treatment, actin 1 (<i>ACT1</i>) and <i>ACT7</i> for cold treatment, <i>ACT1</i> and <i>CYP</i> for heat treatment, and <i>TUA</i> and <i>ACT2</i> for the &#8220;Total&#8221; group. To validate the suitability of the selected reference genes in this study, the Short-Root protein (<i>SHR</i>), Alpha-pinene&#8194;synthase (<i>APS</i>), and Pyrabactin resistance-like protein (<i>PYL</i>) gene expression patterns were analyzed. The expression patterns had significant biases when the most unstable reference genes were used for normalization, compared with when the optimum reference gene or gene combinations were used for normalization. These results will be beneficial for further studies on gene transcription in early-stage, unlignified seedlings of <i>P. massoniana.</i>