Internal Reference Gene Selection under Different Hormone Stresses in Multipurpose Timber Yielding Tree <i>Neolamarckia cadamba</i>

• Main Authors: Deng Zhang, Jingjian Li, Buye Li, Chunmei Li, Xiaoyang Chen, Kunxi Ouyang
• Format: Article
• Language: English
• Published: MDPI AG 2020-09-01
• Series: Forests
• Subjects: <i>Neolamarckia cadamba</i>; RT-qPCR; reference genes; hormone stresses
• Online Access: https://www.mdpi.com/1999-4907/11/9/1014

<i>Neolamarckia cadamba</i>, a member of the Rubiaceae family, is widely distributed throughout South Asia and South China. In order to acquire reliable and repeatable results, the use of a suitable internal reference gene to normalize the RT-qPCR data is essential. In this study, we reported the validation of housekeeping genes to identify the most suitable internal reference gene(s) for normalization of qPCR data obtained among different tissues (bud, leaf, cambium region) under different hormone stresses. Here, ΔCt, geNorm, NormFinder, and BestKeeper analyses were carried out to analyze the normalization of qPCR data of twenty-one reference gene families (<i>ACT, CAC, CYP, EF1α, eIF, FPS1, FBK, GAPDH, RAN, PEPKR1, PP2A, RPL, RPS, RuBP, SAMDC, TEF, Tub-α, Tub-β, UBCE, UBQ, UPL</i>) including 43 genes. The results showed that <i>FPS1</i>, <i>RPL</i>, and <i>FBK</i> were the most stable reference genes across all of the tested samples. In addition, the expression of <i>NcEXPA8</i>, one gene of interest that plays an important role in regulating cell wall extension, under different phytohormone stresses was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene selection under different phytohormone stresses and a foundation for more accurate and widespread use of RT-qPCR in <i>N. cadamba</i>.