Abstract
Gene expression valuated by reverse transcription-quantitative PCR (RT-qPCR) are often applied to study the gene function. To obtain accurate and reliable results, the usage of stable reference genes is essential for RT-qPCR analysis. The traditional southern Chinese medicinal herb, Desmodium styracifolium Merr is well known for its remarkable effect on the treatment of urination disturbance, urolithiasis, edema and jaundice. However, there are no ready-made reference genes identified for D. styracifolium. In this study, 13 novel genes retrieved from transcriptome datasets of four different tissues were reported according to the coefficient of variation (CV) and maximum fold change (MFC) of gene expression. The expression stability of currently used Leguminosae ACT6 was compared to the 13 candidate reference genes in different tissues and 7-day-old seedlings under different experimental conditions, which was evaluated by five statistical algorithms (geNorm/NormFinder/BestKeeper/ΔCT/RefFinder). Our results indicated that the reference gene combinations of PP + UFM1, CCRP4 + BRM and NFD6 + NCLN1 were the most stable reference genes in leaf, stem and root tissues, respectively. The most stable reference gene combination for all tissues was CCRP4 + CUL1. In addition, the most stable reference genes for different experimental conditions were distinct, for instance SMUP1 for MeJA treatment, ERDJ2A + SMUP1 for SA treatment, NCLN1 + ERDJ2A for ABA treatment and SF3B + VAMP721d for salt stress, respectively. Our results lay a foundation for achieving accurate and reliable RT-qPCR results so as to correctly understand the function of genes in D. styracifolium.
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Funding
This work was partially supported by Grants from Key Area R&D Project of Guangdong Province (2020B020221001), Guangdong Provincial Key Laboratory of Applied Botany (AB2018017), Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams, China (NO.2019KJ148), Youth Innovation Promotion Association CAS (2015286).
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This research was designed by FX, SZ and YW; ZW, FY and DS carried out the experiments; ZW performed data analysis; ZW prepared the hormonal treatment and salt stress samples; ZW drafted the manuscript; FX, SZ and YW revised the manuscript. All authors have read and agreed to the published version of the manuscript.
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Supplementary file 1: Supplementary tables. Table S1 The rough candidate genes whose CV was lower than 12%. Table S2 The candidate reference genes selected according to RNA-seq data. Supplementary figures. Figure S1 Electrophoresis analysis of the specificity of primer pairs for RT-PCR amplification (a) and the RNA quality (b). In the Fig. S1(a), ‘1–16’ represent VAMP721d, PP, CCRP4, SF3B, ARI7, BRM, UFM1, NFD6, UN, SMUP1, CUL1, NCLN1, ERDJ2A, ACT6, DsCHS and 2000 bp marker, respectively. In the Fig. S1(b), ‘0’ represent the 1kb ladder marker, and ‘1–21’ represent the RNA from tissues (root, stem and leaf), MeJA, ABA, SA and salt treatment, each sample has three biological repeats. Figure S2 Dissociation curves for the reference genes tested in this study. The dissociation curves for each candidate gene showed a single peak indicated that the primers of each tested gene were specific. (DOCX 297 KB)
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Wang, Z., Yu, F., Shi, D. et al. Selection and validation of reference genes for RT-qPCR analysis in Desmodium styracifolium Merr. 3 Biotech 11, 403 (2021). https://doi.org/10.1007/s13205-021-02954-x
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DOI: https://doi.org/10.1007/s13205-021-02954-x