Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (5): 92-102.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1322

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Screening of Reference Genes for RT-qPCR in Neoscytalidium dimidiatum

YAO Zi-ting1(), CAO Xue-ying2, XIAO Xue2, LI Rui-fang1, WEI Xiao-mei1, ZOU Cheng-wu2(), ZHU Gui-ning1()   

  1. 1. Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Giuangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China Ministry of Agriculture and Rural Affairs, Nanning 530007
    2. College of Agriculture, Guangxi University, Nanning 530004
  • Received:2022-10-26 Online:2023-05-26 Published:2023-06-08
  • Contact: ZOU Cheng-wu, ZHU Gui-ning E-mail:youziting@163.com;zouchengwu@163.com;gnzhu@126.com

Abstract:

The pitaya canker disease is a serious fungal disease caused by Neoscytalidium dimidiatum. In recent years, the outbreak of the pitaya canker disease in the world's pitaya producing areas has seriously affected the pitaya industry development. In order to study the expression level and function of genes responding to stress in N. dimidiatum, it is necessary to screen the reference genes that express stably when the pathogen grows under different cultural conditions. The N. dimidiatum strain LJ02 mycelia cultured in potato dextrose liquid medium(PDW)were taken as the control group, and the mycelia cultured in LB medium, and the mycelia cultured in PDW medium supplemented with hydrogen peroxide, pyrazolyl ester and difenoconazole, respectively, were taken as the treatment groups. The expression stabilities of 18 candidate reference genes(CYT1, SDH2_1, TBCC, SUI1, RPL19, PPH, ATP5B, UBE2_16, RPL13, UBE2_2, PRS17, SUCLA2, ATP5A, TUB1_2, ACT1, EFTU, EF1A and GAPDH)were evaluated by real-time quantitative PCR(RT-qPCR)and stability evaluation software(geNorm, NormFinder, Bestkeeper and RefFinder). Based on the results of RT-qPCR and software analysis, it was found that the Ct values of 18 candidate genes were within 14.80-24.66 under different culture conditions, with moderate expression levels. The reference gene with the highest stability was CYT1, followed by SUI1, while GAPDH and ACT1 were with the worst expression stability. Using at least two reference genes may improve analysis accuracy of RT-qPCR. At this time, SUCLA2 and ATP5A is the most stable reference gene combination. The results provide a theoretical basis for gene expression analysis in the biological process of N. dimidiatum.

Key words: Neoscytalidium dimidiatum, reference gene, real-time quantitative PCR, mycelial growth, expression analysis, abiotic stres, reference gene analysis software