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Selection of suitable reference genes for quantitative real time PCR in different Tulasnella isolates and orchid-fungus symbiotic germination system

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Abstract

Under natural conditions, mycorrhizal symbiosis accompanies nearly the entire life cycle of orchids from seed germination through to flowering and fruiting. Tulasnella-like orchid mycorrhizal fungi are the most common mycorrhizal fungi found in association with orchid species. Presently suitable reference genes have not been systematically selected for the quantification of gene expression via Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). We evaluated 12 candidate Tulasnella genes in nine different Tulasnella isolates and in the Dendrobium-fungal symbiotic germination associations followed by statistical analysis using the programs Bestkeeper, geNorm, and Normfinder to analyze the expression stability of the individual genes. The results showed that the EF2, UBC, and PP2A genes had the highest rankings with relatively stable expression levels across the different genotypes and during the symbiotic seed germination process by the three programs, and may be suitable for RT-qPCR normalization. Furthermore, the gene encoding C-5 Sterol desaturase (C5SD) was selected to verify the reliability of EF2, UBC, and PP2A expression during the Tulasnella-Dendrobium symbiotic seed germination process. This study is the first systematic exploration of optimal reference genes for gene expression studies during the colonization of orchid seeds by the mycorrhizal fungus Tulasnella.

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Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (No. 31700547), and the Beijing Botanical Garden (No. BZ201901).

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  1. Tao Wang and Xiaolu Cao have been equally contributed to this work.

Authors and Affiliations

  1. State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of the National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, China

    Tao Wang, Xiaolu Cao, Xiaojing Wang, Miao Chi, Lubin Li & Na Yao

  2. Beijing Laboratory of Urban and Rural Ecological Environment, Beijing Floriculture Engineering Technology Research Centre, Beijing Botanical Garden, Beijing, 100093, China

    Tao Wang & Miao Chi

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  1. Tao Wang
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Correspondence to Na Yao.

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11033_2020_6085_MOESM1_ESM.pdf

Supplementary file1 (PDF 649 KB) Fig S1. Melting curves of 12 candidate reference genes and a C5SD gene exhibiting only single peaks.

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Wang, T., Cao, X., Wang, X. et al. Selection of suitable reference genes for quantitative real time PCR in different Tulasnella isolates and orchid-fungus symbiotic germination system. Mol Biol Rep 48, 527–538 (2021). https://doi.org/10.1007/s11033-020-06085-z

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