| Summary: | <i>Ginkgo biloba</i>, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in <i>G. biloba</i>. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of <i>Ginkgo biloba</i>. These results showed that elongation factor 1(<i>EF1</i>) and ubiquitin (<i>UBI)</i> were the best choices for samples of different ginkgo genotypes. The expression of <i>UBI</i> and <i>HAS28</i> presented the most stable at different developmental stages of ginkgo, and <i>EIF3I</i> and <i>RPII</i> were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, <i>ACA</i> and <i>ACT</i> were identified as the optimal reference genes. For cold stress treatment, <i>RPII</i> and <i>EIF4E</i> were chosen for the gene expression normalizations. <i>HAS28</i> and <i>GAPDH</i> presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (<i>GbCHS</i>) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.
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