Ginkgo biloba, 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 G. biloba. 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 Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) 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.

WRKY transcription factors are important regulators of diverse plant life processes. Our aim was to clone and characterize GbWRKY20, a WRKY gene of group IIc, derived from Ginkgo biloba. The cDNA sequence of GbWRKY20 was 818 bp long, encoding a 271-amino acid proteins and containing two introns and three exons. The proteinic molecular weight was 30.99 kDa, with a relevant theoretical isoelectric point of 8.15. Subcellular localization analysis confirmed that the GbWRKY20 protein localized to the nucleus. In total, 75 cis-regulatory elements of 19 different types were identified in the GbWRKY20 promoter sequence, including some elements involved in light responsiveness, anaerobic induction and circadian control, low-temperature responsiveness, as well as salicylic acid (SA) and auxin responsiveness. Expression pattern analysis of plant samples from different developmental stages and tissue types, revealed differential GbWRKY20 expression. The GbWRKY20 transcript was downregulated 12 h after heat treatment and at 4–12 h after drought treatment, but was upregulated 12 h after NaCl, cold and methyl jasmonate treatments. For abscisic acid and SA treatments, the GbWRKY20 transcript was upregulated at 24 h. In summary, GbWRKY20 encoded a newly cloned WRKY transcription factor of G. biloba that might be involved in plant growth and plant responses to abiotic stresses and hormones treatments.

DNA methylation plays a vital role in diverse biological processes. DNA methyltransferases (DNMTs) genes and RNA-directed DNA methylation (RdDM)-related genes are key genes responsible for establishing and maintaining genome DNA methylation in plants. In the present study, we systematically identified nine GbDNMTs in Ginkgo biloba, including the three common families of GbMET1a/1b, GbCMT2, and GbDRMa/b/2a/2b/2c, and a fourth family—GbDNMT3—which is absent in most angiosperms. We also identified twenty RdDM-related genes, including four GbDCLs, six GbAGOs, and ten GbRDRs. Expression analysis of the genes showed the different patterns of individual genes, and 15 of 29 genes displayed expression change under five types of abiotic stress. Gene coexpression analysis and weighted gene co-expression network analysis (WGCNA) using 126 public transcriptomic datasets revealed that these genes were clustered into two groups. In group I, genes covered members from all six families which were preferentially expressed in the ovulate strobile and fruit. A gene ontology (GO) enrichment analysis of WGCNA modules indicated that group I genes were most correlated with the biological process of cell proliferation. Group II only consisted of RdDM-related genes, including GbDRMs, GbAGOs, and GbRDRs, but no GbDCLs, and these genes were specifically expressed in the cambium, suggesting that they may function in a dicer-like (DCL)-independent RdDM pathway in specific tissues. The gene module related to group II was most enriched in signal transduction, cell communication, and the response to the stimulus. These results demonstrate that gene family members could be conserved or diverged across species, and multi-member families in the same pathway may cluster into different modules to function differentially. The study provides insight into the DNA methylation genes and their possible functions in G. biloba, laying a foundation for the further study of DNA methylation in gymnosperms.