*4.3. Exogenous GA<sup>3</sup> Treatment on Symbiotic and Asymbiotic Germination of D. o*ffi*cinale*

The concentration of exogenous GAs was selected in preliminary experiments, which spanned 0.05 µM to 1 µM, according to a previous study [19]. Seeds were sown in OMA medium (for SG with fungus) and <sup>1</sup> <sup>2</sup> MS medium (for AG without fungus) with four concentrations of exogenous GA<sup>3</sup> (0.05, 0.1, 0.5, and 1 µM; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). We designed four groups, including seeds in OMA medium (no fungus), fungus in OMA medium (no seeds) with different concentrations of exogenous GA3, and the normal AG group (seeds in 1/2MS without fungus) and SG group (seed in OMA medium with fungus) without exogenous GA3. Next, the petri dishes were incubated at 25 ◦C under a 12-h/12-h light/dark (L/D) cycle. Morphological changes during seed germination were observed daily, under a stereomicroscope and a Leica light microscope DM2500 (Leica Microsystems, Wetzlar, Germany).

#### *4.4. Transcriptome Analysis by RNA-Seq*

Transcriptome analysis of the eight samples was performed in our previous study; the samples corresponded to three different germination stages (stage 2, stage 3, and stage 4) of symbiotic and asymbiotic seeds, respectively, and to free-living mycelium of mycorrhizal fungus and ungerminated seeds [7]. The original transcriptomic data was deposited in the public NCBI and SRA database (accession No. PRJNA279934). Based on this transcriptomic data, we screened the putative genes involved in biosynthesis and signal transduction of plant hormones, the common symbiotic pathway, and specific gene expression in SG of *D. o*ffi*cinale.*

## *4.5. RNA Extraction and Quantitative Real-Time PCR*

RNA extraction and quantitative RT-PCR was performed as described in our prior study [7]. Briefly, total RNA was extracted from 200 mg of seeds using the RNeasy Plant Mini Kit (Qiagen, CA, USA) and treated with an RNase-free DNase I digestion kit (Beijing Aidlab Biotech Company, Beijing, China) to remove any residual genomic DNA. Then 1 µg of RNA was reverse-transcribed to cDNA, using a reverse transcription system (Bio-Rad Laboratories, Inc., Richmond, CA, USA), and the cDNA equivalent to 25 ng of total RNA served as a template for each PCR reaction, carried out using SYBR Green supermix (Bio-Rad Laboratories, Inc., Richmond, CA, USA) with a final concentration of 1.6 mM of each primer. The primer sequences are listed in Table S1. The qRT-PCR experiments were done using a SYBR Premix Ex TaqTM (Takara Biotechnology Co., Ltd., Dalian, China) on the LightCycler 480 machine (Roche Applied, Mannheim, Germany). PCR amplifications of three biological replicates were performed, which also included three distinct technical replicates. A no-template control (i.e., RNase-free water) was included for every qPCR run. Transcript abundance was normalized using the housekeeping gene *EF-1*α and a given gene's expression level amount was calculated by the 2−∆∆C<sup>T</sup> method [44].

#### *4.6. Data Analysis*

The data of hormone content were analyzed with one-way ANOVA and the statistical analysis was performed using software SPSS 11.0. Data were presented as means ± SD from at least three independent experiments. *p* values < 0.05 were considered significant difference.

#### **5. Conclusions**

This study mainly explored the relationship between endogenous hormones and symbiotic germination of orchid *D. o*ffi*cinale* seeds and the effects upon seed germination from exogenous GA3. Endogenous hormonal change regulated the seed germination of *D. o*ffi*cinale* and mycorrhizal fungi invasion can greatly stimulate its host plant's endogenous IAA accumulation. This could explain the faster differentiation of the embryo at the protocorm stage during symbiotic germination. Exogenous GA<sup>3</sup> has a dose-dependent effect on the establishment of the mycorrhizal relationship between the fungus and seeds, such that a high concentration of GA<sup>3</sup> probably acts upon the genes or proteins of the common symbiotic pathway, thereby inhibiting the recognition between orchid seeds and mycorrhizal fungi to further influence seed germination. Gene expression of the putative mycorrhizal-induced and symbiotic signal pathway responds to exogenous GA<sup>3</sup> concentration change, implying GA<sup>3</sup> contributes to the crosstalk between the hormone biosynthetic pathway and common symbiotic signal pathway. This study lays a foundation for the further exploration of seed germination, especially the symbiotic germination mechanism of orchid seeds.

**Supplementary Materials:** The following figures are available online at http://www.mdpi.com/1422-0067/21/17/ 6104/s1. Table S1: Primer sequences used in qRT-PCR amplification. All primers were designed using Primer 3.0 software and synthesized by Genewiz Company (China).

**Author Contributions:** J.C. and S.G. designed the study. J.C. wrote the manuscript. B.Y. performed all experiments. Y.T., Y.X., Y.L. and D.Z. participated in data analysis and reference searching and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (81573527; 81973423) and the CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-I2M-3-013).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
